Human Molecular Genetics, 2000, Vol. 9, No. 5 685-694
© 2000 Oxford University Press
RBMY, a probable human spermatogenesis factor, and other hnRNP G proteins interact with Tra2ß and affect splicing
1Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, UK and 2Medical Research Council Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Received 17 November 1999; Revised and Accepted 28 January 2000.
DDBJ/EMBL/GenBank accession no. AF069682.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis-specific relative, termed hnRNP G-T, interact with Tra2ß, an activator of pre-mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2ß proteins are associated in HeLa nuclear extracts. RBM and Tra2ß co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2ß. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2ß-dependent splicing in spermatocytes.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spermatogenesis is remarkable both for the extent of the changes wrought in cellular morphology and for its universality in animals. In mammals, the diploid stem cells are maintained by mitosis, but a proportion undergo meiosis and then, as haploid syncytial cells, they are subjected to re-packaging and inactivation of the genome, growth of a flagellum, rearrangement of mitochondria and formation of an acrosome. These processes are accompanied by substantial changes in strategies for regulating gene expression, with a marked increase in post-transcriptional regulation (1–3).
A failure in spermatogenesis is a significant cause of human male infertility (4,5), and it appears to be caused in some cases by deletions in one of at least three small regions of the Y chromosome (5–7). The first gene identified as a candidate azoospermia factor in one of these deletion intervals was YRRM (8), now designated RBM or RBMY (9). Although there are numerous RBMY genes on the Y chromosome, only the genes in deletion interval AZFb (and possibly just one of these) produce detectable levels of protein (10), and there is now substantial evidence supporting the importance of RBMY for spermatogenesis (7,8,11–14). There are homologues of RBMY in all mammals (15), and in the mouse partial deletion of the gene family is associated with sperm abnormalities rather than azoospermia (16,17). It has been shown recently that there is an X chromosome homologue of RBMY, termed RBMX, which encodes the widely expressed protein hnRNP G (9,18). HnRNP G is a nuclear protein of unknown function that binds nascent pre-mRNA in vivo and in nuclear extracts (19,20).
Both the sequence and distribution of RBM protein are consistent with a function in nuclear RNA processing during spermatogenesis. It has an RNA-binding domain (RRM) and four tandemly repeated sequences each of 37 amino acids (SRGY boxes) that are rich in the SR/RS dipeptides characteristic of the SR proteins (21), which are involved in constitutive and alternative splicing (22–24). HnRNP G is 60% identical (8,25,26), although it has only one SRGY box. RBM is expressed exclusively in germline cells in the testis, where it is abundant in spermatogonia and spermatocytes but levels decline in later, post-meiotic stages; in contrast, hnRNP G is most abundant in somatic cells and spermatogonia but less so or absent in spermatocytes (27). Both proteins are nuclear; RBM co-localizes with splicing factors in the early stages of meiosis, although it becomes more diffuse in the nucleus at later stages (27). These features suggest that RBM might be a tissue-specific RNA processing factor. Despite considerable progress in defining the sequences in pre-mRNA responsible for mediating tissue-specific splicing, no clear examples of corresponding factors have yet been identified in mammals. In view of the apparent importance and clear tissue specificity of RBM, we have investigated whether RBM interacts with components of known RNA processing pathways and whether these interactions have functional consequences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Yeast two-hybrid methods (28) were used to identify proteins expressed by a human testis cDNA library that could interact with RBM. Positive isolates were tested by re-transformation of their purified plasmids (Fig. 1A). Sequences were derived from 74 independent positives and showed that they derived from 18 proteins, all of which were found by re-screening to require only the SRGY region of RBM (the four SR-rich repeats) for interaction (Fig. 1B); more detailed analysis of the interactions of the three proteins described below showed that any two contiguous SRGY boxes sufficed. Forty-eight of the isolates came from seven proteins with RNA-binding domains. The most common isolate (Fig. 1A) was a novel protein, named T-STAR (29), which is closely related to Sam68. Sam68 is an RNA-binding protein of unknown function that is associated with and phosphorylated by the Src proto-oncogene family during mitosis (30–33). Another abundant isolate was a novel relative of hnRNP G (73% identical), designated hnRNP G-T because it was detected only in testis (Fig. 1C). Neither of these interactions had clear functional implications. Of greater significance was the finding of isolates of Tra2ß (34–36), which is also expressed abundantly in testis (Fig. 1C). The fly homologue, Tra2, regulates sexual differentiation, spermato- genesis and courtship behaviour via alternative splicing (37–41). As with the interaction of Drosophila Tra2 with itself or with the human SR protein SF2/ASF (42), the C-terminal region of Tra2ß (including SR-rich sequences) sufficed for interaction with the SRGY repeats (data not shown).
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The interacting partners detected by the two-hybrid approach were tested in various combinations (Table 1). Most of the proteins fall into three groups: SR or SR-related proteins (Tra2ß, SRp30C and 9G8), the hnRNP G-related proteins (RBM, hnRNP G-T and hnRNP G), and the STAR proteins (T-STAR and Sam68). The interactions fit a pattern in which the hnRNP G family members interact with each other and with the SR proteins and STAR proteins, whereas the STAR proteins and SR proteins do not interact.
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These interactions were tested in vitro (Fig. 2A) by quantifying the binding of 35S-labelled proteins, produced by in vitro translation, to recombinant glutathione S-transferase (GST) fused with either the SRGY repeats of RBM (GST–SRGY) (Fig. 2A, S), the C-terminal part of Tra2ß (GST–TraC: T), the RNA-recognition motif of RBM (GST–RRM: R), or nothing (GST: G). The assays were done in the presence of ribonuclease (see below). The Tra2ß translated in vitro was inferred to behave like the native protein because it bound preferentially (data not shown) to RNA sequences containing GAA repeats (43). The three hnRNP G family members and Tra2ß interacted with each other, whereas neither hnRNP A1 nor luciferase showed any salt-stable interactions with the immobilized proteins. The proportion of input material recovered was markedly lower for the homodimerization of RBM with GST–SRGY and Tra2ß with GST–TraC than for the heterologous interactions of the same immobilized GST fusions. In other experiments we have confirmed the interaction of T-STAR with GST–SRGY and have shown that it interacts with Src-homology domains (29).
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To confirm that the in vitro interactions were not indirect, mediated by residual RNA, the effects of addition of ribonuclease and RNA were tested. Ribonuclease treatment enhanced the binding of 35S-labelled Tra2ß to GST–SRGY (Fig. 2B). Both yeast RNA (data not shown) and an in vitro transcript (Fig. 2C) inhibited the binding of 35S-labelled Tra2ß or RBM to GST–SRGY or GST–TraC, respectively. This confirms that the protein interactions are direct and demonstrates that RNA interferes with these, presumably by non-specific binding to the positively charged RS or SRGY domains of Tra2ß or RBM, respectively. Interactions of STAR proteins are affected similarly (44). Figure 2D summarizes the protein interactions.
Several criteria were used to test whether the interactions of Tra2ß with RBM or hnRNP G occur in vivo: expression of the endogenous proteins in the same cells, co-localization within the nucleus and association in extracts. Immunohistochemical staining of human testis sections (Fig. 3A) showed that Tra2ß was expressed at high levels in the nuclei of spermatocytes, the principal cells in which RBM is expressed (27). The subnuclear location of the proteins in testis was compared indirectly via comparison with a group of splicing factors, including some SR proteins, recognized by the antibody 16H3 (45). These SR proteins were largely localized in two or three major regions in spermatocytes and post-meiotic spermatids. Tra2ß and RBM showed the same pattern in spermatocytes: they accumulated in the SR regions (Fig. 3B: arrows in C and I) but, unlike the SR proteins, also showed a relatively high diffuse nucleoplasmic signal. After meiosis, in round spermatids, RBM became dispersed (Fig. 3B: G and I) but Tra2ß still showed some slight accumulation with the SR proteins (Fig. 3B: D and F). Association of endogenous proteins in extracts was shown by co-precipitation of Tra2ß with hnRNP G from ribonuclease-treated HeLa nuclear extracts (Fig. 3C), with U1-A as a control for non-specific precipitation. Approximately 5% of the input Tra2ß was precipitated, which provides a minimum estimate of the proportion associated in vivo. We conclude that endogenous Tra2ß and hnRNP G family proteins are associated in vivo.
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The functional significance of this interaction was tested by splicing in vitro. The only previous assay for human Tra2 activity used a substrate containing three repeats of a Tra2 SELEX winner sequence, the splicing of which was stimulated by exogenous Tra2 (
or ß) in low concentrations of nuclear extract (43). No natural target has been reported. We have found that splicing of a human s tropomyosin exon requires an enhancer sequence in the exon (46) and that an essential part of this sequence is bound by Tra2ß under splicing conditions (N. Thornton, A.A. Malygin, K.N. Bulygin, D.M. Graifer, G.G. Karpova and I.C. Eperon, manuscript in preparation). The addition to splicing reactions of protein domains that interact with Tra2ß (i.e. the SRGY repeats of RBM and the C-terminal domain of Tra2ß itself) would be predicted to block splicing. As a control, the reactions included also a substrate, derived from rabbit ß-globin exons 2 and 3, that contained two alternative consensus 5' splice sites (C174C) (47). Although the second exon of human ß-globin is known to contain several enhancers (48), there is no evidence that these involve Tra2 or -ß; the rabbit exon contains several GAA sequences, but none of these is present as the tandem repeat preferred for Tra2ß binding. Thus, it would be predicted that splicing of this substrate would be less dependent on Tra2ß activity and so less inhibited by the presence of GST–SRGY. The results (Fig. 4A, quantified in B) show that this prediction was borne out. At the lower concentration of added protein, GST–SRGY abolished splicing of the tropomyosin intron but had no significant effect on splicing of the C174C ß-globin control. In contrast, little or no inhibition was caused by either GST fused with only two of the SRGY repeats (SRGY), the control fusions, a control lysate or GST at either concentration. We conclude that splicing of an exon containing a Tra2ß-binding site in an obligatory enhancer is preferentially abolished by incubation with the Tra2ß-binding domain of RBM.
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The experiment in Figure 4 tested the effect also of incubation with the RNA-binding domain of RBM (GST–RRM), which is 81% identical in amino acid sequence to the RRM of hnRNP G. This appeared to stimulate tropomyosin splicing, but it had two significant effects also on C174C: the second step of splicing was reduced whereas step 1 was unaffected, leading to an accumulation of 5' exon intermediate (Fig. 4B), and splicing shifted to the upstream alternative 5' splice site. Similar effects on C174C were seen with GST–TraC at the higher concentration, but at this concentration tropomyosin splicing was inhibited.
The localization and possibly the activity of Tra2 in Drosophila spermatocytes are affected by an SR protein kinase (49). In mice, an SR protein kinase, SRPK1, is particularly abundant in the testis and specifically in germline cells (50). At a biochemical level, phosphorylation is known to influence RNA-binding proteins by reducing the binding of RNA, increasing its specificity or enhancing protein interactions (43,44,51,52). We observed that ATP enhanced Tra2ß binding to GST–SRGY in the presence of nuclear extract (14% v/v; data not shown), and therefore tested whether this was caused by phosphorylation. The binding of 32P-labelled RNA to immobilized, salt-washed, GST–SRGY was reduced if GST–SRGY had been pre-incubated with two kinases known to phosphorylate SR proteins (53,54), CLK/STY being more effective (Fig. 5A). Incubation with [
-32P]ATP confirmed that these kinases phosphorylated GST–SRGY and GST–TraC (data not shown), and preliminary experiments with GST–TraC showed that phosphorylation reduced binding of RNA to this domain also. A direct effect of phosphorylation on protein–protein interactions was tested by incubation in the presence of protein phosphatase 1. Stringently washed GST–SRGY or GST–TraC beads were incubated with 35S-labelled Tra2ß or RBM. Incubation with phosphatase caused a substantial reduction in binding by Tra2ß, but not by RBM, even in the presence of ribonuclease (Fig. 5B). We conclude that phosphorylation inhibits the binding of interfering RNA to the protein interaction domains and it enhances the direct protein interactions of Tra2ß.
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DISCUSSION |
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The purpose of this investigation was to establish whether RBM could be linked to any nuclear RNA processing pathways via an analysis of its protein interactions. Our results argue that RBM participates in splicing in spermatocytes, for several reasons. The yeast two-hybrid screen produced a number of positive clones, yet none corresponded to proteins known to be involved in any other constitutive RNA processing reactions. Instead, most of the identifiable interacting proteins fell into three major groups: SR-rich splicing activators (primarily Tra2ß), STAR proteins and other hnRNP G proteins. These interactions held up when tested in vitro. Furthermore, RBM was already known to co-localize in two large clusters in spermatocytes with several splicing factors (27), and we have extended this by showing that the same is true for Tra2ß. Thus, the two proteins are present in spermatocytes at the same time and in the same nuclear compartment. It proved to be very difficult to demonstrate that Tra2ß was associated directly with RBM in spermatocytes by co-immunoprecipitation, because RBM was extremely insoluble in testis extracts. However, since hnRNP G is the other member of an X/Y pair of homologues and interacted like RBM in the yeast two-hybrid and in vitro binding assays, it would be predicted, as for RBM, that hnRNP G associates with Tra2ß in cells in which it is expressed. This was tested and confirmed by co-immunoprecipitation of hnRNP G and Tra2ß in HeLa nuclear extracts. We conclude that both products of the X/Y gene pair, RBM and hnRNP G, can and do interact with Tra2ß.
RBM becomes more evenly distributed in post-meiotic spermatid nuclei (Fig. 3) (27), and it is tempting to speculate that this is the result of a weakening association between RBM and Tra2ß caused by changes in the state of phosphorylation of the latter. It is not known yet whether Tra2ß phosphorylation is modulated in vivo, but we note that cyclical changes in phosphorylation are required for the participation of SR proteins in splicing and regulate their subnuclear location in vivo (55–57); protein kinase activity is also required for correct intranuclear localization of Tra2 in Drosophila spermatocytes (49).
The involvement of RBM in splicing could not be tested by addition of RBM to extracts or expression in tissue culture cells, because its specific natural targets (if any) are not known. However, its protein interaction domain alone would be predicted to interfere with protein–protein interactions between Tra2ß and hnRNP G or SR proteins, by analogy with Drosophila Tra2 (42). The results with GST–SRGY bear this out. The inclusion of a ß-globin control in the same reaction mixture as the enhancer-dependent tropomyosin substrate demonstrated that the fusion protein had not inactivated any constitutive splicing reaction components. Although we have not directly demonstrated that splicing of the tropomyosin substrate is dependent on Tra2ß, it is known that Tra2ß is an activator of splicing and that it binds to GAA repeats in an essential enhancer in the substrate. Thus, the results show that the protein interaction domain of RBM can affect the splicing of specific substrates and almost certainly does so by binding Tra2ß. This suggests that azoospermia is caused in some cases by a deficiency in an aspect of splicing in spermatocytes.
The non-specific inhibition by GST–TraC at the higher concentration is likely to be caused by binding to and sequestration of the endogenous Tra2ß and SR proteins. This is absolutely consistent with the shift in C174C splicing towards the 5'-most site; this shift is the converse of that seen with many substrates when most SR protein concentrations are increased, and it would be predicted if the reduction in SR activity weakened U1 snRNP binding (47,58). The RNA-binding domain of RBM had a positive effect on tropomyosin splicing but also shifted C174C and inhibited step 2 of splicing. It is possible that the effects are caused by specific binding to the RNA such that repressors are displaced from the tropomyosin substrate and that SR proteins are displaced from the ß-globin substrate, in particular in exon 2 (48,59).
In Drosophila, Tra2 activates a female-specific exon in dsx by direct binding with other proteins to exon enhancers (60,61), and it is involved in a number of other splicing pathways that regulate sexual differentiation and behaviour (37–41). Its association with each element of the dsx enhancers involves cooperative binding of Tra2 in association with Tra and an SR protein (60,61). Tra is particularly important because its active form is expressed only in females (62), and this determines whether the female-specific exon of dsx is recognized. Tra2 appears to have a distinguishable set of properties in the male germline: it regulates the splicing of several genes (including its own) in the absence of Tra, it forms clumps around partially condensed chromatin in early spermatocytes, and clumping, male fertility and autoregulation are particularly sensitive to alterations of the C-terminal RS domain (63). Our observations that Tra2ß is expressed particularly well in testes, that it forms two large clusters in spermatocyte nuclei (along with RBM and other splicing proteins) and that it interacts with the germline-specific RBM suggest that mammalian Tra2ß may fulfil functions analogous to those of Tra2 in the Drosophila male germline. We have not investigated Tra2, which was not recovered in our screens, but it is possible that its functions may be more analogous to those of Drosophila Tra2 in somatic cells: it was able to rescue the somatic but not the germline functions of Tra2 in transgenic flies (35), and it differs most from Tra2ß in the sequences just prior to the C-terminal RS domain. It would be of interest to know whether Tra2ß can fulfil the male germline functions of Tra2 in Drosophila, and to determine whether Tra2 in male Drosophila interacts with an RBM-like protein instead of Tra.
Unlike Tra, RBM has an RRM domain. If this has any sequence specificity, RBM might divert splicing towards or away from specific targets in spermatocytes, perhaps by re-directing Tra2ß to spermatocyte-specific pre-mRNA targets. Given that Tra2ß interacts with all three members of the hnRNP G family, it might be envisaged that Tra2ß and the widespread hnRNP G are part of a common splicing enhancer complex that is diverted to tissue-specific splicing targets by tissue-specific forms of hnRNP G, such as RBM and hnRNP G-T. This is particularly likely in spermatocytes, where there are high levels of RBM but little or no hnRNP G (27). This model is consistent with preliminary evidence that the tropomyosin enhancer complex contains hnRNP G as well as Tra2ß (A.A. Malygin, unpublished data).
Finally, we note that very little is known yet about tissue-specific factors that regulate pre-mRNA processing in mammals. Although variations in the concentrations of ubiquitous factors influence splicing (64), sequences have been identified in several genes that act only in specific tissues and are presumed to interact with tissue-specific proteins. Candidate tissue-specific factors include Nova-1, a neuronal nuclear RNA-binding protein that binds specifically to two neuronal pre-mRNA sequences (65), and the Hu proteins, RNA-binding proteins that are required for neuronal development (66), but it is not known whether they participate directly in splicing. Several polypyrimidine sequences have been shown to mediate repression of neuron-specific exons in non-neuronal cells, where they are bound by PTB (67–69); repression might be relieved in neuronal cells by a brain-specific counterpart of PTB (69). This suggests an interesting parallel with the complementary distributions of the X/Y homologues, RBM and hnRNP G.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Two-hybrid screens and analysis
Sequences encoding RBM and Tra2ß were cloned in-frame into pAS2.1, a GAL4 DNA-binding domain vector (Clontech, Palo Alto, CA). The sequences cloned were the full RBM coding region (amino acids 1–496), the three-quarters of RBM downstream of the RRM (amino acids 88–226, 225–375 and 374–496) as separate portions and in one fragment, nine combinations of the four SRGY boxes as all possible contigous trimers, dimers and monomers (the three dimers shown in Figure 1D encoded amino acids 220–295, 257–332 and 294–369), and the C-terminal region of Tra2ß (positions 195–288). Eight hundred thousand colonies from a human testis Matchmaker library in pACT (Clontech) were screened against the full RBM sequence by selecting for growth on 25 mM 3-AT and ß-galactosidase activity. Plasmid DNA was prepared from positive colonies by transformation and growth in Escherichia coli, then mixed with the RBM-pAS plasmid or pAS for re-transformation of yeast. Six or more colonies from each transformation were tested by filter lifts for ß-galactosidase activity. The intensity of the blue colour for each colony was assigned a value of 0 (colourless) to 3 (most intense), and the average calculated. An average of >2–3 is shown in Table 1 as +++, >1–2 as ++, >0–1 as +, 0 as –. Positives were tested also in co-transformations with the various combinations of domains of RBM. To test interactions between specific proteins (Table 1) the RBM coding region was cloned in the activation domain vector, pACT2. The full coding region of T-STAR, the corresponding region of Sam68 (residues 97–443) and the full coding region of hnRNP G were cloned into pAS2.1. The pAS and pACT constructs were then co-transfected and assayed as before.
In vitro binding assays
GST fusions of the Tra2ß C-terminus (amino acids 195–288) and the RRM (residues 1–98) and SRGY (residues 220–375) regions of RBM were constructed in pGex2-T and transformed into BL21-DE3. Sonication lysates were cleared and incubated with glutathione–agarose, then washed four times in buffer D.Glu-full (80 mM K glutamate, 20 mM TEA pH 7.8, 5% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.1% Tween) + 1 mM DTT at 4°C (non-stringent conditions) or with the second and third washes containing an extra 0.5 M KCl (stringent conditions); for the experiments in Figure 4B–D, an extra wash in high salt was done at 30°C for 20 min. In vitro-labelled proteins were incubated with beads overnight at 4°C in a final volume of 400 µl of D.Glu-full + 1 mM DTT). Protein binding experiments contained 5 µg of ribonuclease 1A (Pharmacia, Uppsala, Sweden) except where indicated in Figure 4A and D. The efficacy of the ribonuclease was tested by inclusion of labelled transcripts. Beads were then washed as before and boiled in SDS dyes for 12% SDS–PAGE. The proteins were made in vitro from transcripts incorporating the full reading frame by translation in rabbit reticulocyte lysate (Promega, Madison, WI) in the presence of [35S]methionine. The binding of RNA to GST fusions (Fig. 5C) was assayed with 32P-labelled 50mer RNA comprising a random 20mer core and constant flanking regions. This was mixed with 10 µg of yeast RNA and bound to stringently washed fusion proteins overnight, followed by low stringency washes and scintillation counting. Fusion proteins were phosphorylated (Fig. 5C) by incubation for 30 min at 30°C in a volume of 100 µl containing either 14 µl of nuclear extract (CCCC) and 2 mM MgCl2, with or without 25 mM phosphocreatine and 2 mM ATP, or SRP kinase buffer (50 mM Tris pH 7.4, 10 mM MgCl, 1 mM DTT ± 1 mM ATP) with or without 0.15 U of SRPK1 or 0.02 U of CLK/STY kinase. For phosphatase treatment (Fig. 5D), the translation reaction mixtures were incubated with 1 U of protein phosphatase 1 (NEB, Beverly, MA) in 100 µl of supplied buffer for 30 min at 30°C before adding either 5 µg of RNase A or 0.5 µg of RNA (transcribed pCDNA3, as in Fig. 5B) and then addition to stringently washed immobilized GST fusions for 30 min at 30°C. The beads were then washed under non-stringent conditions (but at 30°C) before boiling and loading on a gel. Supernatants from the beads were precipitated with TCA and run on a gel for quantification of unbound protein.
Northern blots
A Multiple Choice RNA blot (Origene, Rockville, MD) was probed with PCR products from hnRNP G-T corresponding to the last 155 nucleotides of the coding region and 227 nucleotides of the 3'-untranslated region, and then with the first 150 nucleotides of the Tra2ß coding region.
Immunolocalization
Surgically removed human testis was fixed overnight in Bouin’s fixative, washed in 70% ethanol and embedded in paraffin wax. Sections (5 µm) were cut and processed (13) using a 1:1000 dilution of anti-Tra2ß antiserum (from Dr S. Stamm, Max-Planck Institute for Psychiatry, Planegg, Germany) and a horseradish peroxidase/diaminobenzidine reaction (brown). Sections were counterstained with haematoxylin. Pre-incubation of the primary antibody with GST–TraC reduced staining intensity substantially. For immunofluorescence, anti-Tra2ß antiserun was diluted 1:200, anti-RBM 1:50 and antibody 16H3 (45) was used at 1:1.
Antibodies and immunoprecipitation
Co-precipitation was done with hnRNP G in HeLa nuclear extract because RBM was insoluble in both testis extracts and after expression in cell lines. Antibodies were raised in rabbits against RBM and a GST fusion of amino acids 1–50 of Tra2ß, and affinity purified with GST–RRM (82% identical to the RRM of hnRNP G) and His-tagged Tra2ß1–50, respectively. For each immunoprecipitation, 50 µl of goat anti-rabbit agarose beads (Sigma, Poole, UK) were incubated with antibody in 400 µl, washed and incubated with 400 µl splicing reaction mixture (40% nuclear extract, pre-incubated with ribonuclease for 30 min at 30°C) at 4°C. Bound components were eluted by two incubations for 30 s at ambient temperature in 50 µl of 1% SDS, 0.5 M NaCl, 50 mM Tris pH 6.8, separated by SDS–PAGE, electroblotted and detected by specific antibodies, protein A peroxidase and enhanced chemiluminescence (Amersham, Little Chalfont, UK). The relative abundance of the proteins in the extract and the binding capacity of the antibody-bound beads were not tested, so the proportion of Tra2ß recovered is a minimum estimate of the proportion associated in the extract.
Splicing assays
GST fusion proteins were prepared from 200 ml cultures in E.coli, with washes at 30°C in 0.5 M KCl before elution in D.Glu-full, 10 mM glutathione. Because GST–SRGY, GST–SRGY2 and GST–TraC preparations contained a number of partial degradation products intermediate in size between the full-length protein and GST itself, the protein concentrations were measured after SDS–PAGE by scanning of Coomassie-stained gels, with a series of bovine serum albumin concentration markers. Aliquots of the proteins were diluted to equal concentrations with a blank protein preparation (from a parallel culture of cells that did not contain an expression plasmid) and concentrated in a Microcon 50 (Amicon, Beverly, MA), followed by measurement of concentrations as above. For splicing reactions, 1 or 2 µl of protein were incubated at 30°C for 1 h in 5.5 µl of splicing reaction mixtures (70,71) in open wells of a 96-well Thermowell C plate (Costar, Cambridge, MA). Half a microlitre of a mixture of the two RNA substrates in buffer D, 0.1% Tween-20 was added and incubation continued for 3 h. The final concentrations included 0.4 mM ATP, 17 mM phosphocreatine, 2.7 mM MgCl2 and 33% nuclear extract (4C, Mons, Belgium). The tropomyosin substrate comprised alternative exons 5NM and 5SK, with the intron between them, modified to improve the branch site and to inactivate the repressor at the 5' end of exon 5SK (46). The enhancer includes nucleotides 16–30 of exon SK and is active in HeLa splicing extracts. Substrate C174C has been described previously (47). After electrophoresis, analysis was by phosphor imager.
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ACKNOWLEDGEMENTS |
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We thank colleagues, especially Drs S.L. Chew and A. Malygin, for advice and encouragement, Dr J. Yeakley for very kind gifts of both SR kinases, Dr S. Stamm for anti-Tra2ß antibody, Dr C.J. Larsen for cDNA encoding hnRNP G, Dr A. Mayeda for cDNA encoding hnRNP A1 and for protocols, and Dr A. Cashmore for advice on yeast. This work was supported by the MRC (UK), the Wellcome Trust and an equipment grant from the Wellcome Trust.
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FOOTNOTES |
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+ Present address: Institut für Molekularbiologie und Tumorforschung, Philipps-Universität, Emil-Mannkopff-Strasse 2, D-35037, Marburg, Germany
§ To whom correspondence should be addressed. Tel: +44 116 2523482; Fax: +44 116 2523369; Email: eci@le.ac.uk
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