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Previous Article | Next Article 
Molecular and Cellular Biology, March 2003, p. 1874-1884, Vol. 23, No. 6
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.6.1874-1884.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Esra Cagavi,1,
and Douglas L. Black1,2*
Department of Microbiology, Immunology and Molecular Genetics,1 Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California 900952
Received 16 August 2002/ Returned for modification 25 September 2002/ Accepted 16 December 2002
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ABSTRACT |
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INTRODUCTION |
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Another large group of proteins that bind to nascent pre-mRNAs are the heterogeneous nuclear ribonucleoproteins (hnRNPs) (19, 30). At least in vitro, spliceosome assembly occurs after hnRNP binding and some hnRNPs are implicated in splicing regulation. Two of these, hnRNP A1 and polypyrimidine tract binding protein (PTB or hnRNP I) bind to exonic splicing silencer or intronic splicing silencer elements and thus repress the splicing of certain alternatively spliced exons (10, 46). The mechanisms that hnRNP A1 and PTB use to mediate splicing repression are not fully understood. Several models have been proposed (10, 50). One mechanism involves direct interactions between positive and negative factors at adjacent binding sites (9, 14, 18, 57). The concentration of the factors and their relative affinities for their binding sites are what determine exon inclusion or skipping. In other systems, the assembly of multimeric A1 or PTB complexes onto sites surrounding an alternatively spliced exon may cause the intervening region of RNA to loop out and thus prevent splice site recognition (2, 17, 22, 52). Another possible mechanism of repression by hnRNP A1 involves binding to a high-affinity site within an exon and then promoting the assembly of additional hnRNP A1 molecules along the pre-mRNA (57). This is thought to create a repressed zone of RNA refractory to the binding of the general splicing machinery at the splice sites. SF2/ASF can block this cooperative propagation of hnRNP A1 along the exon, thereby activating splicing. Both SR proteins and hnRNPs vary in concentration between different cell types, and it is thought that this differential expression of hnRNPs and SR proteins affects the alternative splicing of many pre-mRNAs (7, 25, 28, 37, 38, 56).
The N1 exon of c-src provides a model system for the study of neuron-specific splicing regulation. The 18-nucleotide exon, N1, is included in the mRNA in neurons but is skipped in nonneuronal cells (31, 36). Two intronic sequences are required for regulation of N1 splicing (1, 12, 42, 43). The 3' splice site region upstream of the exon is required for repression of N1 in nonneuronal cells. It has been shown previously that PTB binds to CUCUCU elements (CU) within this sequence and is required for splicing repression in vitro (13, 17). The second intronic sequence, between 17 and 142 nucleotides downstream of N1, acts as a splicing enhancer and is also required for splicing repression (17). Within this element is a highly conserved region between nucleotides 37 and 70 called the downstream control sequence (DCS). The DCS contains a CU element that is required for splicing repression. Flanking this CU element are the elements GGGGGCUG and UGCAUG, which are needed for enhancer activity. Each of these elements binds to specific RNA binding proteins: hnRNP F and hnRNP H bind to the G-rich element (16, 35, 40), KH-type splicing-regulatory protein binds to the UGCAUG element (35, 41), and PTB or its neural homolog, nPTB, binds to the CU element (17, 35).
Studies of splicing regulation, including those on src N1, have generally focused on either intronic elements that bind PTB and other factors or on exonic elements that bind SR proteins, hnRNP A1, and other proteins. However, it is likely that in many systems the exonic and intronic elements are functioning together. Indeed, in addition to the intronic elements, an element within the N1 exon itself also acts as a splicing enhancer element. When this purine-rich sequence from exon N1, GAGGAAGGUG, is placed within a similarly sized test exon of a heterologous minigene, it activates splicing in vivo (42, 43). Splicing of the test exon was strongest when both intronic and exonic elements were placed together in the construct, suggesting that they normally work in combination (42). The enhancement of the test exon by these elements varied between the different neuronal and nonneuronal cell lines tested, but they do not appear to be highly neuron specific.
In this study, we further examine the role of the exonic enhancer in the regulation of exon N1 splicing. We identify the proteins SF2/ASF, hnRNP A1, hnRNP A1B, hnRNP H, and hnRNP F as binding to the element in splicing extracts. We show that SF2/ASF and SC35 can activate splicing of c-src pre-mRNA and that SF2/ASF enhances the splicing pathway leading to inclusion of N1. Both hnRNP A1 and PTB can counteract the activity of the SR proteins to repress splicing but apparently do so by different mechanisms.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro splicing.
Nuclear extracts were prepared and in vitro splicing reactions were conducted as previously described (12), and the reaction mixtures contained either 
100 µg of Weri-1 nuclear extract or 300 µg of Weri-1 S100 extract plus the proteins described in the text in a total volume of 25 µl.
Site-specific labeling and UV cross-linking. Site-specifically labeled c-src pre-mRNA containing a single 32P-labeled guanosine was synthesized (44). The reaction conditions for UV cross-linking were the same as those for splicing. The site-specifically labeled RNAs were incubated between 1 and 120 min. After incubation, heparin sulfate was added to a concentration of 0.25 mg/ml and incubated for an additional 5 min on ice. The samples were then irradiated with 254-nm-wavelength UV light on ice for 15 min by using a handheld UV lamp (UVP). The samples were then treated with 1 µg of RNase A for 30 min at 30°C. The cross-linked proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% or 4 to 20% polyacrylamide) and visualized by PhosphorImager (Molecular Dynamics).
Immunoprecipitation. The immunoprecipitation experiments used rabbit polyclonal antibodies raised against the hnRNP A1 C-terminal peptide, the AK-96 mouse monoclonal SF2/ASF antibody (Zymed), rabbit polyclonal antibodies against the C-terminal peptide of hnRNP H, rabbit polyclonal antibodies against the C-terminal peptide of hnRNP F, rabbit polyclonal antibodies against the C-terminal peptide of U170K, and the 4F4 mouse monoclonal hnRNP C1/C2 (a gift from G. Dreyfuss). In a typical experiment, 20 µl of GammaBind Plus Sepharose (Amersham Pharmacia), preequilibrated with immunoprecipitation buffer (IP buffer) (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X-100) and blocked with 2.5 mg of bovine serum albumin per ml in IP buffer, was preincubated with 5 to 20 µl of antibody for 1 h at 4°C. The beads were washed three times with 1 ml of IP buffer to remove unbound antibody. The UV-irradiated, RNase-digested splicing reaction mixture was incubated with the beads for 1 h at 4°C. The beads were then washed again three times with 1 ml of IP buffer. Proteins were eluted from the beads with SDS-PAGE loading buffer. Twenty microliters of rat anti-mouse beads (Dynal) were used in the mAb104 (a gift from M. Roth) immunoprecipitation experiment.
Protein purification.
hnRNP A1 cDNA (a gift from A. Mayeda) was fused to a six-His tag by using standard PCR methods. hnRNP A1-His was expressed in BL21-pLysS bacteria (Novagen) and was purified by using standard techniques (Qiagen). His-tagged La was expressed in BL21 bacteria (Novagen) and was similarly purified. PTB was purified from 10 ml of HeLa nuclear extract (200 mg of protein) as previously described (35). SC35 and SF2/ASF were isolated by using the baculovirus expression system. SC35 baculovirus (a gift from X.-D. Fu) was used to infect 50 ml of Sf9 cells and was purified as previously described (55). SF2/ASF was cloned into the pFastBac1 insect expression vector (Invitrogen) after PCR amplification from Weri-1 poly(A)+ RNA by using primers 5'-GATCGGATCCATATGTCGGGAGGTGGTGTG-3' and 5'-CTAGGAATTCTTATGTACGAGAGCGAGATCTGCTATG-3'. Baculovirus containing the SF2/ASF cDNA was used to infect Sf9 insect cells. Purification of SF2/ASF from 50 ml of SF9 cell culture was performed as previously described (55). The activity of the SF2/ASF protein varied between preparations and was optimized for each experiment.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify the proteins cross-linking to exon N1, immunoprecipitation experiments were performed after cross-linking. The immunoprecipitated proteins were resolved by SDS-PAGE. The 35- and 38-kDa proteins that cross-link to the RNA labeled at EX3 were immunoprecipitated with antibodies to hnRNP A1 (Fig. 3A, lanes 5 and 13). Thus, the most prominent bands in this size range are likely hnRNP A1 and its longer isoform, hnRNP A1B (5). Antibody to hnRNP H immunoprecipitates the 55-kDa band (Fig. 3A, lanes 4 and 12), and antibody to hnRNP F immunoprecipitates a lighter 53-kDa band (data not shown). Given its activity as a splicing enhancer, it was likely that the exonic element bound to an SR protein. Indeed, immunoprecipitation of the cross-linked proteins identified SF2/ASF as another component of the 33- to 35-kDa band. This band was lighter than that of the comigrating hnRNP A1. This could be due to a lower stoichiometry of binding, poorer cross-linking, or poorer immunoprecipitation with this antibody. The immunoprecipitation experiments were repeated with RNA labeled at position EX8. The proteins cross-linking in the vicinity of this radiolabel were again immunoprecipitated with antibodies to hnRNP A1, SF2/ASF, and hnRNP H (Fig. 3A, lanes 19 to 21 and 24 to 26).
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The binding of SF2/ASF and hnRNPs A1, H, and F to exon N1 was not unexpected. SF2/ASF and hnRNPs A1 and H have been implicated in splicing regulation through exonic elements (9, 15, 18, 27, 47, 49, 50, 57). Moreover, examination of the exon N1 sequence and the nucleotides just upstream reveals the sequence AG/GAGGA. This is identical to an SF2/ASF-dependent exonic enhancer identified in a functional SELEX screen by Liu et al.(33) (Fig. 4). In the c-src pre-mRNA, this SF2/ASF motif is not wholly exonic, overlapping two nucleotides of the 3' splice site. A sequence starting slightly downstream is an 8-of-10-nucleotide match to AGGACRRAGC, another SF2/ASF high-affinity binding site identified by SELEX (49). Interestingly, in the intron upstream of these SF2/ASF binding motifs is an element with a 7-of-8-nucleotide match to the SC35 ESE consensus sequence, GRYYMCYR (R = A/G, Y = C/U, and M = A/C) (32). Overlapping all of these SR protein binding motifs is a series of sequences that each show a 4-of-6-nucleotide match to an hnRNP A1 binding sequence, UAGGGA/U (4). Also, within the exon, there are sequences showing 3-of-4-nucleotide match to the GGGA element identified as an hnRNP H/F binding site (8).
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To demonstrate that these changes in splicing pattern were specific to SR proteins, the RNA binding protein, La, was added to Weri-1 splicing reaction mixtures. The addition of La did not change the splicing of either BS303 or BS304 at any concentration tested (Fig. 5C). These experiments were repeated multiple times in different extracts and with different preparations of SR proteins. Although the overall level of splicing varied between experiments, the same relative stimulation activities of SF2/ASF and SC35 were always observed.
Weri-1 extract contains both SF2/ASF and SC35, as determined by Western blotting (data not shown). S100 extracts are depleted for SR proteins and can be supplemented with individual SR protein family members to activate splicing (29). This complementation assay was used to confirm whether either SF2/ASF or SC35 alone can activate c-src splicing. Increasing concentrations of SF2/ASF were added to Weri-1 S100 reaction mixtures containing either BS303 or BS304. In reaction mixtures containing BS303, SF2/ASF activated splicing pathways leading to both inclusion and skipping of exon N1 (Fig. 6, lanes 3 and 4). In the reactions containing BS304, SF2/ASF activated splicing by the exon-skipping pathway more strongly than N1 exon inclusion (Fig. 6, lanes 7 and 8). The addition of SC35 to Weri-1 S100 activated splicing through both pathways regardless of whether the substrate was BS303 or BS304. These results confirm that the exonic enhancer element affects splicing activation by SF2/ASF.
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PTB is also known to repress N1 splicing (13, 17). Adding PTB instead of hnRNP A1 to the reactions gave similar results. In reactions activated by either SF2/ASF or SC35, PTB repressed splicing of exon N1 (Fig. 7B, lanes 3, 4, 7, and 8). N1 splicing was most easily repressed by PTB in reactions containing BS304 and SF2/ASF. Thus, hnRNP A1 and PTB can each repress N1 exon splicing that has been stimulated by SR proteins. In neither case is the 5' element of the N1 exon required for the repression activity.
hnRNP A1 and PTB use different mechanisms to repress exon N1 splicing. Repression of N1 splicing by PTB requires PTB binding sites upstream and downstream of the exon (13, 17). To demonstrate a difference in activity between PTB and hnRNP A1, we compared the effect of these proteins on BS7 RNA containing all the PTB binding sites with that on BS27 RNA which lacks the upstream sites and is not repressed by PTB. The addition of PTB to reactions containing BS7 repressed splicing as expected (Fig. 8, lanes 2 to 5). Also as expected, PTB did not repress the splicing of BS27 (Fig. 8, lanes 7 to 10). The addition of hnRNP A1 repressed splicing of BS7 (Fig. 8, lanes 12 to 15). In contrast to PTB, hnRNP A1 also repressed splicing of BS27, although at higher concentrations of protein (Fig. 8, lanes 17 to 20). Thus, hnRNP A1 and PTB have different sequence requirements in repressing N1 splicing.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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hnRNP A1 and SR proteins affect N1 splicing in vitro. Cross-linking and immunoprecipitation experiments identified the hnRNPs A1, A1B, F, and H and the SR protein SF2/ASF as binding to the exonic sequence. In spite of extensive previous characterization, SR proteins and hnRNP A1 have not previously been shown to be involved in the regulation of N1 splicing. The effect of SF2/ASF and hnRNP A1 on N1 splicing were examined by adding purified protein to splicing reactions. SF2/ASF and another SR protein, SC35, each stimulate N1 splicing in neuronal nuclear extract, and each is sufficient to activate splicing in S100 extract. SF2/ASF enhanced N1 splicing over the alternate pathway more specifically than did SC35. The SF2/ASF activity was also more dependent on the ESE than was SC35.
In contrast to SF2/ASF, hnRNP A1 repressed splicing of c-src transcripts when added to in vitro splicing reactions. As observed in other systems, SF2/ASF and hnRNP A1 play antagonistic regulatory roles. However, repression of N1 splicing by A1 did not require the exonic element. There are many potential A1 binding sites elsewhere in the src transcript and in most other transcripts that we have looked at. Thus, it may be a negative factor in many different splicing events.
hnRNP A1 and PTB have generally been studied in separate systems. Here, both hnRNP A1 and PTB were found to repress N1-splicing reactions activated by SR proteins, but this activity differed for the two proteins. A c-src pre-mRNA that has no PTB binding sites upstream of N1 is no longer repressed by PTB but is still repressed by hnRNP A1. Moreover, PTB was more specific than hnRNP A1 in inhibiting the N1 inclusion pathway over the exon-skipping pathway. The different sequence requirements and patterns of inhibition of these two proteins likely reflect different mechanisms of action. In other experiments, the two proteins in combination did not seem to synergize in silencing splicing (data not shown).
Models for N1 splicing regulation. Previous results showed that PTB binding to the introns upstream and downstream of N1 prevents splicing in nonneuronal cells, most likely by looping out the RNA and blocking spliceosome assembly (Fig. 9). In neuronal cells, PTB dissociates from the RNA at the 3' splice site, and this may allow proteins binding to the downstream enhancer sequence to activate splicing (17). We now need to fit hnRNP A1 and SF2/ASF into this model.
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One interesting feature of this exonic element in N1 is how closely juxtaposed the elements are. In most other systems, ESEs bound by SR proteins and exonic splicing silencers bound by hnRNP A1 are adjacent but separable and not overlapping sequences. In the N1 exon, the binding sites for all of these proteins are within the same 11 nucleotides. So far, we have not been able to separate the binding sites for the different proteins through mutagenesis or through different site-specific labels, as was done with the DCS (35). For the DCS complex, the binding of the individual proteins to the DCS RNA is strongly affected by the other proteins in the complex (35). Similarly, the PTB binding sites in the DCS also stimulate PTB binding upstream (17). We have not observed these cooperative effects in the binding of the proteins to the ESE. Likewise, purified PTB does not seem to affect the binding of purified hnRNP A1 or SF2/ASF to small RNA probes containing all the binding sites. It is thus not clear yet whether the ESE proteins bind in a single complex to the exon or in separate complexes, although because of the closeness of the binding sites, we favor the idea of separate complexes.
SF2/ASF binds more prominently in Weri-1 extracts, where N1 is spliced, than in HeLa extracts, where it is repressed. Similarly, binding of the A1B isoform of hnRNP A1 is more pronounced in the Weri-1 extract than in the HeLa extract. In vivo and in vitro studies have shown differences in the RNA binding and alternative splicing activities of hnRNP A1 and hnRNP A1B (39, 53). Thus, hnRNP A1B may be a factor in the binding of SF2/ASF and the derepression of N1 splicing in Weri-1 nuclear extracts, but this possibility will require further investigation.
In other systems, hnRNP H acts as either a positive or negative splicing regulator. It binds to enhancer elements in HIV type 1 (HIV-1) tev exon 6D and in the thyroid hormone receptor transcript, whereas its binding in HIV-1 tat exon 2, to exon 7 from the rat ß-tropomyosin pre-mRNA, and to the negative regulator of splicing element from Rous sarcoma virus represses splicing (9, 15, 20, 26, 27). hnRNP F has been shown to interact with the cap binding complex and with the carboxyl-terminal domain of RNA polymerase II, and it is thought that the expression level of hnRNP F plays a role in the regulated use of alternative polyadenylation sites in lymphocytes (21, 51, 54). In the c-src system, hnRNPs H and F are components of the regulatory complex which forms on the DCS. Antibody inhibition and depletion experiments suggest a positive role for these proteins in N1 splicing (16, 40). However, it could not be shown in these earlier studies that the effect of hnRNP H was dependent on the intronic binding site (16). This may be due to the presence of the exonic binding site described here. The addition of purified hnRNP H to splicing reactions slightly reduces splicing activation by added SF2/ASF (data not shown). In general, hnRNP H had a much smaller effect on splicing by either pathway than did the SR proteins or hnRNP A1. Elucidating the role of the exonic H/F proteins will require further study.
In vivo experiments showed that the N1 exonic enhancer and the downstream enhancer work in combination to activate splicing. In experiments with individual elements, the exonic enhancer was weaker than the full downstream enhancer but stronger than a single copy of the core DCS element. In these in vitro experiments, the N1 exonic enhancer is relatively weak compared with other well-studied exonic enhancers. It seems to be a feature of alternative splicing regulatory systems that multiple elements and factors are combined to produce strong effects. A challenge for the future is to understand how these different exonic and intronic components of the regulation work together to produce such highly specific control.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grant GM R01 49662 to D.L.B. D.L.B. is an associate investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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Present address: Rigel, Inc., South San Francisco, CA 94080.
Present address: UCLA ACCESS, Los Angeles, CA 90095.
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