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The introduction of a stable GNRA tetra-loop in one of the hairpins in the JN2D and JN3LH and JN4LH RNAs was expected to increase the folding rates of those hairpins, because of the extra thermodynamic stability of these loops. However, the experimentally determined equal (JN2D) and 2 to 1 (JN3LH and JN4LH) folding ratios clearly showed that this was not the case. Rather, the JN3LH and JN4LH RNAs showed, together with the JN1LH and JN2LH fragments, that the folding of either the rod-like or double-hairpin structure is instead determined largely by its number of nucleation points .
|
16855293_p33
|
16855293
|
DISCUSSION
| 4.187705 |
biomedical
|
Study
|
[
0.999178946018219,
0.00025038610328920186,
0.0005706241354346275
] |
[
0.9993116855621338,
0.0004115590709261596,
0.00021935398399364203,
0.00005748960029450245
] |
en
| 0.999998 |
The fact that the GNRA tetra-loops show no kinetic effects suggests that the additional stabilizing interactions in these loops form after the formation of the first stacking interaction of the stem. This is not difficult to imagine. The specific interactions within the stable tetra-loops require a precise positioning and orientation of the bases, which is not necessarily achieved rapidly. Therefore, the decision to zipper the stem or not could therefore be taken while the loop still has the stability of a regular four-membered loop. Although not tested here, the same might hold true for YNMG tetra-loops as well.
|
16855293_p34
|
16855293
|
DISCUSSION
| 4.035892 |
biomedical
|
Study
|
[
0.9978881478309631,
0.0002930562768597156,
0.0018187768291682005
] |
[
0.9973952770233154,
0.002067886060103774,
0.00045375159243121743,
0.00008312323916470632
] |
en
| 0.999998 |
In the JN6A RNA fragment the folding of the 5′-hairpin loop with the nucleotide composition C UUUCU G is favoured kinetically over the 3′ end C GUGAG G loop. The likely reason is that the 3′-loop region is less flexible because of the rigid G and A stacks at the start of the folding process, hence the 70/30 ratio observed experimentally. The disruption of single-stranded A and G interactions requires ∼0.5 kcal·mol −1 per stack broken in DNA strands ( 32 ). In DNA hairpins, poly(T) loops fold ∼5 times faster than poly(A) loops ( 32 – 34 , 37 – 39 ). If purine–purine stacking also slows down the formation of RNA hairpins, this could explain the consensus CUNUNUG loop sequence in the metastable 5′ end structure of the hok mRNA family ( 38 , 40 ), from which the C UUUCU G loop studied here, was derived. If this loop sequence is particularly prone to form a hairpin loop, then this could provide the driving force behind the rapid folding of these metastable 5′ end hairpins.
|
16855293_p35
|
16855293
|
DISCUSSION
| 4.552338 |
biomedical
|
Study
|
[
0.9991143345832825,
0.0005400650552473962,
0.00034568330738693476
] |
[
0.9985952973365784,
0.0005700619076378644,
0.0006590608973056078,
0.00017563303117640316
] |
en
| 0.999999 |
The JN5A and JN5B RNAs contain a GG mismatch in the 5′ end hairpin stem, which was presumed to favour the regular 3′ end hairpin. However, this was neither observed experimentally, nor predicted at 0°C ( Table 1 ). Apparently, overcoming a GG mismatch in the hairpin does not result in a sufficient delay in the zippering of the remainder of the hairpin to significantly affect the overall folding rate.
|
16855293_p36
|
16855293
|
DISCUSSION
| 4.149236 |
biomedical
|
Study
|
[
0.9992430210113525,
0.0002587487397249788,
0.0004983050748705864
] |
[
0.9991722106933594,
0.0005607464700005949,
0.00019283143046777695,
0.00007424657087540254
] |
en
| 0.999997 |
The equal folding ratios between the 4 and 5 nt containing loops in the JN4A and JN4B RNAs was not expected either, because in DNA hairpins it was shown that the folding time increased with loop size. This loop size effect stems from the increased risk of misalignment of the loop resulting in formation of a closing base pair that does not allow subsequent zippering into a hairpin ( 41 ). Its effect on the folding time was estimated to be L 2.6 (L = loop length), for DNA hairpins ( 33 ). In our RNAs this would lead to an expected 30/70 folding ratio in favour of the four-membered loop, exactly as predicted by the Barriers program using a similar exponential ( Table 1 ). However, this exponential factor L 2.6 was determined for DNA loops of 12–30 nt ( 33 ).
|
16855293_p37
|
16855293
|
DISCUSSION
| 4.184298 |
biomedical
|
Study
|
[
0.9993559718132019,
0.00024800028768368065,
0.00039601221214979887
] |
[
0.9995055198669434,
0.00024703057715669274,
0.00019519191118888557,
0.00005225147833698429
] |
en
| 0.999999 |
The equal folding ratio observed in the JN4A and JN4B RNAs can be explained by the energy cost of bending an RNA chain. In DNA hairpins it decreases by 1/L ( 34 ), potentially favouring 5 over 4 nt loops. It could thus be that the lack of flexibility of a 4 nt loop counteracts the favourable effect of shorter loop lengths, especially for loops containing A and G residues.
|
16855293_p38
|
16855293
|
DISCUSSION
| 4.198582 |
biomedical
|
Study
|
[
0.9994471669197083,
0.0001871905697043985,
0.00036566934431903064
] |
[
0.9985955357551575,
0.0008719228790141642,
0.0004609859606716782,
0.00007155079947551712
] |
en
| 0.999998 |
Changing the closing base pair from a C–G to a U–G was expected to disfavour the formation rate of the U–G containing hairpin, because the C–G pair should be a more stable starting interaction and because the formation of the first stacking interaction is believed to be the rate limiting step in the folding of a hairpin ( 41 ). Unexpectedly, this was not observed in the JN3A and JN3B RNAs, which showed an approximately equal folding ratio for both hairpins. This seems to suggest that it is the rate of formation of the first closing base pair that is important rather than its thermodynamic stability, presumably because after this initial closing of the loop a subsequent efficient zippering of additional base pairs of the stem takes place. If this is the case then it also provides an alternative explanation for the absence of a kinetic effect of the stable tetra-loops on hairpin formation.
|
16855293_p39
|
16855293
|
DISCUSSION
| 4.304406 |
biomedical
|
Study
|
[
0.9993174076080322,
0.0003359396359883249,
0.00034671954927034676
] |
[
0.9991301894187927,
0.0004140832752455026,
0.0003668546269182116,
0.0000888404028955847
] |
en
| 0.999997 |
More in line with expectations is that the stability of the closing base pair does play a role. Then it is likely that not the U–G pair but the subsequent G–C pair acts as the closing base pair, forming a six-membered loop instead of a 4 nt one. This 6 nt loop should then fold with the same rate as the four-membered one of the C–G hairpin. Several observations are in favour of this explanation. The first one is that the 4, 5 and 6 nt loops have nearly identical Δ S contributions ( ), which is one of the barriers that needs to be overcome in hairpin formation ( 42 , 43 ). Second, equal folding ratios were also obtained for the folding of the 5 versus 4 nt loops in JN4A and JN4B. Third, the increased flexibility of the loop could compensate for the negative effects of the two extra nucleotides.
|
16855293_p40
|
16855293
|
DISCUSSION
| 4.299728 |
biomedical
|
Study
|
[
0.9993333220481873,
0.0002633364638313651,
0.0004033422446809709
] |
[
0.9990967512130737,
0.00044100251398049295,
0.00038799966569058597,
0.00007428678509313613
] |
en
| 0.999998 |
The computer predictions at 0 and 37°C with the Barriers program were in reasonable agreement with the experimentally determined folding ratios when the additional tetra-loop energies were excluded as a folding parameter. This effect was most striking for the JN3B and JN4LH sequences ( Table 1 ). Some of the remaining differences between the simulated and experimentally observed folding ratios could be related to the roughness of computational folding landscapes, making it difficult to calculate the correct folding ratio of the two major competing structures. However, the similarities between the simulated and experimental folding ratios showed the validity of the simulation approach, while indicating that not all thermodynamic parameters exert a kinetic effect. This emphasizes the need to experimentally determine additional kinetic parameters, to allow more realistic kinetic simulation of RNA folding landscapes. For instance, the kinetic effects of pyrimidine- and purine-rich loop sequences shown by the JN6A RNA may be incorporated into the Barriers program.
|
16855293_p41
|
16855293
|
DISCUSSION
| 4.15984 |
biomedical
|
Study
|
[
0.999472439289093,
0.00027986979694105685,
0.00024775989004410803
] |
[
0.9991857409477234,
0.00018751676543615758,
0.000555969076231122,
0.00007073553570080549
] |
en
| 0.999995 |
Thus far, little experimental work has been presented on kinetic folding parameters of RNA hairpins ( 16 ), despite growing interest in hairpin formation in DNA ( 32 , 34 , 37 – 39 ). The experimental results presented here give a tentative insight in the parameters that determine the folding rate of an RNA hairpin. We also showed that the current folding program ‘Barriers’ is capable of predicting the folding ratios of competing structures in an RNA chain, especially if the stable tetra-loop energies are not included. However, additional kinetic parameters must be included to allow more accurate predictions, such as those relating to hairpin loop size and nucleotide composition. Our experimental results show that GNRA tetra-loops do not affect the folding rate while pyrimidine-rich loops seem to enhance the folding rate, like in DNA. Such effects can be used by nature to intrinsically guide the folding of an RNA chain into its biologically active conformation and to avoid alternative or misfolded structures.
|
16855293_p42
|
16855293
|
DISCUSSION
| 4.164926 |
biomedical
|
Study
|
[
0.9995064735412598,
0.00026914937188848853,
0.00022434376296587288
] |
[
0.9989948868751526,
0.00018278948846273124,
0.000750360602978617,
0.0000719964227755554
] |
en
| 0.999996 |
The term ‘glycomics’ describes the scientific attempt to identify and study the biological function of all the glycan molecules—the glycome—synthesized by an organism. The aim is to create a cell-by-cell catalogue of glycosyltransferase expression and detected glycan structures ( 1 , 2 ). Sequences for complex carbohydrates differ significantly from the simple linear one-letter code that describes genes and proteins: the number of naturally occurring residues is much larger for glycans, each pair of monosaccharide residues can be linked in several ways, and one residue can be connected to three or four others (branching). Analysis of these carbohydrates has proved difficult in the past due to their structural complexity ( 3 , 4 ). In comparison with the other analytical methods often used for the identification of complex carbohydrates, NMR measurements have the advantage of enabling a complete and unambiguous assignment of all structural features of glycans—the stereochemistry of monosaccharide units, the type of linkage connecting units and even their conformational preferences—using the same experimental setup.
|
16845109_p0
|
16845109
|
INTRODUCTION
| 4.271395 |
biomedical
|
Review
|
[
0.9987751841545105,
0.000569421797990799,
0.0006553541752509773
] |
[
0.3645000159740448,
0.010930933058261871,
0.6237043738365173,
0.0008646861533634365
] |
en
| 0.999997 |
The data produced by NMR experiments are well suited for computational approaches for two reasons. First, each NMR resonance—the so-called chemical shift of an atom given in parts per million relative to an internal or external standard—can often be assigned unambiguously to exactly one atom in a given structure. Second, the exact value of the chemical shift depends on the chemical surroundings of the atom and is essentially influenced by the type of bonds formed with the directly adjacent atoms. The influence of remotely connected atoms decreases with their distance from the focus atom. These characteristics of NMR resonances have prompted computational techniques to be used for the automatic estimation/prediction of the NMR spectra of molecules.
|
16845109_p1
|
16845109
|
INTRODUCTION
| 4.076519 |
biomedical
|
Study
|
[
0.9994537234306335,
0.0001775939017534256,
0.00036858825478702784
] |
[
0.9621784090995789,
0.013001020066440105,
0.024590542539954185,
0.00023005947878118604
] |
en
| 0.999997 |
Increment rule based approaches use the fact that the chemical shifts of glycosyl residues in an oligo- or polysaccharide differ from those in the free monosaccharides in a predictable manner. The glycosylation shifts are additive provided that there are no steric interactions between residues more remote in sequence.
|
16845109_p2
|
16845109
|
INTRODUCTION
| 3.909252 |
biomedical
|
Other
|
[
0.9985283613204956,
0.00037344652810133994,
0.001098241307772696
] |
[
0.47098278999328613,
0.5146713852882385,
0.013650725595653057,
0.0006951207760721445
] |
en
| 0.999996 |
Estimation procedures based on a spherical environment encoding use a canonical linear string describing the spherical environment for each atom which is stored together with the assigned chemical shifts in lookup tables. If this procedure is repeated for a sufficient number of assigned atoms a representative set of canonical linear strings will result, which can be used to estimate shifts of new structures.
|
16845109_p3
|
16845109
|
INTRODUCTION
| 3.976213 |
biomedical
|
Other
|
[
0.9980758428573608,
0.0005153795937076211,
0.0014087774325162172
] |
[
0.4003258943557739,
0.594027578830719,
0.004956219345331192,
0.0006903308094479144
] |
en
| 0.999997 |
Here, we describe a web application which integrates the increment rule based approach implemented in the CASPER ( 5 , 6 ) program and the spherical environment encoding approach put into practice in GlyNest as part of the GLYCOSCIENCES.de portal ( 4 ), the former SWEET-DB ( 7 ). The predictive abilities of both techniques will be discussed and compared.
|
16845109_p4
|
16845109
|
INTRODUCTION
| 3.089812 |
biomedical
|
Study
|
[
0.9952617883682251,
0.0005957389948889613,
0.004142435733228922
] |
[
0.6434664130210876,
0.3511996269226074,
0.0040961033664643764,
0.0012378140818327665
] |
en
| 0.999997 |
A slightly modified version of the so-called extended form recommended by IUPAC ( 8 ) is used for the input of glycan structures in GLYCOSCIENCES.de ( 4 ). Each symbol for a monosaccharide unit is preceded by the anomeric descriptor ( α and β are replaced by a and b) and the configuration symbol (D or L). The ring size is indicated by f for furanose or p for pyranose and so on. The locants of the linkage are given in parentheses between the symbols; a line indicates a linkage between two anomeric positions.
|
16845109_p5
|
16845109
|
Structural encoding of Glycan structures
| 3.988034 |
biomedical
|
Other
|
[
0.9983477592468262,
0.0005482480046339333,
0.0011039709206670523
] |
[
0.2523188889026642,
0.7297198176383972,
0.016791976988315582,
0.0011693445267155766
] |
en
| 0.999997 |
The program CASPER ( 5 , 6 ) has been developed and is hosted by Stockholm University. It is an increment rule based approach that uses chemical shifts of the free reducing monosaccharides which are altered according to attached residues in an oligo- or polysaccharide sequence. Glycosylation shifts for a linkage are obtained from the chemical shifts of a disaccharide by subtracting the chemical shifts of the corresponding monosaccharides. The corrections are obtained in a similar manner by subtracting the monosaccharide and glycosylation shifts from chemical shifts of a trisaccharide. The procedure for estimating chemical shifts will be demonstrated for β- d -Glc p in β-3- O -fucosyl-lactose
|
16845109_p6
|
16845109
|
CASPER
| 4.067967 |
biomedical
|
Study
|
[
0.9991170763969421,
0.0002487082965672016,
0.0006341874250210822
] |
[
0.8532549738883972,
0.14419469237327576,
0.001986538991332054,
0.0005638124421238899
] |
en
| 0.999998 |
To calculate the chemical shifts of a glycan, one starts with the chemical shifts of the individual monosaccharides.
|
16845109_p7
|
16845109
|
CASPER
| 3.610074 |
biomedical
|
Other
|
[
0.9980108141899109,
0.0004940414219163358,
0.001495107775554061
] |
[
0.30068185925483704,
0.6943739652633667,
0.004230829421430826,
0.0007134011248126626
] |
en
| 0.999997 |
In a second step the glycosylation shifts for each linkage are added
|
16845109_p8
|
16845109
|
CASPER
| 2.409189 |
biomedical
|
Other
|
[
0.9781596064567566,
0.0031880633905529976,
0.018652264028787613
] |
[
0.06772278994321823,
0.928425133228302,
0.0023983852006495,
0.0014537250390276313
] |
en
| 0.999996 |
In the third step, corrections for vicinal substitution are added if available.
|
16845109_p9
|
16845109
|
CASPER
| 2.491341 |
biomedical
|
Other
|
[
0.9474730491638184,
0.0032741259783506393,
0.049252863973379135
] |
[
0.27493059635162354,
0.719719648361206,
0.00353979947976768,
0.0018099832814186811
] |
en
| 0.999995 |
The prediction tool GlyNest has been developed and is hosted by the German Cancer Research Centre as part of the GLYCOSCIENCES.de ( 9 ) portal. It estimates chemical shifts of glycans based on a spherical environment encoding scheme. Following the general philosophy in glycobiology to describe carbohydrate structures through the topology of their monosaccharide building blocks rather than through an explicit encoding of the topology of all atoms, a residue-based spherical code was developed. It is based on a slightly modified form of the IUPAC extended form for glycan structures. Since each monosaccharide describes implicitly the stereochemistry of all stereo-centres, the resulting code reflects well the structural specialities of complex carbohydrates as required for NMR shift estimation. Table 1 demonstrates the encoding scheme for each C-atom of β- d -Man p at a branch point of the N-glycan core structure. To reflect the fact that closely connected atoms have a larger impact on the chemical shift of the atom, two rules were applied to order the list of attached residues: (i) the connected carbohydrate residues are ordered according to their increasing distance (in terms of number of bonds) from the atom to be encoded and (ii) if two distances are equal, the residue attached to the C atom with the smaller ring-atom number receives the higher priority (see Table 1 code for atom C2).
|
16845109_p10
|
16845109
|
GlyNest
| 4.22627 |
biomedical
|
Study
|
[
0.9994860887527466,
0.00033925677416846156,
0.00017469389422331005
] |
[
0.9864507913589478,
0.011462918482720852,
0.001699338317848742,
0.00038696074625477195
] |
en
| 0.999997 |
For each atom of all structures of the GLYCOSCIENCES.de database—where assignments for the chemical shifts are available—the corresponding codes are generated and stored together with the shifts in the shift-environment table. It currently contains 27 052 1 H-NMR and 14 129 13 C-NMR shifts.
|
16845109_p11
|
16845109
|
GlyNest
| 3.282453 |
biomedical
|
Other
|
[
0.9969603419303894,
0.0005028197192586958,
0.0025367497000843287
] |
[
0.11086522787809372,
0.8870786428451538,
0.0014531821943819523,
0.0006028585485182703
] |
en
| 0.999996 |
To perform shift estimation, the corresponding codes for each atom of the input molecule are generated and looked up in the shift-environment table. Depending on the completeness of stored codes for the unknown structure, a number of hits with differing shift values can be retrieved from the shift-environment table. The result is reported as the mean value of the stored database entries, including statistics like the standard deviation and the highest and lowest shift value found for the given code. If no match is found for a complete environment code, residues are subsequently removed from the end of the environment code and the search is repeatedly performed until hits are found. The basic requirement for the exchange of NMR data is a common chemical shift reference. GlyNest uses acetone (δH = 2.225, δC = 31.07) which is roughly equivalent to the reference used in CASPER (TSP δH = 0.00, 1,4-dioxane δC = 67.40, D2O at 70°C).
|
16845109_p12
|
16845109
|
GlyNest
| 4.205081 |
biomedical
|
Study
|
[
0.9994028806686401,
0.00030399402021430433,
0.0002930871269199997
] |
[
0.9913739562034607,
0.007727022282779217,
0.0007082803640514612,
0.00019070088455919176
] |
en
| 0.999998 |
CASPER and GlyNest are two services aiming to predict the chemical shifts of glycans, which are based on different scientific approaches and which are implemented on two separate hosts in Stockholm and Heidelberg. Since the World Wide Web is increasingly used for application to application communication using programmatic interfaces, it was one of the goals of this work to develop a set of XML-based descriptions to exchange the data required for NMR shift estimation. The procedure works as follows: First, GlyNest converts the IUPAC extended description to the CASPER line notation, which differ significantly in their details but not in the quality of the structural features, which are encoded. Next, GlyNest sends this description to the CASPER server using the HTTP-get mechanism. CASPER calculates the NMR spectrum, labels residues and atoms according to the nomenclature used in SugaBase ( 10 ) and returns a XML encoding of the assignments that is embedded in the HTML response. GlyNest parses the returned data and integrates the CASPER shifts into the output list.
|
16845109_p13
|
16845109
|
Online connection of the CASPER—GlyNest web service
| 4.085299 |
biomedical
|
Study
|
[
0.9986242055892944,
0.00026214291574433446,
0.0011137244291603565
] |
[
0.9572667479515076,
0.041042882949113846,
0.0014329636469483376,
0.0002573937235865742
] |
en
| 0.999999 |
The input of glycan structures is accomplished using the extended IUPAC description in a free text editor. Table 2 shows the estimated 1 H- and 13 C NMR shifts for the tetrasccharide Lewis X using GlyNest and CASPER. The linkage descriptor lists—starting opposite to the reducing end of the carbohydrate sequence—sequentially the number of all linkage positions (separated by commas) on the non-reducing side of each residue until the looked at residue is reached. In such a way, residues in different branches as well as at various positions within the chain can be easily and unambiguously identified. In addition the basic data used for the GlyNest estimation are displayed.
|
16845109_p14
|
16845109
|
The web-interface implemented in GLYCOSCIENCES.de
| 4.170896 |
biomedical
|
Study
|
[
0.9996137022972107,
0.00020788959227502346,
0.0001784098712960258
] |
[
0.9955224990844727,
0.0034638047218322754,
0.0008377101039513946,
0.00017597312398720533
] |
en
| 0.999998 |
All experimental shifts found for a given environment code can be displayed in a histogram. Figure 1 shows as an example all proton shifts found in the shift-environment table for the H1 atom of terminal β- d -Gal p connected 1-4 to β- d -Glc p NAc (environment code: B-D-GALP [H1:(1-4)B-D-GLCPNAC]). In addition, an option exists to display the individual structures from which a certain shift originates.
|
16845109_p15
|
16845109
|
The web-interface implemented in GLYCOSCIENCES.de
| 3.792182 |
biomedical
|
Study
|
[
0.9969995617866516,
0.00023209954088088125,
0.002768269507214427
] |
[
0.8955293893814087,
0.10316949337720871,
0.0009435631800442934,
0.00035748921800404787
] |
en
| 0.999996 |
To evaluate the predictive ability of both techniques a test set of 155 13 C and 181 1 H spectra were selected form the GLYCOSCIENCES.de NMR database for which completely assigned glycan structures (C1 to C6, and H1 to H6) are available and which could all be estimated using GlyNest as well as CASPER. 1 H spectra. N- and O- Glycans as well as glycosphingolipid head groups and polysaccharides were covered in the test set. For GlyNest estimations, the entries in the shift-environment table originating from the glycan structure to be estimated were removed. Figure 2a–d displays the respective distributions of the deviations and Table 3 reports their corresponding statistical characteristics.
|
16845109_p16
|
16845109
|
Predictive Ability of GlyNest and CASPER
| 4.120744 |
biomedical
|
Study
|
[
0.9995359182357788,
0.00021831222693435848,
0.0002458085073158145
] |
[
0.9993302822113037,
0.00020504863641690463,
0.00040959561010822654,
0.000055181146308314055
] |
en
| 0.999998 |
GlyNest as well as CASPER can calculate accurately 1 H and 13 C shifts of glycans. The standard deviations between experimental and estimated shifts are comparable for both methods. In many cases the discrepancy between calculated and experimental chemical shifts is as low as 0.05 p.p.m./resonance for 1 H and 0.2 p.p.m./resonance for 13 C which is comparable with the differences between measurements from different laboratories resulting from slightly dissimilar experimental conditions. Such a predictive ability may be sufficient to establish the structure of many oligo- and polysaccharides and is in many cases sufficiently accurate to be used for an automatic assignment of NMR-spectra. Since both procedures work efficiently and require computation times in the millisecond range on standard computers, they are well suited for the assignment of NMR spectra in high-throughput glycomics projects.
|
16845109_p17
|
16845109
|
SUMMARY AND DISCUSSION
| 4.165301 |
biomedical
|
Study
|
[
0.9995802044868469,
0.00019118910131510347,
0.00022860766330268234
] |
[
0.9713269472122192,
0.005280949175357819,
0.023159390315413475,
0.0002326612448086962
] |
en
| 0.999996 |
The online connection of two distributed services presented here, which provides access to two methodically different approaches to estimate 1 H and 13 C NMR shifts for glycans, demonstrates the capability of web services to make rapidly accessible distributed scientific data. The established connection will help to find shortcomings in both approaches, detect mistakes in the underlying data and thus improve the quality of both services, which will definitively lead to a better worldwide acceptance of both services within the community of glycoscienists.
|
16845109_p18
|
16845109
|
SUMMARY AND DISCUSSION
| 3.377236 |
biomedical
|
Other
|
[
0.994669497013092,
0.0006138962344266474,
0.0047165872529149055
] |
[
0.07366723567247391,
0.9187762141227722,
0.006883264519274235,
0.0006732238689437509
] |
en
| 0.999997 |
We and others previously described the phenotype of LAT knock-in mice in which tyrosine 136, a consensus binding site for PLC-γ1, is mutated to phenylalanine ( 20 , 21 ). In brief, LAT Y136F m/m mice exhibit a severe block in thymocyte development that is best documented early in life. Thymi from these mice are small and contain mostly CD4 − CD8 − (double negative [DN]) thymocytes. The block is at the DN3 (CD25 + CD44 − ) stage, confirming a requirement for wild-type LAT for pre-TCR signaling. As they age, virtually all LAT Y136F m/m mice develop a lymphoproliferative disease evidenced by markedly enlarged spleens and lymph nodes and lymphocytic infiltration of multiple organs. T cells in LAT Y136F m/m mice arise from polyclonal expansion and consist almost entirely of CD4 + CD3 lo CD62L lo CD44 hi cells, thus showing signs of previous activation. Thymocytes and T cells from LAT Y136F m/m mice are defective in TCR-induced calcium flux, but have near normal levels of Erk activation. To assess the effect of this relatively specific defect in calcium signaling on positive and negative selection, the LAT knock-in mutants were interbred with HY TCR transgenic mice. The HY TCR transgene was chosen because the HY TCR reacts with the male-specific antigen HY, causing negative selection in male mice; however, in female HY TCR transgenic mice, T cells are positively selected by endogenous peptide(s) ( 22 ). Therefore, use of the HY model system allows us to analyze both positive and negative selection in one TCR transgenic system and to investigate if class I–restricted LAT mutant T cells would exhibit a similar phenotype to LAT Y136F CD4 + T cells. As shown in Fig. 1 , HY + LAT Y136F m/m males and females exhibited a phenotype essentially identical to LAT Y136F m/m mice. They also had enlarged spleens and lymph nodes (not depicted). Thymi from the mutant mice displayed varying degrees of infiltration by CD4 + CD3 lo CD62L lo CD44 hi T cells, characteristic of peripheral T cells seen in non-TCR transgenic LAT knock-in mice. These infiltrating peripheral T cells were the only T cells found in the thymus later in life (after ∼2 mo of age; not depicted). Lymph node T cells were largely CD4 + (i.e., they were not MHC class I restricted) and did not stain with antibody to the HY transgenic TCR (T3.70). In contrast, female control HY + LAT +/+ mice contained T3.70 + CD8 hi cells in the periphery, a result of successful positive selection of transgenic T cells . Male control HY + LAT +/+ mice lacked T3.70 + CD8 hi T cells in the periphery, but had T3.70 + CD8 lo transgenic T cells, which have been described previously ( 22 ).
|
15795236_p0
|
15795236
|
The lymphoproliferative disorder in LATY136F m/m mice is not abrogated by introduction of a TCR transgene
| 4.443761 |
biomedical
|
Study
|
[
0.9991933703422546,
0.0005225302302278578,
0.00028414351982064545
] |
[
0.9990172386169434,
0.0003124619834125042,
0.0005228922236710787,
0.0001473773008910939
] |
en
| 0.999996 |
To investigate if the LATY136F m/m lymphoproliferative phenotype would also manifest in other TCR transgenic mouse model systems, we interbred the LAT knock-in mutant with class II–restricted AND TCR transgenic mice ( 23 ). A similar phenotype was observed, i.e., expansion of CD4 + T cells not staining for the transgenic TCR (Vα11 − ) and the presence of large lymph nodes and spleens (not depicted). We speculated that either the LAT Y136F mutation drives all T cells to the same phenotype regardless of the TCR that they express, or that the specificity of the TCRs in these TCR transgenic model systems was changed by the pairing of transgenic TCRβ chains with endogenous TCRα chains, a phenomenon known to occur due to incomplete early allelic exclusion of the TCRα locus ( 24 ). To distinguish between these possibilities, we examined the HY TCR transgenic system in more detail by crossing the LAT mutation onto the RAG knockout background, where TCR specificity would be fixed because contributions from endogenous TCRα chains would be eliminated.
|
15795236_p1
|
15795236
|
The lymphoproliferative disorder in LATY136F m/m mice is not abrogated by introduction of a TCR transgene
| 4.216899 |
biomedical
|
Study
|
[
0.99949049949646,
0.0003010817163158208,
0.00020839195349253714
] |
[
0.999354898929596,
0.0002389689616393298,
0.0003279778466094285,
0.00007811872637830675
] |
en
| 0.999996 |
By breeding onto the RAG-2 null background, we were able to determine whether clonotypic CD8 hi T cells could develop in HY + LAT Y136F knock-in mice by preventing expression of endogenous TCR α or β chains, therefore eliminating competition from CD4 + T cells expressing endogenous TCRα chains. A much different phenotype was observed using the RAG-2 null background. In HY + LAT Y136F m/m RAG-2 −/− female mice, thymi were smaller than those in their LAT +/+ counterparts, about one fifth to one tenth the normal size . This decrease in thymic cellularity, especially in the number of CD4 + CD8 + (double positive [DP]) thymocytes is consistent with a block in the DN→DP transition ( 20 ). Although DP thymocytes are generated, a distinct population of CD8 single positive (SP) thymocytes is missing, indicating a defect in positive selection. DPs from HY + LAT Y136F m/m RAG-2 −/− female mice express lower levels of the HY transgenic TCR as evidenced by staining with the antibody T3.70 (∼60% wild-type levels). Consistent with a lack of positive selection in the thymus, HY + LAT Y136F m/m RAG-2 −/− female mice do not contain T3.70 hi CD8 hi T cells in the periphery .
|
15795236_p2
|
15795236
|
Lack of positive selection in HY + LATY136F m/m RAG −/− female mice
| 4.284062 |
biomedical
|
Study
|
[
0.9993876218795776,
0.0003517243603710085,
0.00026064031408168375
] |
[
0.9992777705192566,
0.00032574933720752597,
0.000302797241602093,
0.00009368528117192909
] |
en
| 0.999997 |
Because of the extensive negative selection of HY clonotypic T cells in male HY + LAT +/+ mice, thymi are small and contain mostly DN thymocytes . In HY + LAT Y136F m/m RAG-2 −/− male mice, thymi are also small; however, T3.70 hi DP and CD8 SP are evident, much like in HY + LAT +/+ RAG-2 −/− female mice , indicating a failure of negative selection and possible conversion to positive selection. In the lymph nodes of HY + LAT Y136F m/m RAG-2 −/− male mice, T3.70 + CD8 hi T cells are evident, again suggesting a failure of negative selection and possible conversion to positive selection . In HY + LAT +/+ RAG-2 −/− male mice, T3.70 + DN and CD8 lo T cells are also observed. These T cells have been hypothesized to be aberrant populations from the γδ T cell lineage ( 25 ). In HY + LAT Y136F m/m RAG-2 −/− male mice, these populations are not present.
|
15795236_p3
|
15795236
|
Impaired negative selection in male HY + LAT Y136F m/m mice
| 4.266462 |
biomedical
|
Study
|
[
0.9994100332260132,
0.0002162960299756378,
0.0003737380902748555
] |
[
0.9991859793663025,
0.00044254728709347546,
0.0003014179819729179,
0.00006993359420448542
] |
en
| 0.999996 |
A possible contribution to the lack of negative selection in HY + LAT Y136F m/m RAG-2 −/− male mice could derive from defects in activation-induced cell death in DP thymocytes. Plate-bound T3.70 and anti-CD28 were used to mimic signals known to induce cell death in DP thymocytes ( 26 ). DP thymocytes from HY + LAT +/+ RAG-2 −/− female mice showed an increase from 22 to 79% annexin V + under these antibody stimulation conditions ( Table I ). However, DP thymocytes from HY + LAT m/m RAG-2 −/− male mice showed no increase in the percentage of annexin V + T cells under these antibody stimulation conditions. As a control, etoposide treatment resulted in 100% annexin V + DPs in both HY + LAT +/+ RAG-2 −/− female and HY + LAT Y136F m/m RAG-2 −/− male mice.
|
15795236_p4
|
15795236
|
Impaired negative selection in male HY + LAT Y136F m/m mice
| 4.158812 |
biomedical
|
Study
|
[
0.9994798302650452,
0.0002488683385308832,
0.0002712890855036676
] |
[
0.9994776844978333,
0.00022549064306076616,
0.00023626281472388655,
0.00006058537110220641
] |
en
| 0.999995 |
We also tested the response of DP thymocytes from HY + LAT Y136F m/m RAG-2 −/− female mice to antibody stimulation. DP thymocytes from HY + LAT Y136F m/m RAG-2 −/− females showed no increase in annexin V staining in response to T3.70 and anti-CD28 treatment. In addition, incubation with HY peptide-loaded APCs did not result in increased annexin V staining above APC controls in the mutants, whereas DP thymocytes from HY + LAT +/+ RAG-2 −/− female mice did have increased annexin V staining. Again, etoposide treatment resulted in high rates of cell death in both HY + LAT +/+ RAG-2 −/− and HY + LAT Y136F m/m RAG-2 −/− female mice. These data indicate a failure of TCR-mediated induction of cell death in thymocytes from both male and female mutant mice.
|
15795236_p5
|
15795236
|
Impaired negative selection in male HY + LAT Y136F m/m mice
| 4.162842 |
biomedical
|
Study
|
[
0.9994864463806152,
0.00026625278405845165,
0.00024717760970816016
] |
[
0.9994367957115173,
0.0002231999096693471,
0.0002740330819506198,
0.00006601357745239511
] |
en
| 0.999997 |
Because defects in TCR-induced calcium influx were described in thymocytes and peripheral T cells from LAT Y136F m/m mice ( 20 ), we also measured calcium influx in T cells from HY + LAT Y136F m/m RAG-2 −/− mice. DP thymocytes and CD8 + lymph node T cells from HY + LAT Y136F m/m RAG-2 −/− male mice had reduced TCR-induced calcium influx compared with DP thymocytes and CD8 + lymph node T cells from C57BL/6 and HY + LAT +/+ RAG-2 −/− female mice . DP thymocytes from HY + LAT Y136F m/m RAG-2 −/− female mice also had diminished levels of calcium influx compared with DP thymocytes from C57BL/6 and HY + LAT +/+ RAG-2 −/− female mice . However, initial calcium influx in DP thymocytes from HY + LAT Y136F m/m RAG-2 −/− male and female mice and in CD8 + lymph node T cells from HY + LAT Y136F m/m RAG-2 −/− male mice was higher than in DP thymocytes and CD4 + lymph node T cells from non-TCR transgenic LAT Y136F knock-in mice . This heightened initial calcium influx could be characteristic of the strength of signal from the HY TCR (compared with the diversity of TCRs found in T cells in LAT Y136F knock-in mice) or due to the higher levels of TCR in HY + LAT Y136F m/m RAG-2 −/− T cells compared with non-TCR transgenic LAT Y136F m/m T cells. In the case of peripheral cells, the difference could also be related to differences between CD4 + and CD8 + T cells. We were unable to obtain data for TCR-induced calcium influx in CD8 + T cells from LAT Y136F knock-in mice to make a direct comparison because so few of those cells exist.
|
15795236_p6
|
15795236
|
Biochemical characterization of T cells from male HY + LAT Y136F m/m mice
| 4.224493 |
biomedical
|
Study
|
[
0.9994445443153381,
0.0002791444421745837,
0.000276298844255507
] |
[
0.9993894100189209,
0.0002455707872286439,
0.0002941326529253274,
0.00007089597784215584
] |
en
| 0.999998 |
We also examined Erk activation in T cells from HY + LAT Y136F m/m RAG-2 −/− mice by measuring levels of phospho-Erk1/2 by flow cytometry. The extent and kinetics of Erk activation was approximately the same in DP thymocytes and CD8 + lymph node T cells (not depicted) from HY + LAT Y136F m/m RAG-2 −/− male mice and HY + LAT +/+ RAG-2 −/− female mice. Erk activation in HY + LAT Y136F m/m RAG-2 −/− female DP thymocytes was observed, although the increase in phospho-Erk levels at 1 min of TCR activation was less pronounced in mutant female (1.4-fold increase) than in mutant male thymocytes (2.2-fold increase). This may reflect differences in levels of ongoing positive selection in the male and female mutant mice. The increase in phospho-Erk staining in male mutant thymocytes at 1 min of activation was similar to that seen in HY + LAT +/+ RAG-2 −/− female thymocytes (2.4-fold). These results are consistent with the small impact of the LAT Y136F mutation on Erk activation in T cells from LAT Y136F non-TCR transgenic mice as observed previously ( 20 ).
|
15795236_p7
|
15795236
|
Biochemical characterization of T cells from male HY + LAT Y136F m/m mice
| 4.17765 |
biomedical
|
Study
|
[
0.9994420409202576,
0.0003069732047151774,
0.00025088374968618155
] |
[
0.9994657635688782,
0.00019706421880982816,
0.00026993989013135433,
0.00006716817006235942
] |
en
| 0.999995 |
To examine the differentiation state of CD8 SP thymocytes and CD8 + lymphocytes from HY + LAT Y136F m/m RAG-2 −/− mice, we measured the expression of several cell surface markers by flow cytometry . Thymocyte maturation is accompanied by down-regulation of CD24 (HSA) and, in the CD8 lineage, up-regulation of β7 integrin ( 27 , 28 ). CD24 is down-regulated and β7 integrin is up-regulated in mature T cells from HY + LAT Y136F m/m RAG-2 −/− male mice as in mature T cells from HY + LAT +/+ RAG-2 −/− female mice. CD69, a marker of maturation and activation, is up-regulated in CD8 SP thymocytes compared with DP thymocytes from both kinds of mice and, in fact, is up-regulated to a slightly greater extent in the mutant male mice, consistent with functional activation of the Ras/Erk pathway in these mice ( 29 ). TCR (T3.70) levels are consistently lower in T cells from the male mutant mice than in female wild-type mice.
|
15795236_p8
|
15795236
|
Evidence of conversion of negative selection to positive selection in male HY + LAT Y136F m/m mice
| 4.266078 |
biomedical
|
Study
|
[
0.9994140863418579,
0.00031925359508022666,
0.00026662833988666534
] |
[
0.9993529915809631,
0.00022801579325459898,
0.00034002031316049397,
0.00007903662481112406
] |
en
| 0.999995 |
To investigate whether bona fide CD8 + T cells develop in HY + LAT Y136F m/m RAG-2 −/− male mice by positive selection, we analyzed the expression of perforin, a differentiation marker of CD8 + cytotoxic T cells. CD8 SP thymocytes were sorted and analyzed by RT-PCR for expression of perforin. As shown in Fig. 5 , CD8 SP thymocytes from HY + LAT Y136F m/m RAG-2 −/− male mice (as well as from HY + LAT +/+ RAG-2 −/− female mice and control C57BL/6 mice) expressed perforin. Perforin expression was also detected by RT-PCR in CD8 + splenocytes from these mice (not depicted).
|
15795236_p9
|
15795236
|
Evidence of conversion of negative selection to positive selection in male HY + LAT Y136F m/m mice
| 4.111631 |
biomedical
|
Study
|
[
0.9995263814926147,
0.00023301731562241912,
0.00024054433743003756
] |
[
0.9994996786117554,
0.0002288756804773584,
0.0002135705144610256,
0.000057970966736320406
] |
en
| 0.999997 |
To investigate whether CD8 + T cells in HY + LAT Y136F m/m RAG-2 −/− male mice were functional (as might be expected if they resulted from normal positive selection), we examined the ability of peripheral CD8 + T cells to proliferate in response to TCR stimulation. Purified CD8 + T cells from HY + LAT Y136F m/m RAG-2 −/− male mice did not proliferate in response to antibody stimulation or treatment with HY peptide-loaded APCs as did CD8 + T cells from HY + LAT +/+ RAG-2 −/− female mice . Even use of high concentrations of antibodies or HY peptide for stimulation did not result in a proliferative response in CD8 + T cells from HY + LAT Y136F m/m RAG-2 −/− male mice (not depicted). Because this proliferation defect could be due to the effects of the LAT Y136F mutation, we included ionomycin in an attempt to bypass the PLC-γ1–mediated Ca 2+ defect in these cells. When ionomycin was included in cultures with CD8 + T cells from HY + LAT Y136F m/m RAG-2 −/− male mice and HY peptide-loaded APCs, proliferation did occur . Ionomycin alone did not induce proliferation (not depicted). Therefore, mutant cells were able to respond to antigen if calcium influx was pharmacologically induced. Under the same conditions, however, interferon γ and IL-2 could not be detected from supernatants of cultures of HY + LAT Y136F m/m RAG-2 −/− male CD8 + T cells and HY peptide-loaded APCs, even in the presence of ionomycin, whereas cytokine secretion was detected after treatment with PMA and ionomycin (not depicted). This suggests that although CD8 + T cells from mutant male mice can respond to antigen, they are not fully functional in vivo, possibly due to defects caused by the LAT Y136F mutation.
|
15795236_p10
|
15795236
|
Evidence of conversion of negative selection to positive selection in male HY + LAT Y136F m/m mice
| 4.36895 |
biomedical
|
Study
|
[
0.999311089515686,
0.00046534789726138115,
0.0002236507716588676
] |
[
0.9991148114204407,
0.00038480438524857163,
0.00036565199843607843,
0.00013469455006998032
] |
en
| 0.999998 |
The LAT Y136F mutation impairs TCR-mediated PLC-γ1 activation in thymocytes and T cells resulting in decreased TCR-induced calcium influx ( 20 , 21 ). We used this LAT mutant to investigate the effect of dampening calcium signaling on thymocyte positive and negative selection in a system that does not use broad-range inhibitors of calcium-dependent enzymes or agents that alter calcium influx triggered by non-LAT–mediated signaling pathways. We examined HY TCR transgenic, RAG-2 knockout mice to evaluate the effect of the LAT Y136F mutation on positive and negative selection. In HY female mice, thymocytes normally undergo positive selection resulting in the generation of HY TCR-specific CD8 hi T cells; however, HY LAT mutant female mice showed a complete absence of positive selection. In HY LAT +/+ male mice, thymocytes undergo negative selection and HY TCR-specific CD8 hi T cells are absent because the HY antigen is male specific. In contrast, T cells from HY LAT mutant male mice do not undergo negative selection, but instead resemble positively selected T cells found in LAT +/+ HY females. The effect of dampening calcium signaling in this system can be interpreted according to a quantitative model of thymocyte differentiation in which very low levels of TCR signal result in nonselection (also called death by neglect), intermediate signals result in positive selection, and strong signals result in negative selection. According to this model, the “window” for positive and negative selection has been shifted in the LAT mutant mice such that thymocytes from male mice, which would otherwise undergo negative selection, undergo positive selection (or a process very much like positive selection), and thymocytes from female mice, which would otherwise undergo positive selection, most likely undergo death by neglect.
|
15795236_p11
|
15795236
|
Discussion
| 4.411161 |
biomedical
|
Study
|
[
0.9992794394493103,
0.00045630973181687295,
0.00026416685432195663
] |
[
0.9990297555923462,
0.0003777658857870847,
0.00046466063940897584,
0.00012784512364305556
] |
en
| 0.999997 |
PLC-γ1, whose activation is defective in LAT Y136F knock-in mice, catalyzes the conversion of phosphatidylinositol ( 4 , 5 ) bisphosphate to diacylglycerol and inositol ( 1 , 4 , 5 ) trisphosphate. An increase in inositol ( 1 , 4 , 5 ) tris phosphate levels results in release of calcium from intracellular stores, whereas increased diacylglycerol results in stimulation of PKC and RasGRP (for review see reference 30 ). Therefore, the effects on selection seen in this study could be because of decreased signaling through PKC and RasGRP as a result of decreased PLC-γ1 activity. However, the downstream endpoint of Erk activation is relatively normal in LAT Y136F mutant T cells, implying that Erk can be activated through mechanisms other than those mediated by RasGRP in these cells. Pharmacologic disruptions of TCR-mediated PLC-γ1 signaling pathways have previously been shown to affect thymocyte selection. Use of inhibitors that block PKC ( 31 ) or of inhibitors of the calcium-dependent phosphatase calcineurin ( 32 ) results in less thymocyte deletion, an in vitro correlate of negative selection. Our results are consistent with the idea that inhibition of downstream mediators of PLC-γ1 action can block negative selection.
|
15795236_p12
|
15795236
|
Discussion
| 4.511635 |
biomedical
|
Study
|
[
0.999342143535614,
0.00039169020601548254,
0.0002661488251760602
] |
[
0.9986991882324219,
0.00040600818465463817,
0.00075419811764732,
0.00014056956570129842
] |
en
| 0.999997 |
Mutations in mice that alter PLC-γ1–mediated signaling have also been shown to affect positive and negative thymocyte selection in ways consistent with results from this study. A spontaneous mutation in ZAP-70, the kinase that phosphorylates LAT, results in decreased LAT phosphorylation, decreased PLC-γ1 phosphorylation, and decreased calcium flux ( 33 ). Erk and p38 activities are also dampened. When this mutation is crossed into the HY TCR transgenic background, positive selection is abrogated in female mice, and in male mice peripheral transgenic CD8 + T cells are observed, similar to the results observed here for the LAT Y136F mutation ( 33 ). In addition, null mutation of TCRζ (which would preclude docking of ZAP-70 to the TCR complex and prevent ZAP-70 activation) resulted in a phenotype resembling positive selection in male HY TCR transgenic mice and nonselection in female HY TCR transgenic mice ( 34 ). The Tec family kinases Itk and Rlk/Txk regulate PLC-γ1 activity in T cells ( 15 ). Null mutation of both Itk and Rlk/Txk results in decreased positive selection in HY female mice and a phenotype suggestive of conversion of negative to positive selection in HY male mice ( 35 ). Null mutation of Itk and Rlk/Txk also resulted in decreased PLC-γ1 activation, decreased calcium flux, and dampened Erk activation ( 35 ). Like the LAT Y136F mutation, all of these mutations decrease activation of PLC-γ1 and activation-induced calcium influx. Unlike the examples above, in HY LAT Y136F knock-in mice, we were able to show antigen-induced proliferation in peripheral CD8 + male T cells by partially correcting for the LAT mutant phenotype by treatment with the calcium ionophore ionomycin, supporting the critical role of calcium in thymocyte selection and T cell function.
|
15795236_p13
|
15795236
|
Discussion
| 4.556213 |
biomedical
|
Study
|
[
0.9991468191146851,
0.0005577583215199411,
0.0002954408701043576
] |
[
0.9984230995178223,
0.0005025547579862177,
0.0008494019857607782,
0.00022491485287901014
] |
en
| 0.999998 |
We have speculated that the lymphoproliferative disease in LAT Y136F knock-in mice could be due to expansion of autoreactive T cells that have escaped negative selection ( 20 ). A similar phenotype was observed in null mutants of the calcium-regulated NFAT transcription family members NFATc2 and NFATc3 ( 36 , 37 ). NFAT transcription factors are downstream targets of calcium-mediated TCR signaling. Recent examples in the literature indicate that point mutations in T cell signaling molecules could result in defects in central (and not just peripheral) tolerance that could eventually lead to autoimmune disease. Examples of point mutations in T cell signaling molecules leading to autoimmune disease, presumably by failure of negative selection, include a spontaneous mutation in the Rasgrp1 gene, which results in a lymphoproliferative disease with symptoms resembling systemic lupus erythematosus ( 38 ). Also, the ZAP-70 spontaneous mutant mentioned earlier ( 33 ) has a syndrome with symptoms similar to autoimmune arthritis. A knock-in mutation of the phosphatase CD45 (E613R) results in a lupus-like nephritis ( 39 ). Possibly, mutations in different molecules will skew the TCR repertoire in different ways, manifesting as different autoimmune syndromes.
|
15795236_p14
|
15795236
|
Discussion
| 4.428995 |
biomedical
|
Study
|
[
0.9994909763336182,
0.00030880552367307246,
0.00020015655900351703
] |
[
0.9959457516670227,
0.00043723758426494896,
0.003460549982264638,
0.0001565247366670519
] |
en
| 0.999999 |
T cell hybridomas made from LAT Y136F peripheral T cells were autoreactive as reported by Aguado et al. ( 21 ). In this study, we have also provided evidence that aberrant negative selection might be contributing to a skewed TCR repertoire in LAT Y136F knock-in mice, which could ultimately lead to proliferation of autoreactive T cells. This conclusion is based on the fact that HY TCR + CD8 hi T cells can develop in LAT Y136F m/m male mice. Lymphoproliferative disease does not ensue, however, in the HY model system, as evidenced by an absence of lymphadenopathy, splenomegaly, and lymphocytic infiltration to lung in HY + LAT Y136F m/m RAG-2 −/− male mice (not depicted). This absence of lymphoproliferative disease from HY + LAT Y136F m/m RAG-2 −/− T cells could be because the strength of signal transduced through the HY TCR may not be sufficient to cause this response, or the HY (self-) antigen may not be presented in the tissues examined. Alternatively, the lymphoproliferative phenotype may only manifest in CD4 + MHC class II–restricted T cells. Future experiments will be aimed at trying to distinguish between these possibilities.
|
15795236_p15
|
15795236
|
Discussion
| 4.215466 |
biomedical
|
Study
|
[
0.9994460940361023,
0.00033228055690415204,
0.0002216365683125332
] |
[
0.9994204044342041,
0.0002276094601256773,
0.000273274868959561,
0.00007872253627283499
] |
en
| 0.999997 |
LAT Y136F knock-in mice were described previously ( 20 ). HY mice, which express an MHC class I–restricted TCR for the male antigen HY ( 22 ), were obtained from NIAID Taconic exchange. RAG-2 knockout mice were also obtained from NIAID Taconic exchange. All mice used in this study were on a C57BL/6 (H-2D b ) background and were housed under specific pathogen-free conditions.
|
15795236_p16
|
15795236
|
Mice.
| 3.60216 |
biomedical
|
Study
|
[
0.9993160963058472,
0.00022675961372442544,
0.0004572024627123028
] |
[
0.9859728813171387,
0.012488720007240772,
0.0012792814522981644,
0.00025914196157827973
] |
en
| 0.999997 |
Single cell suspensions were analyzed by standard flow cytometry as described previously ( 40 ). Flow cytometry was performed using a FACSCalibur and CELLQuest software (Becton Dickinson). Fluorochrome-conjugated antibodies were purchased from BD Biosciences.
|
15795236_p17
|
15795236
|
Flow cytometry.
| 3.379481 |
biomedical
|
Study
|
[
0.9991538524627686,
0.00023226144548971206,
0.0006138281896710396
] |
[
0.9611620306968689,
0.036962106823921204,
0.0015130944084376097,
0.0003626816614996642
] |
en
| 0.999996 |
Thymocytes were harvested in Cellgro complete serum-free medium (Mediatech), washed, and incubated with plate-bound antibodies (5 ug/ml T3.70 and 50 ug/ml anti-CD28) or with APCs and HY peptide. APCs were prepared from ACK-treated splenocytes by treatment with anti-Thy antibody followed by treatment with complement. The T cell–depleted splenocytes were then irradiated at 500 rads to yield APCs. HY peptide (KCSRNRQYL) was synthesized by the FDA core facility. Cells were harvested for flow cytometry after 18 h of incubation. Annexin V-FITC was purchased from BD Biosciences and was used according to the manufacturer's specifications.
|
15795236_p18
|
15795236
|
Thymocyte cell death assays.
| 4.116556 |
biomedical
|
Study
|
[
0.999535322189331,
0.0002170432999264449,
0.0002476493245922029
] |
[
0.9982784986495972,
0.001343614887446165,
0.0003056605637539178,
0.00007227530295494944
] |
en
| 0.999995 |
CD8 + T cells from lymph nodes were purified by negative magnetic bead separation using biotinylated anti-B220, anti–Mac-1, and anti-CD4 antibodies (BD Biosciences) as well as magnetic streptavidin beads (Miltenyi Biotec). Purified cells were loaded with CFSE (Molecular Probes), washed, and incubated with plate-bound antibodies (5 ug/ml T3.70 and 50 ug/ml anti-CD28) or with APCs and HY peptide. Cells were harvested for flow cytometry after 3 d of incubation.
|
15795236_p19
|
15795236
|
Proliferation assays.
| 4.115877 |
biomedical
|
Study
|
[
0.999595582485199,
0.00022439693566411734,
0.00018007686594501138
] |
[
0.9987196922302246,
0.0008265254436992109,
0.00037933990824967623,
0.00007449566328432411
] |
en
| 0.999998 |
Supernatants from the proliferation assays described above were analyzed by ELISA for the presence of interferon γ and IL-2 using reagents and the protocol from BD Biosciences.
|
15795236_p20
|
15795236
|
ELISA.
| 3.827216 |
biomedical
|
Study
|
[
0.9994895458221436,
0.00018133712001144886,
0.00032911685411818326
] |
[
0.9927946925163269,
0.006340126972645521,
0.0007113893516361713,
0.00015372772759292275
] |
en
| 0.999998 |
Thymocytes were stained with anti-CD4 FITC and anti-CD8 PE and were sorted for DP and SP populations using a FACS Vantage SE cell sorter equipped with TurboSort (Becton Dickinson). RNA was prepared from sorted populations using TriZol reagent (Invitrogen). cDNA was synthesized using the SuperScript cDNA synthesis system (Invitrogen). Dilutions of cDNA were made and tested so that amounts used for PCR were in the linear range. Primers for CD3ɛ were (sense) CTGAGAGGATGCGGTGGAACA and (antisense) GACCATCAGCAAGCCCAGAGT. PCR conditions for CD3ɛ were 95°C for 5 min and 30 cycles of 95°C for 45 s, 56°C for 45 s, and 72°C for 1 min. Primers for perforin were (sense) CAAGCAGAAGCACAAGTTCGT and (antisense) CGTGATAAAGTGCGTGCCATA. PCR conditions for perforin were 95°C for 5 min and 30 cycles of 95°C for 45 s, 50°C for 45 s, and 72°C for 1 min.
|
15795236_p21
|
15795236
|
RT-PCR.
| 4.13603 |
biomedical
|
Study
|
[
0.9996082186698914,
0.00019658735254779458,
0.0001951840240508318
] |
[
0.9977522492408752,
0.0017533061327412724,
0.0004043033695779741,
0.00009021989535540342
] |
en
| 0.999995 |
Measurements of calcium flux were performed essentially as described previously ( 41 ). Thymocytes or lymph node cells were stained for CD4 and CD8 and preloaded with indo-1 (Molecular Probes). At 30 s, biotinylated anti-CD3 and anti-CD8 (10 μg each) were added and at 60 s, 25 μg streptavidin was added. Calcium flux, measured as the ratio of FL5 to FL4, was recorded on an LSR I (Becton Dickinson) and is displayed as a function of time.
|
15795236_p22
|
15795236
|
Calcium flux experiments.
| 4.083597 |
biomedical
|
Study
|
[
0.9995287656784058,
0.0002570815267972648,
0.00021416576055344194
] |
[
0.9988614320755005,
0.0007485875394195318,
0.00031768338521942496,
0.0000723155026207678
] |
en
| 0.999998 |
Measurement of levels of phospho-Erk was performed using a modification of the protocol of Priatel et al. ( 42 ). 2 × 10 6 cells were stimulated by the addition of biotinylated anti-CD3 and anti-CD8 (10 ug/ml) followed by the addition of 20 ug/ml streptavidin and incubation at 37°C for the indicated times. Reactions were stopped by the addition of an equal volume of 4% paraformaldehyde followed by cold methanol fixation ( 43 ). Cells were stained with 3 ug/ml anti–p-Erk followed by staining with goat anti–mouse Ig-PE, anti-CD4 PerCP, and anti–Thy 1.2 APC (BD Biosciences). Flow cytometry was performed using a FACSCalibur and CELLQuest software (Becton Dickinson). Induction of phospho-Erk could be blocked by the addition of 10 uM MEK inhibitor U0126 (Calbiochem).
|
15795236_p23
|
15795236
|
Phospho-Erk assays.
| 4.115945 |
biomedical
|
Study
|
[
0.9995406866073608,
0.0002289501135237515,
0.00023049814626574516
] |
[
0.9990021586418152,
0.0006193218287080526,
0.00031707793823443353,
0.00006148383545223624
] |
en
| 0.999999 |
Fig. S1 shows diminished calcium flux in DP thymocytes from female HY + SM88 m/m RAG-2 −/− mice. Fig. S2 shows the cell surface expression of T3.70, CD24 (HSA), CD69, and β7 integrin on DP thymocytes, CD8 SP thymocytes, and CD8 + lymph node T cells from HY + LAT +/+ RAG-2 −/− female and HY + LAT Y136F m/m RAG-2 −/− male mice. Figs. S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20041869/DC1 .
|
15795236_p24
|
15795236
|
Online supplemental material.
| 3.85489 |
biomedical
|
Study
|
[
0.9995483756065369,
0.00015537867147941142,
0.0002962580765597522
] |
[
0.9932097792625427,
0.006063357461243868,
0.0004931722651235759,
0.00023376038006972522
] |
en
| 0.999997 |
The heterogeneous nuclear ribonucleoprotein (hnRNP) H family consists of four highly homologous proteins, namely hnRNP H, hnRNP H′, hnRNP F and hnRNP 2H9. Members of the hnRNP H family specifically recognize poly(G) sequences (G-tracts) that are abundant in both DNA and RNA. These G-tracts are known to form a specific structure, the G-quadruplex, that consists of four guanine bases arranged in a square planar conformation and stabilized by hydrogen bonds [reviewed in ( 1 , 2 )]. In DNA, these structures are mainly located in telomeres and also in some promoter regions. In RNA, G-tracts are frequent splicing recognition elements found both in introns and exons and are crucial for 5′ splice site recognition ( 3 – 5 ). They are also abundant downstream of mammalian polyadenylation signals ( 6 ). hnRNP F and H, through binding to these G-tracts, are responsible for the regulation of polyadenylation ( 6 , 7 ) and the splicing regulation of numerous pre-mRNAs such as the Bcl-x ( 8 ), the rat β-tropomyosin ( 9 ), the Rous sarcoma virus NRS ( 10 ), the HIV type 1 tat ( 11 ), the HIV-1 tev ( 12 ), the HIV-1 p17gag instability ( 13 ) and the c-src ( 14 ) pre-mRNAs. Furthermore, mutations in G-tract splice sites correlate with many diseases [reviewed in ( 15 )] and in some cases, such as the neurofibromatosis type 1 disease involving the NF1 gene ( 16 ), the congenital hypothyroidism involving the TSH-beta subunit gene ( 17 ), or the cystic fibrosis through the CFTR gene ( 18 ), these mutations directly affect (disrupt or enhance) the binding of hnRNP H/F to the pre-mRNA ( 18 , 19 ).
|
16885237_p0
|
16885237
|
INTRODUCTION
| 4.675229 |
biomedical
|
Review
|
[
0.9899408221244812,
0.004225458949804306,
0.005833691917359829
] |
[
0.07363399863243103,
0.0021662614308297634,
0.9232544898986816,
0.0009452748927287757
] |
en
| 0.999998 |
Bcl-x is a member of the Bcl-2 family of apoptotic genes and plays an important role during development by regulating apoptosis of damaged or aged cells. Bcl-x naturally exists in two isoforms, Bcl-x L (233 amino acids) and Bcl-x S (170 amino acids). These two isoforms result from alternative splicing, Bcl-x S containing a truncated exon 2 as compared to Bcl-x L ( 20 ). The effect of these isoforms is antagonistic; Bcl-x L is anti-apoptotic while Bcl-x S is pro-apoptotic. It was shown that in a number of cancer cells, the Bcl-x L isoform is overexpressed, which decreases apoptosis and therefore increases the risk of metastases ( 21 , 22 ). The ratio of Bcl-x S and Bcl-x L in cells depends on two cis -acting elements flanking the 5′ splice site of Bcl-x S ( 23 ) and hnRNP H and F promote the production of the Bcl-x S isoform by recognizing one of these elements that contains three consecutive G-tracts. Mutations of these G-tracts abolish hnRNP F/H binding and thereby the production of the Bcl-x S isoform ( 8 ).
|
16885237_p1
|
16885237
|
INTRODUCTION
| 4.53956 |
biomedical
|
Study
|
[
0.9994990825653076,
0.00023163882724475116,
0.00026929809246212244
] |
[
0.9966381788253784,
0.0006736484938301146,
0.0025574788451194763,
0.00013073139416519552
] |
en
| 0.999998 |
HnRNP H family members contain two (2H9) or three (H, H′ and F) quasi RNA recognition motifs (qRRMs) and one or two glycine rich auxiliary domains and are highly similar in sequence (hnRNP F and H share 78% sequence identity) ( 24 – 26 ). Human hnRNP F is a 45 kDa protein, which consists of two N-terminal qRRMs (residues 1–102 and 111–194) followed by a glycine-rich motif (residues 195–276) and a third qRRM (residues 277–366) . These domains were denoted qRRMs because of the ability of these proteins to bind RNA and the small resemblance between the qRRM and the classical RRM motif. However, two conserved sequences found in all RRMs (RNP 1 and 2, located in the β-strands 3 and 1, respectively) that contain positively charged and aromatic residues involved in RNA binding are poorly conserved in hnRNP H family members ( 24 ).
|
16885237_p2
|
16885237
|
INTRODUCTION
| 4.492541 |
biomedical
|
Study
|
[
0.9994425177574158,
0.0002624315384309739,
0.00029497643117792904
] |
[
0.9978322386741638,
0.0005739028565585613,
0.001475239754654467,
0.00011857134086312726
] |
en
| 0.999998 |
In order to gain insight into G-tract recognition by hnRNP H members, we initiated an NMR study of human hnRNP F. We have determined the solution structure of the isolated qRRMs of hnRNP F and identified the residues that are important for the interaction with the Bcl-x G-tract RNA by NMR chemical shift perturbation and mutagenesis experiments. We show that the structures of the three qRRMs adopt a classical RRM fold and that the two N-terminal qRRMs are responsible for RNA binding. Our data also show that the mode of RNA recognition by hnRNP F differs from classical RNA recognition by RRMs as the β-sheet surface of the qRRM is not engaged in RNA binding.
|
16885237_p3
|
16885237
|
INTRODUCTION
| 4.279696 |
biomedical
|
Study
|
[
0.9994539618492126,
0.00034271524054929614,
0.00020341313211247325
] |
[
0.9993244409561157,
0.0002234938438050449,
0.00035720603773370385,
0.00009491217497270554
] |
en
| 0.999998 |
The DNA sequences encoding the first (residues 1–102), second (residues 103–194), third (residues 277–381) and the first two qRRMs (residues 1–194) of human hnRNP F were cloned into the Xho1/BamH1 site of the Pet28b(+) vector containing an N-terminal hexa-histidine tag and overexpressed in BL21 (DE3) codon plus strains (Stratagene). qRRMs were uniformly labeled by overexpression in M9 minimum medium, containing 15 NH 4 Cl and/or 13 C-glucose as the sole nitrogen and carbon source. Cells were grown at 37°C to OD 600 ∼0.6 and induced by adding isopropyl-β- d -thiogalactopyranoside to a final concentration of 1 mM. Cells were harvested 2 h after induction and centrifuged. Cell pellets were resuspended in lysis buffer (50 mM Na 2 HPO 4 , 1 M NaCl and 10 mM Imidazole, pH 8), and lysed by two passages through a cell cracker (Avestin Inc.). Cell lysates were centrifuged 30 min at 20 000 g . His-tagged proteins were purified using Ni-NTA affinity column, dialyzed against NMR Buffer (25 mM NaH 2 PO 4 , 50 mM NaCl and 10 mM β-mercaptoethanol, pH 6.2) and concentrated to ∼0.4 mM (qRRM1 and qRRM1–2) and 2 mM (qRRM2 and qRRM3).
|
16885237_p4
|
16885237
|
Cloning, expression and purification of hnRNP F subdomains
| 4.181687 |
biomedical
|
Study
|
[
0.9995476603507996,
0.00026503202388994396,
0.0001873388682724908
] |
[
0.9991621971130371,
0.000443987111793831,
0.0003101951442658901,
0.00008369268471142277
] |
en
| 0.999996 |
Mutagenesis experiments were carried out using the Quickchange Kit (Stratagene) following manufacturer's instructions.
|
16885237_p5
|
16885237
|
Cloning, expression and purification of hnRNP F subdomains
| 3.08796 |
biomedical
|
Study
|
[
0.9976639747619629,
0.0005953991785645485,
0.0017406422412022948
] |
[
0.6704830527305603,
0.32426217198371887,
0.0037405153270810843,
0.0015142190968617797
] |
en
| 0.999998 |
RNA oligonucleotides were purchased from Dharmacon Research, deprotected according to manufacturer's instructions, desalted using a G-15 size exclusion column (Amersham), lyophilized and resuspended in NMR buffer.
|
16885237_p6
|
16885237
|
Cloning, expression and purification of hnRNP F subdomains
| 3.516172 |
biomedical
|
Study
|
[
0.9990567564964294,
0.0002941298298537731,
0.0006490259547717869
] |
[
0.8026561737060547,
0.19428198039531708,
0.0022248097229748964,
0.000836942985188216
] |
en
| 0.999996 |
All NMR experiments were carried out at 313K using Bruker DRX-500 MHz equipped with a cryoprobe, DRX-600 MHz and Avance-900 MHz spectrometers. Data were processed using XWINNMR (Bruker) and analyzed with Sparky ( ). Sequence-specific backbone assignments were achieved using 2D ( 15 N- 1 H)-HSQC, 2D ( 13 C- 1 H)-HSQC, 3D HNCA, 3D HNCACB, 3D HN(CO)CA and 3D CBCA(CO)NH experiments [for a review see ( 27 )]. 1 H and 13 C side chain assignments were performed using 3D H(C)CH-TOCSY, 3D (H)CCH-TOCSY, 3D NOESY-( 15 N- 1 H)-HSQC and 3D NOESY-( 13 C- 1 H)-HSQC. NH 2 resonances of Asparagine and Glutamine were identified using 3D NOESY-( 15 N- 1 H)-HSQC. Aromatic proton assignments were performed using 2D-( 1 H- 1 H)-TOCSY and 2D-( 1 H- 1 H)-NOESY in 100% D 2 O. All NOESY spectra were recorded with a mixing time of 150 ms, the 3D TOCSY spectra with a mixing time of 23 ms and the 2D TOCSY with a mixing time of 50 ms.
|
16885237_p7
|
16885237
|
NMR measurement
| 4.266403 |
biomedical
|
Study
|
[
0.9994686245918274,
0.0002791130682453513,
0.00025222066324204206
] |
[
0.9928163290023804,
0.0007008358952589333,
0.006336876656860113,
0.00014596640539821237
] |
en
| 0.999996 |
Relaxation measurements were performed on a BRUKER DRX-600 (heteronuclear NOE) and DRX-500 (T1 and T2) equipped with a cryoprobe ( 1 H frequency of 500.13 MHz,), using a 15 N labeled qRRM1–2 sample with a concentration of ∼0.4 mM. The ( 15 N- 1 H) heteronuclear NOE was recorded in an interleaved fashion, recording alternatively one increment for the reference and one for the NOE spectrum. A relaxation delay of 2 s and a 1 H presaturation delay of 3 s were used in the NOE experiment while a 5 s relaxation delay was used in the reference experiment. 15 N T1 relaxation times were derived from seven spectra with different values for the relaxation delay: 10.01, 75.16, 205.48, 355.84, 506.20, 756.80 and 1007.40 ms and an interscan delay of 3 s. Similarly, 15 N T2 relaxation times were derived from seven CPMG experiments with different values for the relaxation delay: 12.40, 24.81, 43.42, 62.04, 80.66, 105.46 and 124.08 ms and an interscan delay of 3 s. T1 and T2 values were extracted using a curve-fitting subroutine included in the program Sparky. Overall correlation times (τ c ) were estimated from the average T1/T2 ratio of the rigid amide resonances ( 1 H- 15 N NOE > 0.65) that have non-overlapping peaks in the HSQC spectrum.
|
16885237_p8
|
16885237
|
NMR measurement
| 4.25728 |
biomedical
|
Study
|
[
0.9994555115699768,
0.0002968682674691081,
0.000247540301643312
] |
[
0.9992948770523071,
0.0002702643978409469,
0.0003588109102565795,
0.00007605316204717383
] |
en
| 0.999998 |
The automated peak picking, NOE assignment and structure calculation of the three individual qRRMs were performed using the AtnosCandid software ( 28 , 29 ). For each qRRM, peak picking and NOE assignments were performed using the two 3D NOESY ( 15 N- and 13 C- edited) spectra and the 2D homonuclear NOE spectrum recorded in D 2 O. Additionally, H-bond constraints were added based on hydrogen–deuterium exchange experiments on the amide protons. Seven iterations were performed and 100 independent structures were calculated at each iteration steps.
|
16885237_p9
|
16885237
|
Structure calculation
| 4.153301 |
biomedical
|
Study
|
[
0.9994926452636719,
0.00025458517484366894,
0.0002526878088247031
] |
[
0.999376118183136,
0.00026813539443537593,
0.0002971497306134552,
0.00005863089609192684
] |
en
| 0.999996 |
The 20 structures with the lowest target function were refined using the SANDER module of AMBER 7.0 ( 30 ) using the simulated annealing protocol described previously ( 31 ). The 20 final structures were analyzed with PROCHECK ( 32 ).
|
16885237_p10
|
16885237
|
Structure calculation
| 3.973457 |
biomedical
|
Study
|
[
0.9991729855537415,
0.00018632430874276906,
0.0006406704196706414
] |
[
0.99815434217453,
0.001451162388548255,
0.0003231818263884634,
0.00007129419827833772
] |
en
| 0.999997 |
Chemical shifts of qRRM1–2 were deposited previously to the BioMagResBank with entry number 6745 ( 33 ). Coordinates of qRRM1, qRRM2 and qRRM3 have been deposited to the Protein Data Bank with accession numbers 2HGL, 2HGM and 2HGN, respectively.
|
16885237_p11
|
16885237
|
Data Bank accession numbers
| 1.902222 |
biomedical
|
Other
|
[
0.9899759888648987,
0.000934302865061909,
0.00908972043544054
] |
[
0.23520413041114807,
0.7602618932723999,
0.0025684311985969543,
0.0019655770156532526
] |
en
| 0.999997 |
The three qRRMs of hnRNP F were studied separately by NMR. qRRM1, qRRM2 and qRRM3 comprise residues 1–102, 103–194 and 277–381, respectively. Additionally, a construct containing both N-terminal qRRMs (qRRM1–2, residues 1–194) was also studied. We reported previously the resonance assignment of qRRM1–2 ( 33 ). Similarly, resonance assignment of qRRM3 was obtained using classical methods (Materials and Methods). For each qRRM, automated NOE peak picking and assignment were performed using the software AtnosCandid ( 28 , 29 ) based on ( 15 N- 1 H)-NOESY-HSQC spectrum, ( 13 C- 1 H)-NOESY-HSQC spectrum and 2D NOE spectrum recorded in D 2 O. A total of 1548, 1931 and 1818 NOE were extracted for qRRM1, qRRM2 and qRRM3, respectively ( Table 1 ). Additionally, 26 (qRRM1), 22 (qRRM2) and 24 (qRRM3) hydrogen bond restraints derived from slowly exchanging amide protons in presence of D2O were used in the structure calculation. The 20 structures with the lowest target function in the last iteration were refined in implicit solvent.
|
16885237_p12
|
16885237
|
Structure of the three qRRMs of hnRNP F
| 4.195577 |
biomedical
|
Study
|
[
0.9994624257087708,
0.00030353906913660467,
0.00023403484374284744
] |
[
0.9994338154792786,
0.00016128926654346287,
0.00033339473884552717,
0.00007150222518248484
] |
en
| 0.999996 |
The three qRRM structures display a compact β 1 α 1 β 2 β 3 α 2 β 4 fold resulting in a 4-stranded antiparallel β-sheet and two α-helices packed against the β-sheet . This fold is very similar to the classical RRM fold. The core structure consists of residues 11–98 (qRRM1), 111–192 (qRRM2) and 289–362 (qRRM3). The first β-strand comprises residues 12–17, 112–117 and 289–294, for qRRM1, qRRM2 and qRRM3, respectively. It is followed by the first α-helix (residues 24–30, 124–130 and 302–308), the second (residues 43–47, 140–143 and 315–318) and third β-strands (residues 55–61, 153–159 and 330–335), the second α-helix (residues 64–73, 163–171 and 338–345) and the last β-strand (residues 84–89, 182–187 and 357–361). Furthermore, the structures of qRRM1 and qRRM2 are characterized by an additional β-hairpin (β3′ and β3′) located between α2 and β4 (residues 76–78 and 81–83 for qRRM1, and 174–176 and 179–181 for qRRM2) and a C-terminal α-helix (α3) (residues 92–97 and 188–192 for qRRM1 and qRRM2, respectively) that lies on the β-sheet surface . This additional C-terminal α-helix forms a small hydrophobic core involving residues of this helix and of the β-sheet, mainly β1 and β3 . Especially hydrophobic residues of the C-terminal α-helix (M93, V96 and L97 for qRRM1, and V191 for qRRM2) are in contact with hydrophobic and aromatic residues of the β-sheet (V12, H44, I46 and F58 for qRRM1, and F112, T142 and F156 for qRRM2).
|
16885237_p13
|
16885237
|
Structure of the three qRRMs of hnRNP F
| 4.725711 |
biomedical
|
Study
|
[
0.9985470175743103,
0.000965819985140115,
0.00048710915143601596
] |
[
0.9950725436210632,
0.0014417518395930529,
0.00307298987172544,
0.0004126530257053673
] |
en
| 0.999997 |
It was reported previously that two consecutive RRMs can interact with each other ( 34 , 35 ). We therefore studied a longer construct containing the two N-terminal qRRMs (qRRM1–2, residues 1–194) by NMR. The HSQC spectrum of qRRM1–2 is very similar to the HSQC spectra of qRRM1 and qRRM2 indicating that the structures of the individual domains are very similar in the context of qRRM1–2. Some small differences, however, can be observed for some residues located, as expected, at the C-terminus of qRRM1 and the N-terminus of qRRM2, and also in the loop connecting β2 and β3 of the first qRRM (Y47 to S54) suggesting that the two qRRMs might interact with each other . Careful analysis of the NOESY spectra of qRRM1–2, qRRM1 and qRRM2, however, did not provide a clear evidence for the presence of inter-domain NOEs. Similarly, structure calculation of qRRM1–2 generated the proper RRM fold for qRRM1 and qRRM2 but the two domains were completely independent (data not shown). To further investigate whether these two domains are independent in solution or adopt a fixed relative orientation, we measured NMR relaxation experiments and performed a dynamic study of qRRM1–2. ( 1 H- 15 N)-NOE, T1 and T2 were measured . Except for the 10 first residues that are highly flexible [negative ( 1 H- 15 N)-NOE values], the 2 qRRMs possess a rigid core [( 1 H- 15 N)-NOE values higher than 0.7]. The linker region (residues 100–110), however, shows low ( 1 H- 15 N)-NOE values (between 0.19 and 0.5), which indicates that the linker between the two qRRMs is flexible and suggests that the two qRRMs tumble independently in solution. As a confirmation, we calculated the T1/T2 ratio and estimated the overall correlation time (τ c ) of qRRM1–2 and also of qRRM1 and qRRM2 in the context of qRRM1–2 ( Table 2 ). The estimated overall correlation time for qRRM1–2 is 8.3 ± 0.6 ns, which is too short for a compact domain of 21.6 kDa. This value is in agreement with two globular domains of ∼10 kDa that tumble independently (Discussion).
|
16885237_p14
|
16885237
|
Dynamics of qRRM1 and qRRM2
| 4.472528 |
biomedical
|
Study
|
[
0.9991002082824707,
0.0005826702108606696,
0.0003171804128214717
] |
[
0.9990798234939575,
0.0002997437841258943,
0.0004651416966225952,
0.00015534376143477857
] |
en
| 0.999997 |
We used NMR titration experiments to test the ability of each qRRM of hnRNP F to bind G-tract RNA. For this purpose, different RNAs containing one or more G-tracts were used. First, we studied complex formation between a construct containing the two N-terminal qRRMs (qRRM1–2) and CGAU GGG AA (the underlined sequence corresponds to the G-tract, which is the minimum RNA sequence recognized by hnRNP F), which is the HIV-1 p17gag instability (INS) sequence that promotes export of unspliced HIV-1 transcripts ( 13 ). NMR studies of the free RNA show the presence of protected imino protons indicating that intramolecular or intermolecular hydrogen bonds are formed in our NMR conditions. Therefore, the free RNA is not unstructured but adopts a compact conformation that is most likely a G-quadruplex. Our titration experiments show that both qRRM1 and qRRM2 domains are binding RNA, and that two molecules of RNA bind one molecule of qRRM1–2, suggesting that each qRRM binds one G-tract (data not shown). Imino protons that were present in the free RNA are not observed in complex indicating that the RNA unfolds upon binding. Moreover, the complex is in fast exchange on the NMR time scale, which is indicative of a low-binding affinity. We then tested the ability of qRRM1–2 to bind a longer RNA containing two consecutive G-tracts and chose a sequence corresponding to the two first G-tracts of the Bcl-x RNA, C GGG AU GGGG UA . In this case, the complex is in intermediate exchange (peaks disappear during the titration and reappear when a 1:1 complex is formed), which is indicative of a higher binding affinity. As observed with the previous RNA, the free Bcl-x RNA forms a compact structure that is disrupted upon qRRM1–2 binding. To ascertain that these two qRRMs can recognize RNA independently from each other, we titrated the p17gag INS sequence with qRRM1 and qRRM2 and observed the same perturbations as in the context of qRRM1–2, demonstrating that qRRM1 and qRRM2 of hnRNP F can bind and unfold G-tract RNA irrespective of the presence of the other qRRM. We then tested the ability of hnRNP F qRRM3 to bind RNA and chose the third G-tract sequence of the Bcl-x RNA, CU GGGG U . In this case, no changes are observed in the HSQC spectra, which signify that qRRM3 of hnRNP F does not—or very weakly—bind G-tract RNA when isolated. As a consequence, imino protons of the RNA are still observed in the presence of qRRM3. We can therefore conclude that the two N-terminal qRRMs of hnRNP F are primarily responsible for G-tract RNA recognition.
|
16885237_p15
|
16885237
|
NMR studies on hnRNP F binding to G-tract RNA
| 4.476141 |
biomedical
|
Study
|
[
0.9991546869277954,
0.0005212383694015443,
0.0003240782243665308
] |
[
0.9989548921585083,
0.00028328955522738397,
0.0006276670610532165,
0.00013406558718997985
] |
en
| 0.999997 |
In order to identify the minimum RNA sequence recognized by qRRMs, we tested the binding of qRRM2 with GGG and GG RNAs by NMR titration experiments. Complex formation with GGG is very similar to what is observed with CGAU GGGG AA while GG is not recognized (data not shown). This indicates that three consecutive guanosines are necessary and sufficient for hnRNP F binding. We also tested the binding of qRRM2 with another RNA sequence, the UGCAUG Fox RNA-binding site, that does not contain a G-tract ( 31 ). As expected, hnRNP F qRRM2 does not bind this sequence showing that hnRNP F recognition to G-tract RNA is specific (data not shown).
|
16885237_p16
|
16885237
|
NMR studies on hnRNP F binding to G-tract RNA
| 4.143757 |
biomedical
|
Study
|
[
0.9994956254959106,
0.00025637767976149917,
0.0002479628019500524
] |
[
0.9994990825653076,
0.00021881150314584374,
0.00022239894315134734,
0.00005967012111796066
] |
en
| 0.999995 |
Since the complex between qRRM1–2 and Bcl-x G-tract RNA has the highest affinity, we used triple resonance experiments to assign the chemical shifts of qRRM1–2 in complex with Bcl-x RNA. Figure 4C shows the combined chemical shift differences between the free and bound qRRM1–2 amide proton and nitrogen atoms. For each qRRM, the same regions are affected by RNA binding. These are the loop connecting β1 and α2 (residues 16–23 and 115–123 for qRRM1 and qRRM2, respectively), the loop between β2 and β3 (residues 52–57 and 150–154) and the small β-hairpin between α2 and β4 (residues 75–86 and 174–184) .Residues that align with the classical RNP1 and 2 sequences (13–18 and 113–118 for RNP2 and 54–61 and 152–159 for RNP1) do not display a significant chemical shift perturbation upon RNA binding. Resonances corresponding to residues forming the C-terminal α-helix are also not significantly perturbed upon complex formation. Furthermore, NOEs between residues of the C-terminal α-helix and the β-sheet are still present when qRRM is bound to the RNA, indicating that in complex, the hydrophobic cluster between the C-terminal α-helix and the β-sheet is conserved (Supplementary Data 1).Mapping the perturbed residues on the structure of qRRM1 and qRRM2 shows that these two domains bind the RNA through their β-hairpin and the two loops connecting β1−α1 and β2−β3 . These three regions are rich in aromatic and positively charged residues making a suitable RNA-binding platform .
|
16885237_p17
|
16885237
|
NMR studies on hnRNP F binding to G-tract RNA
| 4.449074 |
biomedical
|
Study
|
[
0.9990980625152588,
0.0006479259463958442,
0.00025408933288417757
] |
[
0.9989975094795227,
0.0003319201641716063,
0.0004989597946405411,
0.00017162786389235407
] |
en
| 0.999996 |
To test the importance of the aromatic residues located in the β-hairpin and the β1−α1 loop for G-tract recognition, we performed single mutations of F120, H178 and Y180 of qRRM2 to Alanine. These aromatic residues are at the surface of the protein, solvent exposed in the free structure and display a significant chemical shift perturbation upon RNA binding . As a control, we also mutated F156 to alanine. F156 is located in β3 and corresponds to a very important residue of the RNP1 sequence for RNA binding in classical RRM. In the case of qRRM2, however, this residue is participating in the hydrophobic cluster involving the β-sheet and the C-terminal α-helix . HSQC spectrum of this mutant shows that the F156A mutation drastically affects the fold of qRRM2 since most of the peaks disappear (Supplementary Data 2) most probably due to unfolding or aggregation. This suggests that this phenylalanine is important for proper folding or solubility of qRRM2 most likely due to its interaction with the C-terminal α-helix. The three other mutations (F120A, H178A and Y180A), however, do not affect the fold of qRRM2, which is consistent with the observation that these residues have their side chains exposed to the solvent in the structure of the free qRRM2 and therefore do not participate in the fold of the domain (Supplementary Data 2). We then analyzed the binding ability of the F120A, H178A and Y180A mutants to the CGAU GGG AA RNA sequence by NMR titration experiments. These mutations do not disrupt RNA binding (Supplementary Data 3) but differences with the wild type are observed. In the case of H178A, chemical shift perturbations are similar to those observed for the wild type indicating that this residue does not play a major role in G-tract recognition. F120A and Y180A, however, show a different binding pattern. When F120, located in the β1−α1 loop, is mutated to Ala, peaks corresponding to the three regions of the RNA interface (the β-hairpin, the β1−α1 loop and the β2-β3 loop) display smaller chemical shift perturbations than the wild type, indicative of a lower affinity for RNA. When Y180, located in the β-hairpin, is mutated to Ala, only peaks corresponding to residues in the β-hairpin and β2−α3 loops display a smaller perturbation, while peaks corresponding to the β2−β3 loop are not affected. We then produced the F120A/Y180A double mutant. In this case, the double mutation does not disrupt the fold of the qRRM (Supplementary Data 2) and completely abrogates G-tract binding (Supplementary Data 3). Therefore, we can conclude that, in contrast to classical RRMs, the non-canonical F120 and Y180 residues are crucial for RNA binding.
|
16885237_p18
|
16885237
|
Mutagenesis of hnRNP F qRRM1 and qRRM2 aromatic residues
| 4.435347 |
biomedical
|
Study
|
[
0.9991029500961304,
0.0005786889814771712,
0.0003183534718118608
] |
[
0.9991294741630554,
0.00027444359147921205,
0.0004589512827806175,
0.00013715290697291493
] |
en
| 0.999998 |
The canonical fold of the RRM is well known. To date, >40 structures of this domain free or in complex with RNA have been solved [for a review see ( 34 )]. It consists of ∼90 amino acids and is composed of a 4-stranded anti-parallel β-sheet and two α-helices packing on one surface of the β-sheet. In some cases, extra secondary structure elements could also be observed in addition to the canonical RRM fold, in particular a small β-hairpin located between α-helix 2 and β-strand 4. Sequence conservation between various RRMs is low except for two well-defined regions, RNP 1 and 2, of ∼7–8 amino acids located in the β-strands 1 and 3 that are responsible for RNA binding. hnRNP H family members are able to bind RNA, more precisely poly(G) sequences (G-tract), and three domains of the proteins were identified as possible RNA-binding domains. These domains are ∼90 amino acids and display a small resemblance to the classical RRM motif. The two RNP sequences, however, are not conserved and these domains were therefore termed qRRMs ( 24 ).
|
16885237_p19
|
16885237
|
The three qRRMs of hnRNP F adopt the canonical RRM fold but qRRM1 and qRRM2 display extra secondary structure elements
| 4.548026 |
biomedical
|
Review
|
[
0.9958387613296509,
0.002014610916376114,
0.0021466435864567757
] |
[
0.13881351053714752,
0.0015042927116155624,
0.8586915135383606,
0.0009906922932714224
] |
en
| 0.999997 |
We solved the structures of the three qRRMs of hnRNP F using NMR spectroscopy. The structures are well defined and consist of the canonical β 1 α 1 β 2 β 3 α 2 β 4 RRM fold . For qRRM1 and qRRM2, two additional secondary structure elements are present. A small β-hairpin is formed between α2 and β4 and, more strikingly, the C-terminal part adopts an α-helical conformation and interacts with the β-sheet surface through hydrophobic and aromatic residues . Interestingly, residues of the β-sheet that interact with this C-terminal α-helix correspond to aromatic residues (F58 in qRRM1, and F112 and F156, in qRRM2) that are often essential for RNA interactions in classical RRM–RNA complexes. These residues are buried by the C-terminal α-helix and not solvent exposed as observed in most structures of free RRMs. The presence of a C-terminal α-helix packing against the β-sheet surface is only observed in a few RRM structures, such as the C-terminal RRM of LA protein ( 36 ), the N-terminal RRM of U1A ( 37 ), the N-terminal RRM of CstF-64 ( 38 ) and the p14 spliceosomal protein ( 39 ).
|
16885237_p20
|
16885237
|
The three qRRMs of hnRNP F adopt the canonical RRM fold but qRRM1 and qRRM2 display extra secondary structure elements
| 4.439892 |
biomedical
|
Study
|
[
0.9993366599082947,
0.00044948732829652727,
0.00021379241661634296
] |
[
0.9984649419784546,
0.0003475177218206227,
0.001035991939716041,
0.0001515655021648854
] |
en
| 0.999998 |
An analysis of RRM structures solved to date shows that two consecutive RRMs that are separated by a short linker (10–20 residues) can interact with each other to form a compact fold. This RRM–RRM interaction is often induced by the presence of RNA but can also occur in the absence of RNA ( 34 , 35 ). Since qRRM1 and qRRM2 of hnRNP F are separated by a short linker (residues 100–110), it is possible that these two domains interact with each other. We therefore studied a construct containing the two N-terminal qRRMs (qRRM1–2) by NMR. Relaxation measurements and dynamical studies clearly show that the linker between the two domains is flexible . Furthermore, estimation of the overall correlation time for each qRRM strongly suggests that these two domains tumble independently in our conditions. The estimated overall correlation time for qRRM1–2 is 8.3 ± 0.6 ns, which is too short for a compact domain of 21.6 kDa, while using peaks corresponding to the first qRRM or the second qRRM only, estimations of the overall correlation times are 8.6 ± 0.4 and 7.8 ± 0.5 ns, respectively ( Table 2 ). These values are in agreement with a globular domain of ∼10 kDa. We compared our relaxation analysis with those of RRM3 and RRM4 of the protein PTB. These two domains are interdependent in solution and show a large inter-domain interface involving 27 residues ( 35 ). NMR relaxation measurements and dynamical studies were performed on the wild-type PTB34, as well as on a mutant protein with a disrupted interface. For the wild-type construct, ( 1 H- 15 N)-NOE values of the linker were higher than 0.68 and the estimated overall correlation time was 10.4 ns, while for the mutant, the estimated overall correlation time was 8.0 and 6.9 ns for RRM3 and RRM4, respectively ( 35 ). We can therefore conclude that, in our NMR conditions, qRRM1 and qRRM2 of hnRNP F are independent in solution.
|
16885237_p21
|
16885237
|
The three qRRMs of hnRNP F adopt the canonical RRM fold but qRRM1 and qRRM2 display extra secondary structure elements
| 4.475202 |
biomedical
|
Study
|
[
0.9992571473121643,
0.00045831879833713174,
0.0002844973641913384
] |
[
0.9989811778068542,
0.00029060879023745656,
0.0005908843595534563,
0.0001372856495436281
] |
en
| 0.999998 |
HnRNP F is a key regulator of splicing events and specifically recognizes RNAs containing poly(G) sequences (G-tracts) ( 3 ). It is, however, not clear which part of the protein is responsible for RNA recognition, although the three qRRMs are good candidates. Furthermore, it was postulated previously that the bases flanking the G-tract are important for RNA recognition ( 11 , 13 ). Jacquenet et al . ( 11 ) defined the minimum RNA sequence recognized by the hnRNP H family as UGGG, while Caputi and Zahler ( 13 ) defined the minimum RNA motif as GGGA. In order to identify which part of the protein is involved in RNA recognition and also what is the minimum RNA sequence recognized by hnRNP F, we performed NMR chemical shift perturbation experiments and tested the binding ability of each qRRM with different G-tract RNAs.
|
16885237_p22
|
16885237
|
HnRNP F qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and two consecutive G-tracts are necessary for high-affinity binding
| 4.200388 |
biomedical
|
Study
|
[
0.9994781613349915,
0.0002744600351434201,
0.000247374817263335
] |
[
0.9993013143539429,
0.0001779932208592072,
0.0004531798476818949,
0.00006757387745892629
] |
en
| 0.999998 |
Our data clearly indicate that qRRM1 and qRRM2 are responsible for G-tract recognition . qRRM1–2 is able to bind single G-tracts in a one to two ratio indicating that each qRRM binds one G-tract. Furthermore, we observe that longer RNAs containing two consecutive G-tracts separated by a short linker bind qRRM1–2 with a considerably higher affinity, as was observed previously ( 40 ). Our results also show that the minimum RNA sequence recognized by qRRM1 and qRRM2 is GGG, which indicate that three consecutive guanosines are important for hnRNP F binding while the bases flanking the G-tract are not. We could not detect binding of qRRM3 with G-tract RNA. These results are striking since the three qRRMs of hnRNP F display a high sequence similarity . The structure of qRRM3, however, differs slightly from the two N-terminal qRRM structures. qRRM3 adopts the canonical RRM fold and does not exhibit additional secondary structure elements like the small β-hairpin and the C-terminal α-helix. Since in qRRM1 and qRRM2, the β-hairpin is involved in G-tract recognition (see below), its absence in the qRRM3 structure might prevent RNA binding, although most of the residues that seem to be involved in RNA binding by qRRM1 and qRRM2 are conserved in qRRM3. It should, however, be noticed that, although no clear interaction between qRRM3 and G-tract could be observed, few peaks tend to disappear during the NMR titration and these residues are also located in the β-hairpin region.
|
16885237_p23
|
16885237
|
HnRNP F qRRM1 and qRRM2 but not qRRM3 are responsible for G-tract recognition and two consecutive G-tracts are necessary for high-affinity binding
| 4.404282 |
biomedical
|
Study
|
[
0.999254047870636,
0.00047668328625150025,
0.00026915711350739
] |
[
0.9991558790206909,
0.0002802476810757071,
0.0004238342517055571,
0.00014009332517161965
] |
en
| 0.999998 |
Based on the numerous structures of RRM-RNA complexes, it is now well established that the RNA recognition by RRM is mediated by amino acids present at the surface of the β-sheet, in particular two sequences located in the central β3 (RNP1) and β1 (RNP2) strands [reviewed in ( 34 )]. These mainly positively charged (R, K) and aromatic residues (F, Y) are crucial for RNA binding through H-bond formation and base stacking. In the case of hnRNP F qRRMs, these RNP sequences are not canonical, especially a positively charged residue of RNP1 is changed to a Serine (S54 in qRRM1) or a Threonine (T152 and T328 in qRRM2 and 3) and two highly conserved aromatic residues are changed to a Glutamate in all qRRMs (E56, E154 and E330) and to a positively charged residues in qRRM1 (K14) and in qRRM2 (R114) . Furthermore, the structures of qRRM1 and qRRM2 of hnRNP F show that a C-terminal α-helix that is unusual for the RRM fold packs against the β-sheet, forming a hydrophobic core with residues of RNP1 and RNP2, therefore masking the classical RNA-binding site .
|
16885237_p24
|
16885237
|
qRRM1 and qRRM2 of HnRNP F recognize G-tracts by an unusual binding surface
| 4.640725 |
biomedical
|
Study
|
[
0.9976414442062378,
0.001360688591375947,
0.0009979282040148973
] |
[
0.5085223913192749,
0.0022566188126802444,
0.48769962787628174,
0.0015213738661259413
] |
en
| 0.999997 |
We identified the residues of qRRM1–2 that are important for Bcl-x G-tract binding using NMR chemical shift perturbation experiments. Strikingly, the residues that show the highest perturbations are not found in the β-sheet, but are located in the extra β-hairpin and in the β1−α1 and β2−β3 loops. Furthermore, the C-terminal α-helix that interacts with the canonical RNP1 and RNP2 residues is still present in the complex (Supplementary Data 1), which provides strong evidence that hnRNP F qRRMs bind RNA in a way distinct from the canonical RRM. It was reported previously that in some RRMs, a C-terminal α-helix is present in the free form and makes hydrophobic interactions with the β-sheet. However, these RRMs either do not bind RNA (LA C-terminal domain) or when they do, the β-sheet surface is the primary binding surface (U1A N-terminal RRM, CstF-64). In the case of U1A, the C-terminal α-helix rotates away from the β-sheet, and repositions in a way that the β-sheet surface binds to the RNA ( 41 ). For CstF-64, no structure in complex with RNA is available but NMR titration and relaxation studies show that the C-terminal α-helix unfold upon RNA binding and that the β-sheet RNP1 and RNP2 residues are responsible for RNA binding ( 38 , 42 ).
|
16885237_p25
|
16885237
|
qRRM1 and qRRM2 of HnRNP F recognize G-tracts by an unusual binding surface
| 4.398888 |
biomedical
|
Study
|
[
0.9993127584457397,
0.00045829839655198157,
0.00022890925174579024
] |
[
0.9990851879119873,
0.0002629712107591331,
0.0005224385531619191,
0.00012935003906022757
] |
en
| 0.999996 |
Analysis of the NMR chemical shift perturbation data show that many of the residues that are perturbed upon RNA binding are positively charged or aromatic (R16, W20, R52, R75, H80, R81, Y82 and F86 for qRRM1, and R116, F120, K150, R175, H178, R179, Y180 and F184 for qRRM2) and hence make a favorable binding platform for a negatively charged RNA molecule . Correspondingly, analysis of the electrostatic potential at the surface of qRRM1 and qRRM2 shows that these regions form a highly positively charged surface . Mutagenesis experiments of aromatic residues located in this novel RNA-binding regions confirm their importance for RNA recognition. Our data show that F120, located in the β1−α1 loop, and Y180, located in the β-hairpin, of qRRM2 (corresponding to W20 and Y82 of qRRM1) are crucial for hnRNP F to bind G-tracts since mutation of these two residues to alanine completely abolish RNA binding (Supplementary Data 3). The involvement of the β1−α1 loop and the β-hairpin in RNA binding has been described earlier ( 31 , 43 ). In the structure of the Fox-1–RNA complex, the RNA lies on top of the β-sheet as observed for other RRM domains and the β1−α1 is also involved in binding. In particular, mutagenesis of an aromatic residue (located at the same position as W20 and F120 of qRRM1 and qRRM2 of hnRNP F) decreases significantly the affinity of Fox-1 for RNA ( 31 ). The structure of the tcUBP1 protein also contains the β-hairpin observed in hnRNP F qRRM1 and qRRM2, and NMR chemical shift perturbation data showed that RNA binding involves the β-sheet surface but also the β-hairpin extending the RNA binding surface ( 43 ). In both cases, however, the RNP1 and RNP2 sequences are involved in RNA binding and the β1−α1 or β-hairpin constitute an additional binding area. Our NMR chemical shift perturbation and mutagenesis data therefore define a novel RNA recognition mode involving the β-hairpin, the β1−α1 and the β2−β3 loops but not the β-sheet surface. Classical RRMs are known to bind single-stranded RNAs. The fact that qRRM1 and qRRM2 of hnRNP F recognize G-quadruplex structures and not single-stranded RNAs might explain the novel interaction interface that we observe.
|
16885237_p26
|
16885237
|
qRRM1 and qRRM2 of HnRNP F recognize G-tracts by an unusual binding surface
| 4.67779 |
biomedical
|
Study
|
[
0.9986357092857361,
0.0009762840927578509,
0.00038796637090854347
] |
[
0.9977996945381165,
0.0007646689191460609,
0.001038967864587903,
0.0003966193471569568
] |
en
| 0.999997 |
Our data demonstrate that qRRM1 and qRRM2 of hnRNP F bind G-tract RNA via a novel mode of recognition, in which the RNP1 and RNP2 sequences are not involved. This is consistent with previous analyses showing that the amino acid composition of hnRNP H family members in these sequences was different from the classical RRM. We observe that residues in the β-hairpin, the β1−α1 and the β2−β3 loops of hnRNP F qRRM1 and qRRM2 are involved in RNA binding. We therefore performed a sequence alignment of hnRNP F from different species and human hnRNP H, H′ proteins. Full-length human hnRNP F, H and H′ share 40% identity (56% homology) but the sequence identity in the qRRM domains is higher (77% identity and 94% homology for the two first qRRMs). Furthermore, residues of human hnRNP F having a significant chemical shift perturbation upon G-tract RNA binding are strictly conserved between the three proteins. Similarly, full-length human, monkey, dog, bull, mouse and rat hnRNP F share 96% sequence identity (99% similarity) and all residues that show a high chemical shift perturbation upon RNA binding are strictly conserved. We also compared human hnRNP F with glorund, a drosophila homolog ( 44 ). These two proteins share 50% sequence homology. When considering the first two RRM domains only (residues 1–194 of human hnRNP F), the homology increases to 78% (42% identity) and residues of hnRNP F that are involved in RNA binding are strictly conserved. This is striking since glorund was shown to interact with a stem loop that does not contain G-tracts. The interaction of glorund with G-tracts, however, was not investigated.
|
16885237_p27
|
16885237
|
Biological implication for RNA metabolism
| 4.444007 |
biomedical
|
Study
|
[
0.9992583394050598,
0.00046626394032500684,
0.0002754218876361847
] |
[
0.9990220069885254,
0.0002591844240669161,
0.0005797969060949981,
0.00013906163803767413
] |
en
| 0.999998 |
G-tracts found both upstream and downstream of introns cooperate to allow intron definition ( 4 , 45 ). Since hnRNP H family members are able to form homodimers ( 46 ), it was proposed that a dimer of hnRNP F or H can simultaneously recognize the two upstream and downstream G-tracts, looping out the intron, therefore facilitating intron definition ( 45 ). This model is similar to the one proposed previously for the protein PTB in which PTB dimerizes to loop out alternatively spliced exons ( 47 ). In our laboratory, however, we reported recently an alternative model in which one monomer of PTB is sufficient to loop out an exon through the unique organization of the RRM3 and RRM4 domains ( 48 ). In this case, the two RRMs interact extensively with one another and each RRM bind one polypyrimidine tract. Our data indicate that both qRRM1 and qRRM2 of hnRNP F are able to bind a G-tract independently. We therefore considered whether each qRRM could bind G-tracts located upstream and downstream of the intron looping it out. Our results, however, are very distinct from what was observed for PTB. In contrast to PTB RRM3–4, hnRNP F qRRM1 and qRRM2 are independent in solution. Furthermore, qRRM1–2 is able to bind two consecutive G-tracts separated by a short linker with high affinity, while PTB RRM3–4 can only bind two polypyrimidine tracts separated by at least 15 bases ( 48 ). It is therefore unlikely that one molecule of hnRNP F is sufficient for looping out the intron and a dimer of hnRNP F is probably necessary. Further investigation on full-length hnRNP F should clarify which region of the protein is responsible for dimerization.
|
16885237_p28
|
16885237
|
Biological implication for RNA metabolism
| 4.541139 |
biomedical
|
Study
|
[
0.9991163611412048,
0.0005218886071816087,
0.00036170496605336666
] |
[
0.9987578392028809,
0.0005103863659314811,
0.0005555712268687785,
0.0001762943429639563
] |
en
| 0.999997 |
G-tract RNAs are frequent splicing recognition elements found downstream of splice sites ( 4 , 5 ). They are also present downstream of polyadenylation sites ( 6 ). McCullough and Berget ( 4 ) analyzed the base composition of small human introns. They observed that many introns contain a high frequency of G triplets near the splice sites, as illustrated with the 129 nt intron 2 of the human α-globin gene that contains seven G-tracts. Six of them are grouped in pairs with the generic sequence GGG(N) 2–4 GGG and are important for splicing efficiency and exon–intron definition. Similarly, the intron region near the Bcl-x S 5′ splice site contains three consecutive G-tracts ( 8 ). Mutation of one G-tract does not have a tremendous effect on splicing while mutation of two of these G-tracts, leaving one G-tract intact, prevents hnRNP F binding and completely abrogates Bcl-x S production ( 8 ). In combination with our data, we can expect that two consecutive G-tracts are important for hnRNP F qRRM1–2 recognition and function. Furthermore, an analysis of different introns containing consecutive G-tracts shows that the length of the linker between two G-tracts is variable. Our dynamical studies of hnRNP F qRRM1–2 demonstrate that the linker between the two domains is flexible and that the two qRRMs do not adopt a fixed conformation relative to each other. This flexibility might be important for the adaptation of qRRM1–2 to bind two consecutive G-tracts separated by linkers of variable length.
|
16885237_p29
|
16885237
|
Biological implication for RNA metabolism
| 4.397859 |
biomedical
|
Study
|
[
0.9993808269500732,
0.0003732513578142971,
0.00024584776838310063
] |
[
0.9990524649620056,
0.0002709747932385653,
0.0005551696522161365,
0.00012128326488891616
] |
en
| 0.999999 |
G-tracts have been shown to form special structures called G-quadruplexes. In RNA, G-tracts are responsible for recruitment of hnRNP H family members ( 45 , 49 ). In our NMR study, we observe that free G-tracts adopt a G-quadruplex compact fold, and this structure is disrupted upon HnRNP F binding. This is similar to what was observed for hnRNP A1 and hnRNP D. These two proteins, that specifically recognize telomeric DNA with the sequence TTAGGG, unfold the G-quadruplex structure stimulating telomerase activity ( 50 ). A model for the role of hnRNP F in alternative splicing and polyadenylation could therefore reside in the unfolding of the RNA, allowing other nearby RNA sites, such as 5′ splice sites or polyadenylation signals, to be available for other proteins. In the case of Bcl-x splicing, the G-tracts are located 20 residues downstream of the Bcl-x S 5′ splice site. Formation of a G-quadruplex might therefore prevent the accessibility of this site for the spliceosome leading to production of the Bcl-x L isoform. When hnRNP H members bind to the G-tract, however, they might prevent G-quadruplex formation and therefore make the Bcl-x S 5′ splice site available for the spliceosome.
|
16885237_p30
|
16885237
|
Biological implication for RNA metabolism
| 4.48861 |
biomedical
|
Study
|
[
0.9992460012435913,
0.0005077086971141398,
0.0002463572018314153
] |
[
0.9988399147987366,
0.00044221937423571944,
0.0005511213676072657,
0.00016673936625011265
] |
en
| 0.999998 |
Proteins that have been demonstrated to bind specifically to methylated DNA belong to two characterized families. The methyl-CpG-binding domain (MBD) family comprises five polypeptides in mammals, four of which MBD1, MBD2, MBD4 and MeCP2 have been shown to preferentially bind a symmetrically methylated CpG motif ( 1 – 3 ). These proteins share the MBD which confers specific DNA binding ( 4 ). The unrelated Kaiso protein family binds to DNA sequences containing methyl-CpGs via a distinctive zinc finger motif ( 5 , 6 ). In keeping with evidence that DNA methylation is a signal for transcriptional repression, MeCP2, MBD1, MBD2 and Kaiso all repress transcription in vitro and associate with co-repressors ( 1 , 5 , 7 – 10 ).
|
16893950_p0
|
16893950
|
INTRODUCTION
| 4.40658 |
biomedical
|
Study
|
[
0.9993975162506104,
0.0002669589885044843,
0.00033551239175722003
] |
[
0.9842425584793091,
0.0005496835801750422,
0.01507432758808136,
0.000133409645059146
] |
en
| 0.999996 |
The aim of the present study was to harness the affinity of the MBD to create a superior reagent for the detection of methyl-CpG in vitro and in vivo . Assessment of global levels of DNA methylation currently relies on mass spectrometry ( 11 ), ‘nearest neighbour’ methods ( 12 ), enzymatic methyl-group acceptance assays ( 13 ) or HPLC analysis ( 14 ). These quantitative measures are complemented by the use of antibodies against 5-methylcytosine (m 5 C), which in addition to quantification permit visualization of m 5 C distribution within DNA or chromosomes ( 15 , 16 ). A disadvantage of anti-m 5 C antibodies is that the epitope is best exposed when DNA is single-stranded and DNA must therefore normally be denatured before detection. The MBD, on the other hand, is specific for native double-stranded DNA. Another potential benefit of the MBD reagent would be its specificity for m 5 C in the context of a symmetrically methylated CpG sequence ( 4 ).
|
16893950_p1
|
16893950
|
INTRODUCTION
| 4.212447 |
biomedical
|
Study
|
[
0.9995144605636597,
0.00028154405299574137,
0.000203982213861309
] |
[
0.9993013143539429,
0.0002547510957811028,
0.00037273255293257535,
0.00007121674570953473
] |
en
| 0.999996 |
To test the MBD reagent, we designed a series of artificial methyl-CpG-binding proteins based on multimerization of the MBD of Mbd1. The resulting polypeptides bound strongly to methylated DNA with high specificity and could compete with other methyl-CpG-binding proteins for DNA binding in vitro . When expressed in vivo , the poly-MBD protein localized to the methyl-CpG-rich heterochromatic foci of mouse cells and could recruit a functional domain specifically to methylated DNA sequences. Poly-MBD proteins proved to be sensitive and specific reagents for the detection of methyl-CpGs in immobilized DNA and cytological preparations.
|
16893950_p2
|
16893950
|
INTRODUCTION
| 4.186425 |
biomedical
|
Study
|
[
0.9995595812797546,
0.00023493268236052245,
0.00020548432075884193
] |
[
0.9992352724075317,
0.0003329564060550183,
0.00036168075166642666,
0.00007014825678197667
] |
en
| 0.999997 |
To generate constructs encoding the poly-MBD with varying numbers of MBDs, two PCR fragments (PCR-A and PCR-B) were amplified from each of the templates pFlag-mMBD1 or pFlag-mMBD1-R22A ( 17 ) to produce the wild-type and R22A control constructs, respectively. PCR-A was amplified using primers 5′-GCGAATTCGATGCCAAAAAAGAAGAGAAAGGTAGATATCATGGCTGAGTCCTGGCAGGACTGCC-3′ and 5′-GCTCTAGACACGTGTTAAGCGTAGTCTGGGACGTCGTATGGGTACTTGGGAATGGGATGGCATAAGG-3′. It encodes a N-terminal nuclear localization signal (NLS), cDNA from mouse Mbd1 corresponding to amino acids 1–75 and a C-terminal HA-tag. In addition, it has a 5′ EcoRI site, a EcoRV site just downstream of the NLS, a PmlI site just downstream of the HA-tag and a 3′ XbaI site. PCR-B was amplified using primers 5′-AAGGTAGATATCATGGCTGAGTCCTGGCAGGACTGCC-3′ and 5′-CTAGACACGTGAGAACCACCACCACCAGAACCACCACCACCCTTGGGAATGGGATGGCATAAGG-3′. It encodes a mouse Mbd1 fragment corresponding to amino acids 1–75 followed by a flexible linker [(Gly 4 -Ser) 2 ] with a 5′ EcoRV and a 3′ PmlI site. The pTRE-1xMBD construct was made by inserting EcoRI/XbaI digested PCR-A into EcoRI/XbaI opened pTRE (Clontech). Poly-MBD constructs were made by inserting PmlI/EcoRV digested PCR-B into an EcoRV cut MBD construct. A PCR fragment encoding the activation domain of VP16 was inserted between the NLS and the first MBD using the EcoRV site, creating pTRE-NxMBD-VP16. For expression, the inserts were excised using EcoRI/XbaI and transferred into pET30b+ (pET-NxMBD, bacterial expression), pcDNA3 (p-NxMBD, mammalian expression) or pLZRS-MS (pLZRS-NxMBD, retroviral delivery). The retroviral shuttle vector has been described previously ( 18 ). Reporter plasmids (pRL-TK and pGL2-control) are from Promega.
|
16893950_p3
|
16893950
|
Plasmids
| 4.332409 |
biomedical
|
Study
|
[
0.999437153339386,
0.0002809751022141427,
0.0002819010696839541
] |
[
0.9984993934631348,
0.0007678078836761415,
0.0006275504711084068,
0.00010531269072089344
] |
en
| 0.999995 |
DNA methylation deficient Dnmt1 n/n ( 19 ), p53 −/− mouse embryonic fibroblasts [described in ( 17 )] were maintained in DMEM (Gibco) supplemented with 15% bovine calf serum, non-essential amino acids, sodium pyruvate and antibiotics (Gibco). The presence of a homozygous p53 mutation allows survival of Dnmt1 -deficient cells ( 20 ). Phoenix packaging cells were grown in DMEM and wild-type mouse fibroblasts in MEM alpha, all supplemented with 10% bovine calf serum and antibiotics (Gibco). Production of virus particles and infection of adherent cells was performed as described ( ). Cell lines expressing N-MBD proteins were made by retroviral infection.
|
16893950_p4
|
16893950
|
Cell lines and retroviral infection
| 4.155869 |
biomedical
|
Study
|
[
0.999612033367157,
0.0001819884346332401,
0.00020603668235708028
] |
[
0.9979650974273682,
0.0015490148216485977,
0.00039341827505268157,
0.00009252704330720007
] |
en
| 0.999996 |
Reporter constructs were either M. SssI or mock methylated and co-transfected with p-NxMBD expression constructs plus a non-modified internal transfection control. The amount of expression construct was kept constant by adding empty vector (pcDNA3). Cells were transfected using Lipofectamine (Invitrogen) or JetPEI (QBiogene) according to the manufacturer's instructions. Reporter gene activity and activity of the internal transfection control was measured 40–48 h after transfection using the Dual Luciferase system (Promega) according to the manufacturer's instructions. Each transfection was performed in triplicate and repeated at least twice. Reporter activity is expressed relative to the internal control activity to correct for differences in transfection efficiency.
|
16893950_p5
|
16893950
|
Transfection and reporter gene activity
| 4.111222 |
biomedical
|
Study
|
[
0.9995591044425964,
0.00022921781055629253,
0.0002116034011123702
] |
[
0.9991325736045837,
0.00045125785982236266,
0.00035680498695001006,
0.000059334775869501755
] |
en
| 0.999997 |
Recombinant His 6 -tagged NxMBD proteins were purified from 750 ml induced BL21(DE3) cultures on Ni-NTA agarose (Qiagen) using denaturation and on column renaturation cycles in accordance with the manufacturer's instructions. Recombinant GST-MeCP2 was made as described previously ( 4 ). Nuclear extracts were made from mouse fibroblasts as described previously ( 21 ).
|
16893950_p6
|
16893950
|
Recombinant proteins
| 4.063748 |
biomedical
|
Study
|
[
0.9996167421340942,
0.00014613631356041878,
0.00023718138982076198
] |
[
0.9978354573249817,
0.001621781848371029,
0.0004524939286056906,
0.00009024015162140131
] |
en
| 0.999998 |
Binding reactions including purified His-tagged NxMBD protein, recombinant MeCP2 or nuclear extract in 20 mM HEPES, pH 7.9, 3 mM MgCl 2 , 10% glycerol, 1 mM dithiothreitol, 100 mM KCl and 0.05 µg/µl sonicated Escherichia coli DNA (Sigma) were pre-incubated 10 min at room temperature before the addition of 25 fmol end-labelled double-stranded probe. For supershift reactions anti-His 6 (Santa Cruz, G-18) or anti-MBD3 (Santa Cruz, C-18) was added. After a further 25 min incubation at room temperature, the reactions were loaded on to either 6% polyacrylamide/0.5× TBE gels and run for 2 h at 240 V (4°C) or 1.3% agarose/0.5× TBE gels and run at 6 V/cm (4°C). Polyacrylamide gels were dried on to 3 mm Whatman paper and agarose gels on to DE81 (Whatman). Radioactivity was detected using a phosphor-screen and a Storm 840. All oligonucleotide probes (3ME, 2ME, 1ME, 0ME) used for bandshift are based on the sequence 5′-ATCAGA CG TTCGCCGG CG GATTGGCTTGGCTG CG AAGAAGATA-3′ and the complementary strand. In 3ME all the underlined CpGs are symmetrically methylated, in 2ME C17 and C33 are methylated and in 1ME C17 is methylated whereas all CpG sites in 0ME are left unmethylated. The oligonucleotides were annealed in 10 mM Tris–HCl, pH 8, 1 mM EDTA and 50 mM NaCl. The CG11 probe is described previously ( 22 ), the methylated version is methylated at all CpG dinucleotides by M. Sss I .
|
16893950_p7
|
16893950
|
Bandshifts
| 4.244296 |
biomedical
|
Study
|
[
0.9994080066680908,
0.0003466990892775357,
0.0002453066117595881
] |
[
0.9987232089042664,
0.0008208869840018451,
0.0003593894944060594,
0.00009645050158724189
] |
en
| 0.999997 |
Cells were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature followed by permeabilization in 0.2% Triton X-100 in PBS. Slides were blocked in 3% BSA/PBS before incubation with primary antibody for 60 min. After washing and incubation with Alexa-594 coupled anti-mouse antibody , slides were washed and mounted in Vectashield with DAPI (Vector). Images were obtained using a Zeiss microscope fitted with a CCD camera and processed using Adobe PhotoShop or a Delta Vision deconvolution microscope and SoftWorx software.
|
16893950_p8
|
16893950
|
Immunostaining
| 3.933032 |
biomedical
|
Other
|
[
0.9981876015663147,
0.0010054500307887793,
0.0008068843162618577
] |
[
0.3801078796386719,
0.6163967847824097,
0.0021910308860242367,
0.0013043744256719947
] |
en
| 0.999995 |
Native double-stranded mock- or M. SssI -methylated lambda phage DNA was slot-blotted on to Nytran Supercharge membrane (Schleicher & Schuell) and immobilized by UV crosslinking. Alternatively, DNA was denatured by incubation at 95°C for 10 min, transferred and immobilized on Optitran membrane (Schleicher & Schuell). The filters were blocked 2 h at room temperature or ON at 4°C and incubated with purified recombinant NxMBD proteins (10 µg/ml) for 2 h at room temperature. After washing, the bound protein was detected by incubation with first anti-HA antibody (1:500, F-7, Santa Cruz), then horseradish peroxidase coupled anti-mouse antibody . For detection using anti-m 5 C antibody, the NxMBD and anti-HA incubations were substituted by a 1 h incubation with anti-m 5 C antibody (1:500, Eurogentec). All incubations were in TBS-T supplemented with 5% skimmed milk powder. The blots were developed by enhanced chemiluminescence and exposed to film.
|
16893950_p9
|
16893950
|
Staining purified DNA using NxMBD proteins
| 4.167025 |
biomedical
|
Study
|
[
0.9995065927505493,
0.00027142546605318785,
0.0002220087917521596
] |
[
0.9977611303329468,
0.0016948055708780885,
0.00043783598812296987,
0.00010619996464811265
] |
en
| 0.999996 |
Attached mouse fibroblasts were permeabilized for 2 min on ice in 0.2% Triton X-100 in PBS and fixed 15 min at room temperature in 4% paraformaldehyde in PBS. The cells were incubated with purified recombinant NxMBD protein diluted in PBS/10% goat serum for 2 h, washed and fixed again for 15 min with 4% paraformaldehyde in PBS. The NxMBD protein was detected by incubation with first anti-HA antibody (1:500, F-7, Santa Cruz), then Alexa-594 coupled anti-mouse antibody . The cells were counterstained with DAPI and mounted in Vectashield (Vector). The cells were examined using a DeltaVision deconvolution microscope with SoftWorx software. Metaphase spreads were obtained from mouse fibroblasts arrested 2 h with 0.1 µg/ml colcimid. Trypsinized cells were swollen 10 min in 75 mM KCl, and adjusted to 0.1% Tween-20 before spinning 20 000 cells on to slides [cytospin 3 (Shandon), 800 r.p.m., 4 min]. After air drying, the slides were equilibrated with KCM buffer (10 mM Tris–HCl, pH 8, 120 mM KCl, 20 mM NaCl, 0.5 mM EDTA and 0.1% Triton X-100) before incubating with purified recombinant NxMBD protein diluted in KCM plus 10% goat serum for 1–2 h. After washing, the cells were fixed in 3% formaldehyde in KCM. The NxMBD protein was detected by incubation with first anti-HA antibody (1:500, F-7, Santa Cruz), then Alexa-594 coupled anti-mouse antibody . The slides were fixed in 3% formaldehyde in KCM followed by counterstaining and mounting in Vectashield with DAPI (Vector). The chromosomes were examined as above.
|
16893950_p10
|
16893950
|
Staining cells using NxMBD proteins
| 4.256895 |
biomedical
|
Study
|
[
0.9991876482963562,
0.0005111635546199977,
0.00030124548356980085
] |
[
0.9928060173988342,
0.006419249344617128,
0.0005303958314470947,
0.0002443174598738551
] |
en
| 0.999997 |
To create a high-affinity methyl-CpG-binding protein, we chose the MBD of Mbd1 as it is well characterized biochemically and structurally and is known to recognize a single methylated CpG ( 1 , 23 ). Moreover, the transcriptional repression domain and the recently identified DNA-binding CXXC domain of Mbd1 could be easily excluded from the construct as both map to regions distant from the MBD ( 17 , 24 ). To increase the binding affinity, multiple copies of the MBD were linked together, connected by flexible linker peptides, to generate ‘NxMBD’ polypeptides, where N is the number of MBDs. Increased binding of a poly-MBD protein to a methylated DNA molecule is expected for two reasons. (i) Each protein molecule has a higher concentration of DNA-binding sites than does a single MBD and, therefore, the probability of a stable interaction resulting from a DNA–protein collision is increased. Also, when a complex dissociates, the local concentration of MBDs is higher than for one MBD, favouring rebinding. (ii) The poly-MBD can interact with several sites on a DNA molecule with multiple mCpGs, thereby increasing the stability of the complex. Multimerization of a DNA-binding domain has previously been shown to create a high-affinity multi-AT-hook protein ( 25 ). In addition, the poly-MBD protein included an N-terminal NLS and was equipped with a C-terminal HA-tag for detection purposes . Negative control proteins in which each MBD carried the R22A point mutation that disrupts DNA binding ( 23 ) were created in parallel (NxMBD–R22A).
|
16893950_p11
|
16893950
|
Multimerization of MBDs increases their affinity for methylated DNA
| 4.372415 |
biomedical
|
Study
|
[
0.9993415474891663,
0.0004037494654767215,
0.0002546644536778331
] |
[
0.999249279499054,
0.0002764520177152008,
0.000361358659574762,
0.00011285632353974506
] |
en
| 0.999996 |
Recombinant His 6 -tagged 1xMBD and 4xMBD proteins were expressed in bacteria, purified to homogeneity and tested for in vitro DNA-binding. Bandshift analysis demonstrated that the wild-type proteins specifically form complexes with a methylated probe containing 27 methylated CpGs, but no binding of the mutant control proteins was observed . The ladder of complexes reflects the varying number of proteins associated with each DNA probe molecule ( 4 ). The hypothesis that the multimerization of this domain would enhance its binding affinity was tested by titrating identical weights of monomeric (1xMBD) or tetrameric (4xMBD) proteins into binding reactions that contain oligonucleotide probes with 0–3 methylated CpGs. Based on the amounts of each protein required to complex ∼50% of the probe, we calculated the dissociation constants for 1xMBD and 4xMBD proteins ( Table 1 ). The results showed that 4xMBD has a 50–80-fold higher affinity for substrates containing 1, 2 or 3 methylated CpGs than does 1xMBD. The affinity of 4xMBD for the probe with 3 methyl-CpG moieties was ∼20 nM. To test whether the multimeric protein competes with a wild-type methyl-CpG-binding protein for binding to methylated DNA, increasing amounts of 4xMBD were added to bandshift reactions that contained purified recombinant MeCP2. As shown in Figure 2C , DNA–MeCP2 complexes were competed away by the purified wild-type 4xMBD protein, whereas the mutant R22A protein did not compete. A similar result was obtained using nuclear extracts as a source of MBPs (data not shown).
|
16893950_p12
|
16893950
|
Multimerization of MBDs increases their affinity for methylated DNA
| 4.269654 |
biomedical
|
Study
|
[
0.999402642250061,
0.0003874021931551397,
0.00020997427054680884
] |
[
0.9992766976356506,
0.00022116870968602598,
0.000398516800487414,
0.00010365573689341545
] |
en
| 0.999997 |
We examined the sensitivity of methyl-CpG-binding in vitro by incubating recombinant 1xMBD and 4xMBD plus their mutant counterparts with nylon membranes carrying either M. SssI -methylated or non-methylated bacteriophage lambda DNA. The results showed that 4xMBD was able to detect 0.1 ng of immobilized double-stranded DNA, whereas 1xMBD was more than 20-fold less sensitive in this assay . Binding to methylated DNA was lost in the R22A mutant proteins. No background staining of non-methylated DNA (up to 250 ng) was detected with the poly-MBD protein probes. For comparison, we probed equivalent slot-blots with a commercially available anti-m 5 C antibody. The antibody was ∼100-fold less sensitive than 4xMBD when probed against native double-stranded methylated DNA, but gave a comparable signal level when the immobilized DNA was denatured. Denaturation abolished binding of the MBD proteins to methylated DNA (data not shown).
|
16893950_p13
|
16893950
|
Detection of methylated DNA by NxMBD proteins in vitro
| 4.185744 |
biomedical
|
Study
|
[
0.9994505047798157,
0.0003096287546213716,
0.00023984852305147797
] |
[
0.999422550201416,
0.00018891242507379502,
0.0003202922234777361,
0.00006834317900938913
] |
en
| 0.999997 |
We then asked whether the artificial MBD proteins were able to access densely methylated heterochromatic foci in fixed mouse fibroblasts. After incubation with NxMBD proteins, bound proteins were immobilized by fixation to prevent washout during the subsequent procedures. Incubation with the wild-type protein resulted in characteristic staining of DAPI-positive heterochromatin . The increased affinity resulting from multimerization was apparent, as 4 µg/ml of the tetrameric 4xMBD protein stained more strongly than 50 µg/ml of monomeric 1xMBD . The 4xMBD protein also stained metaphase chromosomes at centromeric regions and along the chromosome arms . No signal on metaphase chromosomes was detected using 1xMBD or the 1x or 4xMBD–R22A negative control proteins. Taken together with the slot-blot data , these results indicate that 4xMBD is a sensitive and specific reagent for the detection of CpG methylation of double-stranded genomic DNA in the genome. Multimerization of the MBD does not compromise the specificity of the MBD, as non-methylated DNA is not detectably recognized.
|
16893950_p14
|
16893950
|
Detection of methylated DNA by NxMBD proteins in vitro
| 4.255147 |
biomedical
|
Study
|
[
0.9995079040527344,
0.000326091714669019,
0.0001659971458138898
] |
[
0.9990804195404053,
0.00031995089375413954,
0.0005021155229769647,
0.00009749487071530893
] |
en
| 0.999998 |
Subsets and Splits
Clinical Cases Sample
Retrieves 100 samples of clinical cases, providing a basic overview of this specific document type.
High-Score Clinical Cases
The query retrieves a limited set of clinical case documents with a high educational score, providing a basic filtered view of the dataset.