Nucleic Acids Research

Figure 5. 3A and 3B sequences act as ESEs in vitro. (A) Nucleotide sequences of T, 3A, 3B and 3S RNAs. (B) Schematic representation of the model wild-type human [beta]-globin pre-mRNA and its derivatives containing the T, 3A, 3B or 3S sequence. Sequences derived from [beta]-globin exon 1 or exon 2 are boxed in white and grey, respectively, and black boxes represent non-[beta]-globin sequences (3A, 3B and 3S). Numbers on top indicate the length of the corresponding exonic and intronic regions and the angled lines represent the splicing events. (C) 3A and 3B sequences enhance splicing of a [beta]-globin intron in vitro. Splicing reactions were performed under standard conditions with 30% HeLa nuclear extract and 50 fmol of the radiolabeled pre-mRNA indicated. Splicing products (depicted on either side of the panel) were analyzed on a 7% denaturing polyacrylamide gel and revealed by autoradiography. (D) 3A and 3B sequences promote exon inclusion in vitro. Reactions were performed as in Figure 5C with the indicated model three-exons [beta]-globin pre-mRNAs. Splicing intermediates and products are depicted on either side of the panel.

Given that RSF1 has a modular structure with an N-terminal RRM domain and C-terminal GRS domain, both of which are capable of binding homopolymers (data not shown), we decided to determine the contribution of individual domains for the binding of RSF1 to cognate RNAs. RSF1-RNA interaction probably involves the RRM domain of RSF1, because a truncated version of RSF1 in which the RRM domain was selectively removed (GST-RSF1[Delta]N, Fig. 1B, lane 4) failed to bind 3A (Fig. 3A, panel I, lanes 7-9) or 3B (Fig. 3A, panel II, lanes 7-9) sequence. In contrast, a RSF1 mutant lacking the GRS domain (GST-RSF1[Delta]C, Fig. 1B, lane 3) efficiently bound to these sequences (Fig. 3A, panels I and II, lanes 4-6, respectively), implying that the GRS domain does not contribute to RSF1-RNA interaction. Critical comparison of the band shift patterns obtained with GST-RSF1 and GST-RSF1[Delta]C showed that the latter formed three complexes of different mobilities and the former only one. This result could be explained if the 3A or 3B sequence binds three molecules of GST-RSF1[Delta]C simultaneously (Fig. 3A, panels I and II, lanes 4-6, respectively) but only one molecule of GST-RSF1. These data validate the SELEX experiments and further indicate that the RRM per se, with very few neighboring residues, is necessary and sufficient to interact with high affinity to single nonamer sequences.

The similarity of RSF1 binding sites with purine-rich ESEs, the fact that RSF1 and a range of SR proteins share closely related RRM domain at their N-terminal ends (Fig. 2B) and the co-localization of RSF1 with the splicing factors at transcriptionally active chromosomal sites (data not shown), prompted us to investigate whether representative GST-RSF1 ligands might be target sites for splicing factors. We initially assessed the ability of 3A or 3B RNA to titrate splicing factors from HeLa nuclear extract. We therefore supplemented HeLa nuclear extract with increasing concentrations of cold 3A or 3B RNA and tested their capacity to support the splicing of a model substrate containing the first two exons and first intron of the wild-type human [beta]-globin gene. No inhibition of splicing was observed on adding up to a 100-fold molar excess of 3B RNA over ³²P-labeled [beta]-globin substrate (Fig. 4A, lanes 13). In contrast, the splicing efficiency was reduced by 50% when only a 10-fold molar excess of 3A RNA was used (Fig. 4A, lane 5) and a 100-fold excess of 3A RNA led to the complete inhibition of splicing (Fig. 4A, lane 7). These observations support the notion that the 3A RNA could be a high-affinity binding site for trans-acting splicing factors. Analysis of splicing reactions by native gel electrophoresis revealed that the 3A RNA interferes with the formation of A and B splicing complexes (data not shown), implying that 3A RNA was titrating factors acting at the earliest stages of spliceosome assembly.

To identify the factor(s) that interact(s) with GST-RSF1 target sequences, we used UV cross-linking assays. Radiolabeled probes corresponding to the 3A or 3B RNA were cross-linked in the presence of HeLa nuclear extract, and labeled proteins were analyzed by electrophoresis on SDS-polyacrylamide gels and visualized by autoradiography. The 3A RNA transferred label predominantly to protein species with apparent molecular weights of ~20, 30, 40, 55, 75 and 130 kDa (Fig. 4B, panel II, lane 4). Allowing for the 130 kDa protein which was only seen in nuclear extract, protein bands with identical mobility also appeared when cross-linking experiments were performed with either purified SR proteins (Fig. 4B, panel II, lane 1) or nuclear extract supplemented with purified SR proteins (Fig. 4B, panel II, lane 6). This result indicates that the 20, 30, 40, 55 and 75 kDa bands are probably SRp20, SRp30, SRp40, SRp55 and SRp75, respectively. Although the amount of cross-linking of these proteins correlated with their abundance in the extract, indicating that the 3A RNA had no preference for binding any of the SR proteins, a very strong signal was observed at the level of SRp30. Since at least four SR proteins are expected to have the same electrophoretic behavior at 30 kDa, it is possible that one or several SRp30 species make strong contact with 3A RNA. A likely candidate could be 9G8 protein from human, because a recent SELEX performed with this protein resulted in purine-rich sequences that resemble the 3A RNA consisting mainly of a repetition of three GAC triplets (39). Accordingly, comparison of the RRM domain of RSF1 with those of SR proteins revealed that the highest homology score (56% identity and 68% similarity) was detected over the RRM of 9G8 from human (Fig. 2B). The cross-linking of the 3A RNA to SR proteins was likely to be specific, because it was not competed out with a large excess of nuclear proteins (Fig. 4B, panel II, lanes 4 and 6), whereas it was effectively competed by increasing amounts of an unlabeled 3A RNA (Fig. 4C, lanes 4 and 5). Interestingly, cross-linking to SR proteins was no longer detected when the nuclear extract was supplemented with purified GST-RSF1 (Fig. 4B, panel II, lane 8). Instead, two strong signals were produced by purified GST-RSF1 (Fig. 4B, panel II, compare lanes 3 and 8). The concentration of GST-RSF1 required to achieve this was almost identical to that of SR proteins in nuclear extract (quantitation of the amount of SR proteins in the extract was made by comparison with known amounts of purified SRp30), suggesting that the 3A RNA displays higher affinity for GST-RSF1 than for SR proteins. However, GST-RSF1 yields much less cross-linked product than SR proteins in HeLa extract (Fig. 4B, panel II, compare lanes 4 and 8). Given that GST-RSF1 also gives very poor cross-linking when purified proteins were used (compare lanes 1 and 3), it is possible that amino acids of GST-RSF1 contacting the 3A RNA are moderately reactive after excitation of the nucleic bases by UV light. The failure of GST-RSF1[Delta]N to compete with cross-linking of SR proteins to 3A RNA (Fig. 4B, panel II, lane 7) makes it very likely that the competition effect was mediated by RSF1's RRM domain. Consistent with this, purified GST-RSF1[Delta]N did not show any cross-linking to 3A RNA (Fig. 4B, panel II, lane 2).

Although 3B RNA was efficiently cross-linked to purified individual SR proteins (Fig. 4B, panel III, lane 1), a large excess of cold 3B RNA failed to compete with the cross-linking of SR proteins to 3A RNA in nuclear extract (Fig. 4C, lanes 6 and 7). Labeled 3B RNA produced no signal by SR proteins in nuclear extract (Fig. 4B, panel III, lane 4) but, instead, a smear plus a strong signal at the level of high-molecular-weight protein species. Neither purified SR proteins (Fig. 4B, panel III, lane 6) nor purified RSF1 (Fig. 4B, panel III, lane 8) could compete efficiently with the cross-linking of these proteins, indicating that the 3B RNA may display higher affinity for unknown masking proteins present in the nuclear extract. Among those the 130 kDa protein was a likely candidate. Failure of SR proteins to bind the 3B RNA in nuclear extract offers a plausible explanation of why splicing of [beta]-globin pre-mRNA was not affected by a large excess of this RNA.

Purine-rich sequences that bind SR proteins have been shown to act as cis-acting regulatory elements for splicing (13,14,40,41). To better understand the meaning of the binding of SR proteins to the 3A RNA, we asked whether this site could function as a splicing enhancer. We used a [beta]-globin derivative with a short second exon (Fig. 5B, [beta]-T) as a model substrate. Though it has the wild-type [beta]-globin splice sites, this substrate was very poorly spliced in nuclear extract containing a low amount of SR proteins (Fig. 5C, lane 2). However, splicing efficiency could be altered if a splicing enhancer was introduced downstream from the 3[prime] splice site. Substitution in the second exon of an optimal binding site for SF2/ASF (Fig. 5B, [beta]-3S), previously shown to constitute a powerful splicing enhancer in vitro (14), in place of [beta]-globin sequences enhanced splicing dramatically (Fig. 5C, compare lanes 2, 5 and 7). An identical enhancing effect was also observed with the 3A sequence (Fig. 5C, lane 3), indicating that the latter can function as a strong splicing enhancer sequence when inserted within an exon. However, the 3B sequence, which did not inhibit splicing in trans, was less efficient at stimulating splicing in cis (Fig. 5C, lane 4). It is significant that 3S and 3A have comparable splicing enhancing activities (Fig. 5C, compare lanes 3 and 5), suggesting that they could bind trans-acting factors with similar affinity. Consistent with this view, cross-linking of isolated copies of the 3A sequence to SR proteins in nuclear extract was competed to the same extent with increasing amounts of unlabeled 3A or 3S competitor RNA (Fig. 4C, lanes 2-5).

To determine whether the GST-RSF1 winner sequences might be required for the regulation of alternative splicing, we used model pre-mRNAs containing two introns and three exons, derived from the human [beta]-globin gene. These substrates have been successfully used to demonstrate ESE-dependent inclusion of a small internal exon (42). Therefore, four nucleotides of the first exon adjacent to the 5[prime] splice site, the first intron and the second exon of [beta]-globin were inserted downstream of [beta]-T, [beta]-3A, [beta]-3B or [beta]-3S, generating four constructs each containing a 51-nt middle exon flanked by two identical introns, termed DUP51 T, DUP51 3A, DUP51 3B and DUP51 3S, respectively (Fig. 5B). Our constructs differ from those described by Dominski and Kole (42) as the middle exon contained more sequences from the second exon of [beta]-globin. Importantly all four splice sites, as well as their immediately adjacent sequences, were the same in all the pre-mRNAs studied. Thus, splice site usage should reflect solely the influence of the introduced sequences. Splicing was assayed in vitro in HeLa nuclear extracts using ³²P-labeled SP6 transcripts and in vivo during transient expression in transfected cells of the same constructs driven by a [beta]-actin promoter. As previously observed for single-intron containing substrates, both DUP51 T and DUP51 3B were very poorly spliced in nuclear extract containing low levels of SR proteins (Fig. 5D, lanes 1 and 2). Interestingly, the patterns of splicing for these transcripts were characterized by the existence of slowly-migrating large superlariat structures which represent intermediates of middle exon skipping reaction, as depicted on the left in Figure 5D. Although skipping was the predominant reaction for DUP51 T (lane 1), yielding a final product with the same size as the product of splicing for monointronic [beta]-globin (Fig. 5C, lane 7), splicing of DUP51 3B generated, in addition, the final product containing three exons (Fig. 5D, lane 2). Quantitative analysis showed that half of the spliced product from DUP51 3B contains the internal exon, suggesting that the 3B sequence acts as a positive element for the selection of immediately adjacent splice sites.

Very strong enhancement of in vitro splicing efficiency was observed when the internal exon contained either 3A or 3S sequence (Fig. 5D, lanes 3 and 4). Strikingly, both DUP51 3A and DUP51 3S were spliced by a pathway that generates only a fully spliced product containing all three exons. No band corresponding to the superlariat containing the middle exon was detected. The number of RNA bands generated during splicing of the two transcripts was identical and correlated well with the electrophoretic properties of the intermediates and final products predicted for the regular splicing pathway of a two-intronic pre-mRNA. Their structures are depicted on the right in Figure 5D.

Figure 6. 3A and 3B sequences are functional ESEs in human and Drosophila living cells. (A) HeLa cells were transfected with the plasmid construct coding for either DUP51 3S, T, 3B or 3A [beta]-globin pre-mRNA (lanes 3-10) or with the empty plasmid (lanes 1 and 2). The splicing products were detected by RT-PCR using total RNAs prepared from transfected cells and primers specific to upstream and downstream [beta]-globin exon sequences. For each experiment, 1/10 or 1/50 of the RT reactions were used for 20 cycles of PCR amplification in the presence of 2 µCi [[alpha]-³²P]dCTP. Amplified fragments were separated on a 5% denaturing polyacrylamide gel and revealed by autoradiography. As an internal control, PCR reactions were performed in the presence of two additional primers designed against human GAPDH mRNA. The DNA bands corresponding to unspliced pre-mRNA (651 bp), spliced mRNA (391 bp), skipped mRNA (340 bp) or GAPDH mRNA (228 bp) are marked on the right. (B) Same experiments using total RNA purified from transfected Drosophila S2 cells. As a control, PCR with two oligonucleotides specific for copia mRNA are also shown for each experiment. The DNA bands corresponding to [beta]-globin pre-mRNA, its splicing products and copia mRNA are marked on the right.

Patterns of splicing in vitro and in vivo may differ significantly and splice site selection in vitro may be affected by the concentration of the nuclear splicing factors in the extract. We therefore performed in vivo studies in cultured cells to ascertain the splicing pattern of the tested pre-mRNAs. The various DUP51 constructs, inserted within a suitable expression vector, were transfected in HeLa cells or Drosophila S2 cells and total RNAs were prepared 24 h post-transfection to avoid reduction of mRNA levels owing to cell death. The mRNA products of each transfected construct were examined by RT-PCR and normalized to GAPDH mRNA in HeLa cells or to copia retrotransposon mRNA in S2 cells. As shown in Figure 6, internal exons containing either a control [beta]-globin sequence (T) or 3B were skipped (Fig. 6A and B, lanes 3-6) and only the 3B sequence allowed a very low level of exon inclusion in HeLa cells (Fig. 6A, lanes 5 and 6) but not S2 cells (Fig. 6B, lanes 5 and 6). In sharp contrast, both the 3A and 3S sequences conferred exclusive inclusion of the middle exon in HeLa cells (Fig. 6A, lanes 7-10) and half of these exons were skipped in S2 cells (Fig. 6B, lanes 7-10), consistent with the role of these sequences as powerful and functional ESEs in living cells. Failure to observe exclusive inclusion of exons containing the 3S or 3A sequence following transfection of constructs in S2 cells was attributable to lower levels of SR proteins. Quantitation by western blot analysis revealed that extracts from S2 cells contained at least six times less SR proteins than extract from HeLa cells (data not shown). Thus, the presence in S2 cells of RSF1 and/or other as yet unidentified SR protein antagonists at a concentration that may easily displace the association of SR proteins with exon containing 3A, 3B or 3S sequences, could inhibit exon inclusion during splicing of these reporter pre-mRNAs. This, together with results described by Labourier et al. (29), is consistent with the idea that RSF1 acts as a sequence-specific pre-mRNA splicing repressor in vivo.

DISCUSSION

In this paper we have presented several lines of evidence suggesting that representative RSF1 RNA ligands could serve as exon recognition signals that promote exon inclusion. Two purine-rich sequences, A and B, were identified from a random sequence pool as high-affinity binding sites for RSF1. Mobility shift data obtained with full-length and truncated versions of recombinant RSF1 were consistent with the results of the SELEX experiment and demonstrated that its RRM domain was essential and sufficient to mediate specific interaction with these sequences. The finding that RSF1 bound much less efficiently SF2/ASF-selected sequences (data not shown), which were also purine-rich (14), makes it unlikely that RSF1 binds any polypurine-rich sequences but in fact recognizes short distinct sequences. However, there are differences in the behavior of RSF1-selected sequences. Three copies of the A-type sequence display specific cross-linking to SR proteins in HeLa nuclear extract, efficiently stimulate splicing of reporter pre-mRNAs and promote exon inclusion. Significantly, both in vitro and in transfected cells the reiterated A-type sequence acts in a manner indistinguishable from similarly arranged SF2/ASF high-affinity binding sites when similarly positioned within a model pre-mRNA. In sharp contrast three copies of the B-type sequence only had slight effects on these splicing events. Since purified SR proteins do bind the B-type sequence, we assume that in HeLa nuclear extract there are factors that bind the B-type sequence with higher affinity than SR proteins and thereby antagonize the function of SR proteins. These factors could be related to RSF1 because complementing HeLa nuclear extract with purified RSF1 displaces the binding of SR proteins to A-type sequences and impedes splicing of reporter pre-mRNAs (29). Experiments are currently underway to identify the human homolog of RSF1 in HeLa cell nuclear extract depleted of SR proteins, which could specifically interact with 3A or 3B targets.

Although additional work is needed to establish whether the sequences described here occur naturally in pre-mRNAs subject to alternative splicing, it is striking that several splicing silencer sequences (SSEs), including HIV regulatory sequences, contain motifs closely related to those selected by GST-RSF1 (43,44). Interestingly, in some cases the SSE sequences were found to be juxtaposed to ESEs, forming bipartite splicing regulatory elements that may control the overall efficiency of proximal splice site usage (18,45,46). Therefore, the binding of RSF1 to ESEs shown here could be a way to mediate splicing repression to prevent activation of cryptic splice sites within exonic sequences. In agreement with this hypothesis, it has recently been shown that RSFc, a protein in the dipteran Chironomus tentans, highly homologous to RSF1, binds extensively to newly transcribed BR1 and BR2 pre-mRNAs, which contain a huge exon 4 of 30-35 kb but much less to the BR3 pre-mRNA, which largely consists of intron sequences (L. Wieslander, personal communication). Thus, while ESE elements are required to assure that constitutive splicing does take place, an appropriate balance of splicing repressors and splicing activators, expected to bind these sequences, controls splice site usage of suboptimal splicing signals.

How might RSF1 mediate splicing repression? Binding of this protein may interfere directly with the binding of SR proteins and/or their interactions with constitutive splicing factors. Several lines of evidence support a model for initial splice-site recognition in which multiple protein-RNA and protein-protein interactions between SR proteins bound to the exon and the 5[prime] and 3[prime] splice sites led to the formation of a stable complex (6,26-28). SR proteins have been shown to enhance interactions between the U1 snRNP and the 5[prime] splice site (27,47,48), interactions between U2AF and the 3[prime] splice site as well as bridging the 5[prime] and 3[prime] splice sites (27,28). They are also expected to bind ESE sequences to stimulate splicing of the upstream intron or inclusion of an internal exon (5,12,13,40,49-51). Accordingly, ESE sequences have been found in many cellular and viral pre-mRNAs which are subject to alternative splicing (13,15-21) as well as constitutively spliced pre-mRNAs (22,50,51). The finding that one such sequence also serves as a high-affinity binding site for RSF1, a splicing repressor, provides a framework for understanding the mechanism by which splicing repression can be mediated. The binding of RSF1 to ESEs will prevent recruitment of constitutive splicing factors (U1 snRNP and U2AF) to splice sites, and as a result will abrogate the formation of stable enhancer complexes. This view is consistent with the finding that RSF1 induces a dose-sensitive inhibition of splicing for several reporter pre-mRNAs, an inhibition that occurs at the level of early splicing complexes formation (29). The results of UV cross-linking assays with HeLa nuclear extracts (Fig. 5B) clearly show that the binding of SR proteins to an enhancer sequence can be displaced by addition of recombinant RSF1 in the extract. The amount of RSF1 required to achieve this displacement was comparable with the amount of SR proteins in the extract, implying that the binding of both factors to this enhancer sequence is of the same strength. The fact that RSF1 interacts directly, through its GRS domain, with the RS domain of SR proteins (29) makes it likely that protein-protein interactions are also involved in splicing repression. These protein-protein interactions could, indeed, interfere with those occurring between SR proteins and constitutive factors. At least in vitro, we have shown that RSF1 prevents cooperative interaction between SF2/ASF and U1-snRNP 70 kDa protein and thus prevents the stable binding of U1-snRNP at the 5[prime] splice site (29).

In vitro splicing studies revealed that complementation of HeLa nuclear extract with recombinant GST-RSF1 inhibits splicing of reporter pre-mRNAs, whether they contain the A-type sequences or not (29), indicating that RSF1 inhibition of splicing does not require high-affinity RSF1 binding sites. Therefore, it is possible that, in vitro, RSF1 might act either by binding RNA with low affinity and/or by binding free SR proteins. In keeping with this possibility, recent studies employing in vitro selection for functional splicing enhancers that bind specific SR proteins (12,52) revealed that both SELEX-isolated high-affinity consensus binding sites (10,14,39) and more degenerate (and possibly lower-affinity) binding sites (12,52) can function as exonic splicing enhancers. Thus, some reporter pre-mRNAs, used for in vitro splicing studies, might contain potential targets for RSF1 which have escaped our notice, because the iterative protocol is designed to yield the highest affinity sequences for purified RSF1, but not target sequences involving interaction of RSF1 with other splicing components. These potential targets could also be enhancer sequences, since it has been demonstrated recently that the second exon of [beta]-globin pre-mRNA, a constituvely spliced pre-mRNA, harbors multiple distinct splicing enhancers (22).

Numerous studies contributed to the view that SR proteins can modulate splice site selection in a concentration-dependent manner (9,10). One imaginable consequence of this is that a wide-variety of pre-mRNAs can be controlled by a small set of evolutionarily conserved antagonistic sequence-specific splicing factors. Consistent with this view, targeted expression in Drosophila of transgenes encoding either the SR protein B52 (SRp55) or RSF1 led to pronounced deleterious effects on development (29,53). However, when both proteins were overexpressed together in the same tissue, partial or complete rescue of normal phenotypes were observed (29), implying that RSF1 primarily functions as a SR protein antagonist. According to our model RSF1 and B52 may affect splicing of several pre-mRNAs in a given tissue; it is, therefore, not surprising that overexpression of either factor would be detrimental for the correct development of this tissue. For instance, cryptic splice sites may be activated if the level of B52 is high; conversely, weak splice sites will not be activated if the level of RSF1 is high and in either case this will lead to a default of splicing. Given that RSF1 (Labourier et al., unpublished data) and SR proteins co-localized with transcriptionally active sites of RNA polymerase II on polytene chromosomes, it is tempting to propose that a recruiting mechanism may exist to regulate the local concentrations of splicing activators and splicing repressors at the sites of transcription in the cell nucleus. The elucidation of this mechanism will be important in understanding the general principles that underline gene regulation by alternative splicing.

ACKNOWLEDGEMENTS

We thank A. Krainer for providing pSP64H[beta][Delta]6 construct, H. Land for pJ6[Omega] plasmid and P. Bouvet for the random oligonucleotide pool. H.-M.B. acknowledges F. Amalric and A. Vincent for their interest and logistical support in the initial part of this work. We thank D. Cribbs for support, scientific interest and critical review of the manuscript. A special thanks to A.-M. Duprat for her constant support. We also thank Y. De Preval for synthesizing a number of oligonucleotides. This work was supported by an ATIPE grant from the CNRS (to H.-M.B) and a grant from the CNRS and the ARC (to J.T.). E.L., E.A. and S.B. were supported by graduate fellowships from the Ministère de l'Enseignement Supérieur et de la Recherche (MESR); E.L. and S.B. also benefited from graduate training fellowships from the ARC.

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*To whom correspondence should be addressed. Tel: +33 4 67 61 36 85; Fax: +33 4 67 04 02 31; Email: tazi@igm.cnrs-mop.fr
⁺Present address: Department of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Road, Manchester M13 9PT, UK

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