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Department of Biological Sciences,1 Department of Microbiology and Immunology, Vanderbilt University, Nashville, Tennessee 372352
Received 29 April 2003/ Returned for modification 2 June 2003/ Accepted 29 July 2003
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ABSTRACT |
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INTRODUCTION |
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Alternative splicing can be regulated by both cis-acting RNA elements, such as splicing enhancers and splicing silencers, and trans-acting factors, of which the SR protein family is perhaps the best studied. SR proteins are characterized by one or two RNA recognition motifs at the N terminus and a C-terminal region rich in arginine-serine dipeptides (RS domain). They are essential splicing factors that participate in multiple steps of splicing (19, 21, 38). In early spliceosome formation (E complex), SR proteins enhance the binding of U1 snRNP to 5' splice sites through interaction with U1 snRNP U1-70K (30, 73). At later steps, SR proteins escort the U4/U6•U5 tri-snRNP into the spliceosome (50) and they play important roles in forming bridge complexes across exons and introns by mediating essential protein-protein interaction across splice sites (1, 29, 49, 66). For alternative splicing, SR proteins most often bind enhancer elements to facilitate the recognition and activation of weak splice sites (5).
The functions of SR proteins partially overlap in that most individual SR proteins can complement splicing-deficient S100 extracts. Consistent with partial redundancy, RNA interference-mediated knockdown of several SR proteins in Caenorhabditis elegans led to no observable phenotype (36). However, targeted disruption of ASF/SF2 in chicken DT40 cells (64), RNA interference with ASF/SF2 in C. elegans (36), and null alleles of Drosophila SR protein B52 all resulted in lethality (48). In addition, SR proteins display substrate specificity and have distinct functions in alternative splicing (19, 23, 61). Therefore, SR proteins do not simply have redundant functions but can act in a variety of ways to regulate splicing.
As key regulators of splicing, SR proteins themselves need to be regulated. The expression levels of some SR proteins are transcriptionally regulated (2, 52), but posttranscriptional control is also important. Many SR protein genes are themselves subject to alternative splicing, often in an autoregulatory manner (25, 59). Phosphorylation-dephosphorylation of the RS domain is also known to affect the activity and subnuclear localization of SR proteins (9, 28, 31, 40, 60, 62, 67, 68, 70). In addition, the effects of SR proteins can be counteracted by the action of other proteins, including the hnRNP A/B proteins (16, 39, 69), p32 (44), RSF1 (33), and two recently identified SR superfamily members, SRrp35 and SRrp40 (13, 53). These proteins inhibit SR proteins either by interfering with RNA binding or by disrupting crucial protein-protein interactions. The important point is that the activity of SR proteins is tightly controlled at multiple levels.
We have been studying SRrp86, a novel SR-related protein that can regulate the activity of other SR proteins both negatively and positively (3, 4, 34). In both in vitro and in vivo splicing assays, SRrp86 inhibits ASF/SF2, SC35, and SRp55 while activating SRp20. Domain analysis revealed that the C-terminal RS-EK-RS domain is required for full activity while the unique EK domain acts as a splicing inhibitor (4, 34). It appears that SRrp86 regulates SR proteins through direct protein-protein interaction, and we have identified some of the specific interaction targets, but the full range of proteins that interact with SRrp86 remain unknown.
To identify proteins that interact with SRrp86 in vivo, we performed a yeast two-hybrid library screen and coimmunoprecipitation experiments coupled to mass spectrometry. Consistent with the regulation of other SR proteins, we found that all of the core SR family members associated with SRrp86. In contrast, only SRp20 and SRp75 associated with a construct lacking the EK domain, indicating that this domain is an important modulator of protein-protein interaction. Besides SR proteins, other splicing factors and RNA-processing factors were also found to associate with SRrp86. In this study, five such proteins, SAF-B (scaffold attachment factor B), hnRNP G, YB-1, p72, and 9G8, were further investigated because they have been previously implicated in splicing regulation (11, 24, 41, 56, 63). By using a CD44 v5 minigene as a splicing reporter, we show that SAF-B, hnRNP G, and 9G8 antagonize the ability of SRrp86 to activate v5 inclusion. In the same assay, YB1 and p72 were found to have no effect. Our findings support the hypothesis that protein-protein interaction underlies the function of SRrp86 and reinforce the notion that combinatorial control of splicing by multiple factors allows precise regulation of alternative splicing.
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MATERIALS AND METHODS |
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Plasmid constructs.
pFlag-SRrp86, pFlag-SRrp86
EK, and pFlag-SRrp86RS were subcloned from pcDNA-SRrp86, pcDNA-SRrp86EK, and pcDNA-SRrp86RS (4, 34) into the pFlag-CMV2 vector.
The CD44 v5 minigene, SAF-B, and hnRNP G cDNA clones for in vitro translation and in vivo expression were generously provided by S. Stamm (University of Erlangen, Erlangen, Germany). The 9G8 cDNA clone for in vitro translation was a gift from J. Stévenin (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France). The 9G8 cDNA clone for in vivo expression was a gift from J. F. Cáceres (MRC Human Genetics Unit, Edinburgh, United Kingdom). The YB-1 and p72 cDNA clones for both in vitro translation and in vivo expression were gifts from S. M. Berget (Baylor College of Medicine) and S. Kato (University of Tokyo).
Immunoprecipitation.
Human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Transfections were performed on 100-mm-diameter plates at 70% confluency with 10 µg of pFlag, pFlag-SRrp86, pFlag-SRrp86EK, or pFlag-SRrp86RS and 30 µl of Trans IT-LT2 (Mirus). At 24 h after transfection, cells were lysed in IP buffer (50 mM Tris [pH 7.4], 100 mM KCl, 5 mM MgCl2, 10% glycerol, 0.1% NP-40, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin per ml, 10 µg of aprotinin per ml) for 20 min on ice. About 850 µg of total cell lysate was incubated with 20 µl of M2 anti-Flag-conjugated agarose beads (Sigma) for 2 h at 4°C and washed three times in IP washing buffer (50 mM Tris [pH 7.4], 100 mM KCl, 0.1% NP-40). Proteins were eluted with 8 M urea-50 mM Tris, pH 8.0. One-third of the eluate was analyzed on 15% sodium dodecyl sulfate (SDS) gels; the rest was analyzed by mass spectrometry. For high-salt washes, 250 mM KCl was included.
Western blot assays. Proteins eluted from agarose beads were separated on 15% SDS gels and transferred to nitrocellulose membranes (Millipore). Western analyses were performed as previously described (4), except that the ani-SRp20 antibody (Zymed) was diluted 1:4,000.
Mass spectrometry. Direct analysis of large protein complexes was used to identify proteins copurifying with SRrp86 as described previously (51). Acquired tandem mass spectral data were searched against a human subset of the National Center for Biotechnology Information nonredundant protein database. Data processing of the SEQUEST output files into a list of proteins copurifying with SRrp86 was performed as described previously (35).
Protein-protein interaction in vitro. Pulldown assays were performed as described previously (3). Briefly, 20 µl of rabbit immunoglobulin G (IgG)-conjugated agarose beads (Sigma) was incubated with in vitro-translated, 35S-labeled proteins in the absence or presence of 2 µg of an SRrp86 construct containing two IgG binding sites from protein A fused to the carboxy terminus (47). Bound proteins were extensively washed and eluted in Laemmli loading buffer and resolved on 12% SDS gels.
In vivo splicing assays. COS7 cells were grown in DMEM/F12 medium with 10% FBS. Transfections were performed on 60-mm-diameter plates at 80% confluency with 1 µg of the CD44 v5 minigene (32) and the indicated cDNAs with 2 µl of Lipofectamine and 1 µl of plus reagent in accordance with the manufacturer's (Invitrogen) instructions. Cells were harvested after 48 h, and total RNA was isolated with Tri Reagent (Molecular Research Center). Reverse transcription (RT)-PCR was performed as previously described (3). Similarly, HeLa cells (grown in DMEM-10% FBS) were transfected with 1 µg of pMTE1A (71) and 200 ng of the indicated cDNA construct. Primer sequences and PCR conditions were the same as those previously described (10, 32), except that the PCR was limited to 18 cycles (CD44 v5) and 23 cycles (E1A) with end-labeled radioactive 5' primers. The cycle numbers and starting RNA amounts for analysis of CD44 splicing were optimized in separate titration experiments (data not shown). Analysis of E1A splicing was done as previously described (8, 39). Products were resolved on 6% denaturing gels, visualized, and quantitated with a phosphorimager.
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RESULTS |
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EK) and RS-EK-RS (Flag-SRrp86RS) domains (Fig. 1A). Addition of the Flag tag did not affect the function of these proteins, since all three constructs showed the same activity in splicing assays as did untagged versions (data not shown). To identify associated proteins, HEK 293T cells were transfected and immunoprecipitation experiments were carried out with anti-Flag M2-conjugated agarose beads. All three constructs were expressed at approximately similar levels, but it was clear that more proteins associated with SRrp86 and SRrp86EK than with SRrp86RS (Fig. 1B). Because we had previously shown that SRrp86, but not SRrp86RS, interacts with and activates SRp20, we performed Western blot assays of the proteins associated with the epitope-tagged versions of SRrp86 with antibodies against SRp20. As shown in Fig. 1C, SRp20 associated with SRrp86 and SRrp86EK but did not associate with SRrp86RS. Thus, the coimmunoprecipitation experiments with epitope tags recapitulated previous results, enabling the use of mass spectrometry to further identify SRrp86-interacting proteins.
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RS construct or the empty vector (data not shown), even under low-salt washing conditions. In contrast, many proteins were found to associate with full-length SRrp86. Some of these proteins were background proteins that could be detected even with the empty vector, whereas several other proteins were obviously unrelated to splicing or RNA processing and were often identified through the presence of only a single peptide. Multiple ribosomal proteins were also identified, although their relevance is unknown. Ribosomal proteins have also been identified in purified spliceosomes (27, 45). For our purposes, those SRrp86-associated proteins best supported by the tandem mass spectral data, including those identified in the two-hybrid screen or other assays (3), are shown in Table 2 grouped into seven classes: snRNPs, hnRNPs, SR proteins, helicases, RNA-processing factors, transcription factors, and kinases. Many of the associated proteins are either core SR proteins or members of the hnRNP family. These two groups of proteins are the most extensively studied proteins related to the control of alternative splicing. Their identification provides valuable support for the hypothesis that SRrp86 regulates splicing through protein-protein interaction with other regulatory proteins. Also, as described above, phosphorylation of SR proteins can dramatically alter protein activity and a well-characterized SR protein kinase (SRPK1) was also found to associate with SRrp86.
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Protein-protein interactions involving the EK domain.
Since the EK domain was shown to inhibit splicing by apparently titrating one or more splicing factors (34), we compared proteins associated with the SRrp86 and SRrp86EK constructs. Strikingly, only 15 proteins (Table 2, footnote a) were found to associate with SRrp86EK under low-salt washing conditions. In contrast to the situation with the full-length protein, only two SR core proteins (SRp20 and SRp75) and three hnRNPs associated with SRrp86EK. Interestingly, one protein, Tra2
, was found to interact with only the SRrp86EK construct whereas the closely related protein Tra2ß3 was identified with SRrp86. The change in the number and identity of proteins that associate with the full-length and EK constructs suggests that the EK domain might serve in some way to modulate the interaction of SRrp86 with splicing regulatory proteins. Incubation of splicing extracts with peptides derived from the EK region inhibited splicing (34), and it appears that such inhibition is due to titration of one or more SR proteins.
SRrp86 stably associates with SAF-B, hnRNP G, YB-1, p72, SRp20, and 9G8. The large number of proteins found to interact with SRrp86 under low-salt conditions suggests that SRrp86 might be involved in multiple pathways but could also reflect possible nonspecific interactions or multiple weak interactions. To increase the stringency required for interaction, immunoprecipitation pellets were washed in buffer containing 250 mM KCl. As expected, fewer proteins and lower overall concentrations of each protein were found to associate with SRrp86 under these conditions (Table 3). Nevertheless, even when identified with only a single peptide, each of the proteins shown in Table 3 showed a statistically significant association with SRrp86. Among these, six proteins (SAF-B, hnRNP G, YB-1, p72, SRp20, and 9G8) were especially interesting because they have been previously implicated in splicing regulation (11, 15, 24, 41, 56). Before proceeding with functional splicing assays, we first confirmed the immunoprecipitation results with in vitro pulldown assays with an epitope-tagged version of SRrp86 containing two copies of an IgG binding site from protein A (47). Individual 35S-labeled proteins were incubated with recombinant, tagged SRrp86, and interacting proteins were captured with IgG-conjugated beads. As shown in Fig. 2, no interaction between the IgG-conjugated beads and any of the constructs could be detected (beads alone), and further, no interaction was detected between SRrp86 and a luciferase control protein. In contrast, SAF-B, hnRNP G, YB-1, and 9G8 all interacted strongly with SRrp86 TAP while p72 interacted somewhat more weakly. The weaker association between p72 and SRrp86 is consistent with the fact that even under low-salt washing conditions, only a single p72 peptide was identified. This suggests that p72 does indeed interact but that the association is weak, barely above the background. None of the interactions described above appear to be mediated by RNA, since pretreatment of extracts with RNase A had little effect (data not shown). Note that only the C termini of hnRNP G and SAF-B were used in the pulldown assays since they were more efficiently translated (personal communication), and most importantly, the C terminus of SAF-B had previously been shown to mediate the interaction with RNA polymerase II-CTD and a subset of SR proteins in both yeast two-hybrid and in vitro pulldown assays (41). The C-terminal domains of both SAF-B and hnRNP G also interact with SRrp86.
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RS construct, even at much higher concentrations (Fig. 3C). In contrast, deletion of the EK domain (EK) reduced, but did not abolish, such activity.
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The effect of hnRNP G on CD44 v5 inclusion was very similar to that of 9G8, except that only 5 ng of hnRNP G cDNA was needed to inhibit v5 inclusion by about 50% (Fig. 4C). Cotransfection of SRrp86 and hnRNP G showed that hnRNP G also antagonized the effect of SRrp86 in a dose-dependent manner, with almost complete inhibition observed at only 50 ng of hnRNP G. To test whether such inhibition could be reversed by adding more SRrp86, increasing amounts of SRrp86 cDNA were cotransfected with 50 ng of hnRNP G (Fig. 4D), and as expected, more v5 inclusion was observed as more and more SRrp86 was cotransfected. Therefore, among the different proteins tested, CD44 v5 inclusion seems most sensitive to the levels of hnRNP G.
Antagonism between SRrp86 and 9G8 is not substrate specific. The finding that 9G8 could antagonize SRrp86 was very interesting since we previously showed that SRrp86 could negatively regulate SR proteins whereas the converse had not been observed. To examine whether such regulation of SRrp86 might be substrate specific, we used a second substrate. The E1A pre-mRNA contains three alternative 5' splice sites leading to 13S, 12S, and 9S mRNA. As shown in Fig. 5, cotransfection of increasing amounts of SRrp86 caused a steady increase in the level of 9S mRNA because of increasing use of the most distal 5' splice site. Concomitantly, use of the most proximal 5' splice site was reduced, as reflected by the 12S/13S ratio.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SRrp86-SR protein interaction. To identify the full range of proteins that might interact with SRrp86, we first set up a yeast two-hybrid cDNA library screen with full-length SRrp86 as bait. Three SR proteins were identified, SC35, 9G8, and SRp46, although the overwhelming interacting partner was clearly SC35. Interestingly, of the few non-SR proteins identified in the two-hybrid screen, most contained extensive RS repeats, implying that such regions are central to protein-protein interactions involving SRrp86. Consistent with this, all of the core SR proteins were identified by coimmunoprecipitation coupled to mass spectrometry. An open question is whether all of these interactions are direct or possibly mediated by RNA. We favor the former because treatment with RNase did not abolish in vitro interaction and previous experiments have also supported direct interaction (3, 4). Nevertheless, it remains possible that additional interactions mediated by RNA could expand the range of proteins that interact with SRrp86.
A network of splicing regulators. Six proteins (SAF-B, hnRNP G, YB-1, p72, SRp20, and 9G8) that interact with SRrp86 were examined in more detail since they have been proposed to be involved in splicing regulation. SAF-B was first identified on the basis of binding to scaffold or matrix attachment regions (S/MAR) (46). It colocalizes with splicing factors in nuclear speckles and interacts with several SR proteins through its C-terminal domain (41). YB-1 is a multifunctional protein that binds to promoter regions in the nucleus to activate transcription (42), binds to mRNA in the cytoplasm, regulating stability and translation efficiency (17, 18), and can also bind splicing enhancer elements to stimulate CD44 exon v5 inclusion (56). hnRNP G is a member of the hnRNP family and has been shown to interact with Tra2ß and SRp30c (15). p72, although it interacted the weakest with SRrp86, is an ATP-dependent DEAD-box RNA helicase that can activate inclusion of CD44 variant exons 4 and 5 (24). Both SRp20 and 9G8 are canonical SR proteins that play multiple roles in both constitutive and alternative splicing (11, 26). With the exception of YB-1 and p72, we show that coexpression of each of these proteins with SRrp86 leads to differential effects on alternative splicing. SAF-B, hnRNP G, and 9G8 were all able to inhibit SRrp86, whereas SRrp86 activates SRp20 (3). Altogether, the data suggest a network of protein-protein interactions that lead to precise fine tuning of alternative splicing events.
Mechanistically, determination of the exact function of SRrp86 requires additional work but it appears to modulate protein-protein interactions that define bridge complexes across introns and exons. Such complexes have been proposed to help define exons and introns linking protein complexes across splice sites. For alternative splicing events, defining such exons can be difficult because the accompanying splice sites are often weak. Splicing enhancers apparently exert their effects by helping to overcome weak splicing signals through the recruitment of SR proteins, ultimately enabling tissue-, cell-, or development-specific activation of splicing. SRrp86 may exert its effects by activating or repressing the formation of bridge complexes by modulating interactions between splicing regulatory proteins at specific splice sites and/or enhancer elements.
SRrp86: a component of the spliceosome? Under low-salt washing conditions, we found that a few snRNPs associated with SRrp86 although when we lowered our standards to include single peptide hits, many more were identified (data not shown). Several recent studies have analyzed the protein composition of purified spliceosomes by mass spectrometry (27, 45, 72). Although many SR proteins have been identified in active spliceosomes, only one group identified SRrp508, the human homolog of SRrp86 (45). It is possible that SRrp86 only transiently associates with the spliceosome. We favor this possibility because it is supported by the fact that antibodies against SRrp86 are not able to immunoprecipitate any of the U snRNAs (data not shown).
The EK domain modulates protein-protein interaction.
Previously, we found that deletion of the EK domain converted SRrp86 from a splicing inhibitor to a splicing activator (34). The EK domain is highly polar, with alternating positively and negatively charged amino acids mimicking hyperphosphorylated RS domains. Similar domains have been found in proteins involved in splicing, such as the RNA helicase Hel117 (58), U1-70K (55), YT521-B (22), and SAF-B (41). However, none of these proteins are EK rich and instead are DR or ER rich. The exact function of D/ER regions is not known, but it has been shown that they contribute to protein-protein interactions, at least for YT521-B and SAF-B (22, 41). Consistent with this, SAF-B was found to interact with SRrp86, but not with SRrp86EK. The EK domain might provide a secondary level of regulation to define the scope and strength of protein-protein interactions involving SRrp86.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grant GM62487.
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FOOTNOTES |
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