Ser/Arg-rich Protein-mediated Communication between U1 and U2 Small Nuclear Ribonucleoprotein Particles*

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5 (cid:1) splice site, can positively influence utilization of an upstream 3 (cid:1) splice site via exon definition in both trans - and cis -splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5 (cid:1) splice site on trans -splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5 (cid:1) splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis -spliced intron. Weaken-ing of the 3 (cid:1) splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between

Pre-mRNA splicing is a process whereby introns are removed, and the exons are linked together to produce mature mRNA molecules. Central to this reaction are the small nuclear ribonucleoprotein particles (snRNPs) 1 including U1, U2, U4/ U6, and U5 in addition to a multitude of protein factors (1). Each of the snRNPs consists of a small RNA complexed with both common as well as specific proteins (2). Functions have been assigned to the snRNPs involved in splicing including the interaction of U1 with the 5Ј splice site, interaction of U2 snRNP with the branch site sequence, U5 interaction with the free 5Ј exon generated after the first step of the reaction, and contact between U6 and the 5Ј splice site region subsequent to the interaction with U1.
The trans-splicing reaction occurring in a growing list of lower eukaryotes, including trypanosomatids (3,4), nematodes (5), trematodes (6), euglenoids (7), ascidians (8), and cnidarians (9) is characterized by essential similarities as well as fundamental differences with standard cis-splicing of pre-mRNA introns (for reviews, see Refs. 10 and 11). The most striking feature of trans-splicing is the fact that the 5Ј exon is donated from a separate RNA, the spliced leader RNA (SL RNA) (12,13). The SL RNA, when complexed with proteins in the functional SL RNP, has essential characteristics of the Sm snRNPs involved in numerous RNA-processing reactions (14 -16). In nematodes, these traits include a 5Ј trimethylguanosine cap, binding of the Sm core proteins, and the presence of snRNPspecific proteins (17). It has been pointed out in numerous reviews on the subject (for example 10) that essential features of the actual catalysis of the cis-and trans-splicing reactions are presumed to be very similar. This similarity extends to the requirements for the U2, U4/U6, and U5 snRNPs (18,19) as well for the Ser/Arg-rich (SR) family of splicing factors (20). The clearest difference in the reaction schemes for trans-and cissplicing relates to the apparent ability of the SL RNP to identify its own 5Ј splice site without the necessity for the U1 snRNP (18). Although U1 snRNP is not required for the transsplicing reaction per se, it can influence the efficiency of the reaction via the process of exon definition (21). Exon definition involves the interaction of U1 snRNP with a 5Ј splice site leading to the enhanced utilization of an upstream 3Ј splice site (for review, see Ref. 22).
In addition to the snRNPs, SR proteins are also required for trans-splicing (20). The SR proteins are a family of non-snRNP splicing factors that have been implicated in a variety of discrete functions intimately involved with splicing reactions (for review, see Ref. 23). In general, SR protein architecture includes one or two RNA recognition motif-type RNA binding domains in the N terminus followed by a region rich in arginine/serine dipeptides at the C terminus. These two regions within the proteins are critical for RNA binding and proteinprotein interactions, respectively. SR proteins have been shown to stimulate pre-mRNA splicing through their interaction with individual, short sequence elements termed exonic splicing enhancers (ESEs). Stimulation can be effected by activation of either 5Ј splice sites or 3Ј splice sites. In fact, a single ESE can activate both a weak 5Ј splice site and a weak 3Ј splice site simultaneously (24). Splicing enhancers have since been demonstrated to exist in numerous alternatively spliced messages as well as in constitutively processed transcripts (for reviews, see Refs. 25 and 26). In fact, it has recently been suggested that many mutations contributing to human disease may manifest themselves in the disruption of ESE (27)(28)(29) or the generation of exonic splicing silencers (30) and the result-ing alterations in pre-mRNA splicing. The potential for therapeutic intervention via modulation of alternative pre-mRNA splicing by synthetic trans-acting factors has recently been proven effective in vitro (31).
Earlier work postulated that intermediary factors might be involved in the enhanced utilization of 3Ј splice sites seen upon binding of U1 snRNP downstream (32). In fact, several studies have demonstrated that SR proteins interacting with an ESE can recruit splicing factors to the 3Ј splice site (33)(34)(35)(36). In this study, we addressed the longstanding question of whether or not SR proteins play a role in mediating exon definition. We employed a system in which trans-and cis-splicing activity are developmentally regulated via the state of phosphorylation of SR proteins (37). We reasoned that we could assay the nature of the interaction between U1 and U2 snRNP in early, two-cellstage extracts derived from embryos of the nematode Ascaris lumbricoides, which are devoid of active SR proteins. Upon the addition of functional SR proteins, we were able to monitor their effects on both U1 and U2 snRNP binding. As a first step, we examined the interaction between U1 snRNP and a 5Ј splice site involved in exon definition in inactive two-cell-stage extracts. Even though this extract is not competent for splicing due to the phosphorylation state of the SR proteins, U1 snRNP interacted with the 5Ј splice site-like enhancer without supplementation with active SR proteins. In and of itself, this is interesting as there have been numerous reports of SR proteinassisted U1 snRNP interaction with authentic 5Ј splice sites (for example, see Ref. 38). Having established that U1 binds to the 5Ј splice site-like enhancer in inactive two-cell-stage extracts, we next examined the loading of U2 snRNP onto the branch site. Even though U1 snRNP was bound downstream, no U2 interaction with the branch point sequence was seen. Only when the reaction was supplemented with active SR proteins was U2 addition observed. We have, therefore, determined that in order for U1 snRNP bound in a downstream position to promote U2 binding SR proteins must play an intermediary role. We next extended this work to include an examination of the ability of U1 snRNP to promote U2 addition from an upstream 5Ј splice site, which is in cis-splicing. As with exon definition, although U1 was bound to the 5Ј splice site, robust U2 interaction with the branch point sequence was only observed in two-cell-stage extracts upon the addition of active SR proteins. Our results demonstrate that SR proteins mediate the interaction between U1 and U2 snRNPs whether U1 is bound in an upstream or downstream position.

EXPERIMENTAL PROCEDURES
Substrates-The trans-splicing constructs have been described previously including WT trans and 5Јss enh. trans. These correspond to the WT and 7.2 (5Јss) clones examined through in vitro selection experiments (21). The WT cis substrate was also previously described (21). The 5Јss enh. cis substrate was generated by substituting a 69-nucleotide BglII-HindIII fragment (from ϩ82 to ϩ151, relative to the 3Ј splice site) from the 5Јss enh. trans construct into the WT cis clone. The cis py and cis py ϩenh constructs were generated by overlap PCR (39), introducing a two-base substitution (UUUUUUAG 3 UUAAUUAG) at positions Ϫ5 and Ϫ6 relative to the 3Ј splice site.
Extracts, SR Proteins, and in Vitro Splicing Assays-A. lumbricoides whole cell extracts were prepared from developmentally staged embryos allowed to develop to either the two-cell stage (inactive SR proteins) or the 32-cell stage (active SR proteins) (37). Extracts and in vitro pre-mRNA splicing were as previously described (40) with the splicing reactions resolved on 5% denaturing acrylamide gels and visualized by phosphorimaging analysis. Uniformly labeled pre-mRNA substrates for in vitro splicing assays were prepared as described (40). SR proteins were purified from developmentally staged embryos as previously described (20).
Cross-linking Assays-All cross-linking assays were performed on body-labeled RNA substrates essentially as described (21) except that the reactions were performed in two-cell-stage, inactive whole cell extracts. In reactions containing 2Ј-O-methyl oligonucleotides (Dharmacon), 200 ng of the U2-specific or 100 ng of the U1-specific oligonucleotides were included in the reaction before the addition of the substrate. SR proteins (0.5 g) were added to the cross-linking reactions as indicated. Reactions were resolved on 5% (Fig. 2) or 4% (Fig. 4) denaturing acrylamide gels and visualized by phosphorimaging analysis.
Micrococcal Nuclease Protection Assays-Site-specifically labeled substrates were generated as described (41) with the label positioned at the 3Ј splice site A[ 32 P]G dinucleotide. Nuclease protection assays were conducted as previously described (42) including SR protein supplementation and 2Ј-O-methyl oligonucleotide blocking targeting either U1 or U2 snRNP. Reactions were resolved on 8% denaturing acrylamide gels and visualized by phosphorimaging analysis.

U1 snRNP Interacts with a 5Ј Splice Site-like Enhancer Element in the Absence of Functional SR Proteins-Many groups
have documented the ability of SR proteins to participate in U1 addition to the 5Ј splice site (38,(43)(44)(45)(46). In our own previous work we performed in vitro selection experiments designed to uncover splicing enhancers that were able to promote transsplicing (21). One family of selected enhancers included 5Ј splice site-like elements that interact with U1 snRNP. These selection experiments were conducted in whole cell extracts derived from developing embryos of the nematode A. lumbricoides at a stage (ϳ32-cell stage) where the SR proteins are in an active state of phosphorylation (37). Thus, in these experiments we were not able to discern what possible role SR proteins might play in promoting this exon definition-like effect since SR proteins might be needed for efficient U1 interaction with the enhancer as opposed to potentially playing a role in mediating an interaction between U1 and U2 snRNPs. We employed several constructs to address these questions including the wild type weak trans-splicing substrate (Fig. 1, WT  trans), which was the starting material in the experiments in the previous selection (21). In addition, we also used a selected winner substrate containing a 5Ј splice site-like element ϳ100 nucleotides downstream from the 3Ј splice site (5Јss enh. trans). Finally, we also generated full-length cis-splicing substrates that include the same 3Ј splice site/3Ј exons as the indicates the presence of the 5Ј splice site-like enhancer element that was derived from in vitro selection experiments (21). This sequence contains a perfect 9/9 match to the 5Ј end of U1 snRNP.
trans-splicing constructs (WT cis and 5Јss enh. cis). Each of these pre-mRNA substrates required SR proteins to splice (either trans-or cis-) when incubated in inactive two-cell-stage extracts (data not shown).
Initially, we asked whether the interaction of U1 snRNP with the 5Ј splice site-like enhancer element was SR proteindependent. We assayed U1 binding to this element in inactive two-cell-stage extracts via psoralen cross-linking. We were able to determine whether U1 or U2 snRNP was present in the cross-linked species by preblocking the extract with 2Ј-Omethyl oligonucleotides specific for the 5Ј end of U1 or the branch site interaction region of U2. In the absence of added psoralen, no specific cross-links were observed under any of the conditions examined (Fig. 2, lanes 2-4). When psoralen was included in the reactions, however, a specific low mobilitycross-linked species was generated (lane 5). This species was unaffected by preincubation of the two-cell-stage extract with the 2Ј-O-methyl oligonucleotide targeting U2 (lane 7). In contrast, when the U1-specific 2Ј-O-methyl oligonucleotide was included, the cross-linked species failed to be produced (compare lanes 5 and 7 to lane 6). If these same reactions were conducted in the presence of additional active SR proteins (at a level that allows for trans-splicing activation), the cross-linking profile under the different blocking conditions was exactly the same (lanes 8 -10). Although the psoralen cross-linking assay does not detect subtle changes in the levels of U1 snRNP bound to mutant 5Ј splice sites altered in positions that do not impart a change in splicing efficiency (data not shown), it does allow for the assessment of U1 snRNP interaction at a gross level. Substrates containing an SR protein-dependent enhancer in place of the 5Ј splice site-like element do not cross-link to U1 (21). Therefore, the interaction of U1 snRNP with the 5Ј splice site-like enhancer was independent of the presence of active SR proteins. Interestingly, although the splicing of this substrate requires both the interaction of U1 with the exon and SR proteins, the role of the SR proteins is not to assist in the binding of U1 to the 5Ј splice site-like element. Alternatively, the hyperphosphorylated SR proteins that are present in the early developmental extract do not promote the splicing reaction itself, they may promote U1 snRNP binding to the 5Ј splice site-like enhancer.

U2 snRNP Addition to the Branch Site Promoted by U1
Interaction Downstream Requires SR Proteins-We next investigated whether SR proteins played a role in the communication between U1 and U2. Because we determined that U1 snRNP bound to the 5Ј splice site-like enhancer in the absence of SR proteins, we wanted to ask whether the addition of U2 to the branch site was dependent upon SR proteins in a bridging capacity. We employed micrococcal nuclease protection coupled with transcripts site-specifically labeled at the 3Ј splice site (41). When combined with the addition of 2Ј-O-methyl oligonucleotides specific for U2, this assay allows for the direct assessment of U2 addition to the branch site (35,42,47).
We examined the addition of U2 to the branch site in twocell-stage extracts on the WT trans and 5Јss enh. trans substrates. In both cases, only background levels of U2 snRNP addition to the branch site were observed (Fig. 3, lane 2, WT trans, and lane 6, 5Јss enh. trans). No substantial differences in the protection patterns were seen upon preincubation with the U2-specific 2Ј-O-methyl oligonucleotide (lanes 3 and 7). Upon supplementation of the two-cell-stage extract with SR proteins, a trace level of U2-dependent protection was observed with the WT trans substrate (lanes 4 and 5). This correlates with a low level of trans-splicing activity under these conditions (data not shown). When the 5Јss enh. trans substrate was examined after the addition of SR proteins, a dramatic difference was seen. The addition of SR proteins led to a large increase in the level of U2 specific protection (lanes 8 and 9). This protection is not the result of direct SR protein stimulation of U2 snRNP binding to the substrate as micrococcal nuclease protection experiments had previously demonstrated that U2 snRNP binding to this substrate is absolutely dependent on the availability of U1, even in extract containing high levels of active SR proteins (35). Therefore, the presence of U1 snRNP bound to the downstream exon was not sufficient to allow for the exon definition-like effect but, rather, was promoted in the presence of SR proteins.

SR Proteins Play an Intermediary Role in U2 Addition to the Branch Site via U1 snRNP Interaction across an Intron-Hav-
ing established that SR proteins play a direct role in the communication between U1 and U2 in mediating the loading of U2 onto the branch site, we next wanted to assess the involvement of SR proteins in the communication between U1 and U2 across an intron in cis-splicing. Previous work had shown that the SR protein SC35 could participate in an interaction between U1 and U2 at the 3Ј splice site (48) and that SR proteins were present in a complex with U1 assembled on a 3Ј splice site (49). Also, SR proteins were shown to be required for the splicing of mammalian pre-mRNA substrates that had been separated into 5Ј and 3Ј halves (50). However, the question of whether the effect of U1 bound to an upstream 5Ј splice site in loading U2 snRNP onto the branch site is mediated by SR proteins had not been directly addressed.
In a manner analogous to our investigation of the interaction of U1 snRNP with the 5Ј splice site-like enhancer, we began by examining the interaction of U1 with the 5Ј splice site of a cis-spliced pre-mRNA (Fig. 1, WT cis). U1 binding to the 5Ј splice site was assayed by psoralen cross-linking in inactive two-cell-stage extracts. When reactions were conducted in the absence of psoralen, no cross-linked RNAs were seen (Fig. 4,  lanes 2-4). Upon the addition of psoralen, a specific slow migrating band was seen (lane 5). We determined that this crosslinked species represented an interaction between the substrate pre-mRNA and U1 snRNP by preincubation of the reaction with a 2Ј-O-methyl oligonucleotide that targets the 5Ј end of U1. Under these conditions, the indicated cross-linked complex is eliminated (lane 6). As a control we also preincubated the reactions with a 2Ј-O-methyl oligonucleotide that targets the branch point interaction region of U2. In this case, the appearance of the cross-linked species was unaffected (lane

7)
. Upon the addition of active SR proteins, the identical results were obtained (lanes 8 -10). As a control we also generated a mutant cis-splicing substrate containing the identical 5Ј splice site sequence as the 5Ј splice site-like enhancer (WT ϭ AG/ GUGAGAU 3 5Ј ss UP mutant ϭ AG/GUAAGUU). Both pre-mRNA splicing as well as U1-cross-linking experiments conducted with this substrate gave identical results to the WT cis construct (data not shown). Thus, as we had observed with the 5Ј splice site-like enhancer present in the downstream exon, an active 5Ј splice site upstream of the branch point sequence can be occupied by U1 snRNP in the absence of SR proteins that are active in the promotion of splicing activity.
We next assayed the interaction of U2 with the branch site on the WT cis substrate in two-cell-stage extracts. As with the trans-splicing substrates, the cis-splicing substrate was sitespecifically labeled at the 3Ј splice site, and U2 snRNP addition to the branch site was monitored as before. Very little U2specific protection was observed with the cis-spliced substrate in these inactive extracts (Fig. 5, lanes 2 and 3, corresponding to no block and U2 block). When SR proteins were added to the reactions, a striking increase in U2 loading onto the branch site was observed (lanes 4 and 5, corresponding to no block and U2 block). Thus active SR proteins are required for the positive effect of U1 at an upstream 5Ј splice site in terms of U2 addition to the branch site.
Functional Inequality of 5Ј Splice Sites Positioned either Upstream or Downstream of a 3Ј Splice Site-We had shown that SR proteins were able to play a role in the functional interaction between U1 and U2 in terms of U2 addition to the branch site and subsequent splicing activity. Although we had determined that this positive interaction was able to occur when the 5Ј splice site was positioned either upstream or downstream of the 3Ј splice site, we wanted to ask whether the strength of these activities was also equivalent. To address this issue, we introduced a mutation into the polypyrimidine tract (UUUUUUAG 3 UUAAUUAG) of both the WT cis and 5Јss enh. cis substrates, generating the cis py and cis py ϩ enh. constructs. Pre-mRNA splicing activity of the WT cis and 5Јss enh. cis substrates in active ϳ32-cell-stage extract is shown for comparison (Fig. 6A, lanes 2-5). The addition of the 5Ј splice site-like enhancer stimulates pre-mRNA splicing activity ϳ2fold (compare lane 3 to lane 5). Upon mutation of the polypyrimidine tract, cis-splicing activity was lost (Fig. 6B, lanes 2  and 3). If however, the polypyrimidine tract mutation was combined with the 5Ј splice site-like enhancer downstream, robust splicing activity was regained (lanes 4 and 5). Therefore,  although there is a functional 5Ј splice site upstream, no splicing activity was seen unless a 5Ј splice site-like element was added downstream. Thus, there is functional inequality in terms of splicing activity when U1 is bound at an upstream position versus when U1 is bound at a downstream position even though both can promote U2 addition when there is a wild type polypyrimidine tract. DISCUSSION There are numerous factors that play roles in the addition of U2 snRNP to the branch site sequence. Early in spliceosome assembly, the large (65 kDa) and small (35 kDa) subunits of U2 auxiliary factor (U2AF) interact with the polypyrimidine tract (51) and the 3Ј splice site AG dinucleotide, respectively (47,52,53). The interaction of U2AF with the pre-mRNA 3Ј splice site region occurs in a cooperative manner with the branch point recognition protein SF1/BBP (54). In addition, the heterodimeric complex of the splicing factors SRp54 and PUF60 has also been shown to recognize this same region (55). In fact, recent work demonstrated that the Drosophila melanogaster protein Half pint, which plays a role in developmentally regulated pre-mRNA splicing, is the orthologue of mammalian PUF60 (56). Each of these factors along with several more (i.e. UAP56 (57) and the complexes SF3a and SF3b (for review, see Ref. 58) have all been shown to play roles in the association of U2 snRNP with the branch site.
With this as the context, the question then arises, How does U1 snRNP positively affect the binding of U2 to the branch site? Here, we demonstrated that SR proteins play the role of intermediary in this interaction. The potential interplay of U1 and SR proteins with the multitude of factors described above remains unclear. The ability of certain splicing factors to interact with each other has suggested models for discrete networks of interactions. These include the interaction of the U1 snRNPspecific 70-kDa protein with SR proteins as well as the interaction between SR proteins and the 35-kDa U2AF subunit (38,59). The interaction of 3Ј splice site-recognizing factors as well as U1 with the forming spliceosome occurs early, before the first ATP-dependent step, the stable association of U2 with the branch site. Although U2 was recently shown to be a component of the ATP-independent early or E complex, it was suggested that stable binding was only achieved in the ATP-dependent A complex (60).
With respect to the trans-splicing reaction in nematodes, one potential scenario for U2 addition involves the interaction between U1 snRNP (presumably via the 70-kDa protein), SR proteins, and the branch site recognition factor SF1/BBP. Mammalian and yeast SF1/BBP both contain U2AF interaction and RNA binding domains, with the yeast protein also containing a region that allows for communication with the U1 snRNP protein Prp40p (61). The interaction of mammalian SF1/BBP with the 65-kDa U2AF and subsequent spliceosome assembly is regulated by phosphorylation of a conserved serine (62). In both nematodes as well as D. melanogaster, SF1/BBP contains an additional domain that encodes an N-terminal RS region (63,64). If U1 binds downstream of a 3Ј splice site, interactions between 70 kDa (SR proteins) and the RS domain of SF1/BBP would allow for the establishment of an early complex, poised to promote the interaction of U2 with the branch site. In addition, A. lumbricoides SF1/BBP can also interact with the 30-kDa protein specific to the SL RNP, which donates the 5Ј exon in trans-splicing (64). Thus, the positive effect of U1 bound downstream from a 3Ј splice site involved in trans-splicing (21) may be mediated by a complex of proteins all interacting early in trans-spliceosome assembly (see the model, Fig. 7). This model could also be drawn to contain SR proteins bound to an ESE in place of U1 interacting with the downstream 5Ј splice site. Both ESEbound SR proteins as well as 5Ј splice site-bound U1 can promote U2 snRNP addition and subsequent trans-splicing in vitro (21,35). The additional RS domain in nematode SF1/ BBP may promote the establishment of a more stable 3Ј complex, allowing for the association of the SL RNP containing the 5Ј splice site in trans.
The results described here demonstrate that SR proteins play a critical role in the communication between factors bound at either end of an exon. In addition, they are also required for interactions between the 5Ј and 3Ј ends of an intron. Mechanistically, SR proteins are indispensable for the U1-mediated addition of U2 snRNP onto the branch site sequence whether this activity occurs across exons or introns. This process may well involve additional factors that have been shown to interact with the information-rich 3Ј splice site region. Undoubtedly, more functionally relevant interactions between specific factors remain to be uncovered.