In the present study, we show that a subset of pre-mRNA splicing factors including 17S U2 associate with WW domains derived from two spliceosomal proteins, CA150 and FBP11. We also demonstrate that CA150 is critical for in vivo splicing and can modulate exon selection. According to our observations, we assume that WW domain-containing proteins may nucleate the assembly of splicing factors at the 3′ splice site of the intron, which in turn facilitates splicing or exon selection (Fig. ). Since these factors also interact with phosphorylated RNA Pol II mainly via their FF domains (19
), they likely serve a role in regulating cotranscriptional pre-mRNA splicing (Fig. ).
FIG. 7. Model for a role of WW/FF domain-containing factors in pre-mRNA splicing. WW/FF-proteins bind to the phosphorylated CTD of the largest subunit of RNA Pol II (20) and recruit splicing factors such as 17S U2 snRNP, SF1, and U2AF to facilitate 3′ (more ...)
The WW domains of CA150 and FBP11 showed potential association with a number of factors in vitro, including spliceosomal components that recognize the intron elements near the 3′ splice site as well as several additional proteins (Table ). In particular, we detected the functional 17S U2 snRNP (Fig. ) consisting of the 12S U2 core and the heteromeric complex SF3a/b (6
). Additionally, Western blotting revealed SF1 and U2AF65
(Fig. ). SF1 may bind directly to the WW domains via its proline-rich motif (19
), whereas the nature of U2AF interactions with WW domains is unclear. U2AF65
can interact via its noncanonical RRM3 with SF1 or component p155 of SF3b (42
), suggesting that the association between U2AF and WW domains can be mediated by either factor, although direct interactions still remain possible. At present, it is unclear whether the identified WW-associating factors concomitantly assemble into a large complex or only form subcomplexes that in turn interact individually with a WW domain. SF1 indeed interacts with U2AF in a transient complex to bind to the 3′ end of the intron (5
). Moreover, a report that the 17S U2 snRNP associates with substoichiometric levels of U2AF subunits (50
) suggests preassembly of subcomplexes prior to splicing. Evidence of subcomplexes is also supported by the mutually exclusive interaction of SF1 with U2AF and a 17S U2 snRNP protein and by the sequential binding of SF1 and U2 to the branch site (48
). Therefore, WW domain-containing proteins probably serve as a platform for exchange of these early splicing factors on 3′ intron sequences.
Depletion of CA150 had no effect on in vitro splicing but reduced efficiency of in vivo splicing (Figs. and ). This result implies that CA150 serves a role necessary for in vivo but not for in vitro splicing. The splicing of pre-mRNA in vivo occurs in concert with transcription (4
). RNA Pol II conducts cotranscriptional splicing possibly via the interaction of the CTD with splicing factors such as SR proteins and snRNPs (4
). Our data indicate a critical role of CTD-interacting CA150 in splicing in vivo, emphasizing a very close relationship between transcription and pre-mRNA splicing. Moreover, WW and FF domain-containing CA150 polypeptides activated in vivo splicing or exon inclusion but failed to drive alternative 5′ splice site selection (Fig. and ). The critical role of the WW2 domain in activation of exon inclusion is apparently consistent with the assumption that WW domain proteins recruit the factors that facilitate recognition of elements near the 3′ splice site (Table and Fig. ). However, the possibility remains that WW-associating factors bind to exonic elements, thereby altering splicing outcome. Finally, CA150 has been implicated in transcriptional regulation via specific promoters (43
), and therefore it may be useful to investigate whether CA150 associates with the transcriptional machinery and concomitantly regulates splicing in a gene-specific manner.
We also identified several hnRNP proteins in association with WW domains (Table ). The hnRNP M protein contains an unusual repeat region rich in methionine and arginine residues that resembles a component of the cleavage stimulation factor (CstF) involved in polyadenylation (13
). Since hnRNP M is transiently associated with the pre-mRNA at early stages of spliceosome assembly (27
), it may thus act concurrently with several other WW domain-associated splicing factors. We observed that hnRNP M overexpression enhances exon 6 inclusion of the β-TM (data not shown), consistent with the role predicted for WW-associated splicing factors. Several different functions have been assigned to hnRNP F, including activation of c-src
exon N1 inclusion (10
). It will be interesting to investigate whether hnRNP F functions in conjunction with WW domains and/or their associating factors in alternative exon selection or in any other steps of mRNA processing.
Hsp70 proteins function as molecular chaperones to assist protein folding. Three members (HSP70, HSP71, and GRP78) were previously identified as H complex components that bind to pre-mRNA prior to spliceosome assembly (53
). These three proteins were identified in the WW column eluates (Table ), consistent with a role for WW-associating factors in early splicing complex formation. It is noteworthy that the DnaJ domain-containing protein SPF31 was recently shown to interact with the 17S U2 snRNP (50
). Our data also revealed different DnaJ homologs that associate with CA150 (Table ) or FBP11 (data not shown). The DnaJ cochaperones can regulate the activity of Hsp70 with respect to substrate binding and ATP hydrolysis (21
). It is thus possible that these chaperone factors are involved in protein folding or remodeling during spliceosome assembly.
Finally, we identified two additional WW domain- or splicing factor-associated proteins with potential chromatin remodeling activities (Table ). ATP-dependent chromatin assembly factor acts on histone deposition into periodic nucleosome arrays by hydrolyzing ATP (17
). Another protein identified is a novel member of the family of chromatin remodeling, helicase, and DNA-binding proteins that is a part of the nucleosome remodeling and histone deacetylation (NuRD) complex (41
). CA150 represses RNA Pol II transcription by inhibiting transcriptional elongation via its first two WW domains (44
). Thus, it will be interesting to investigate whether the NuRD complex plays any role in CA150-mediated transcriptional inhibition. Moreover, the spliceosome-associated kinase hPRP4 was recently shown to form a complex with the N-CoR histone deacetylase complexes (16
), suggesting a possible link between pre-mRNA splicing and chromatin remodeling during mRNA synthesis. Here, our data reveal a possibility that WW domains coordinate these two events; thus, whether WW-containing proteins influence exon selection also by modulating the rate of transcription remains to be determined.