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Biology Articles » Molecular Biology » RNA polymerase II and the integration of nuclear events » Splicing and transcription

Splicing and transcription
- RNA polymerase II and the integration of nuclear events

Splicing of mRNA precursors takes place in a large macromolecular complex called the spliceosome, which is composed of small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP proteins including members of the serine/arginine-rich (SR) protein family (for reviews, see Moore et al. 1993; Kramer 1996; Manley and Tacke 1996). Although cytological studies have suggested that splicing can occur cotranscriptionally (see, for example, Beyer and Osheim 1988; Bauren and Wieslander 1994), and factors required for splicing can be found localized at sites of active transcription (Zhang et al. 1994), functional coupling between transcription and splicing is not obligatory because splicing can be reconstituted in vitro with pretranscribed RNA and splicing-competent cell extracts. Both biochemical and in vivo studies have provided support for the existence of functional interactions between RNAP II, especially the CTD, and the splicing apparatus (see Figs. 1 and3). RNAP IIO, but not RNAP IIA, has been found to associate with splicing factors, and this isoform has also been detected in active spliceosomes (Chabot et al. 1995; Mortillaro et al. 1996; Yuryev et al. 1996; Kim et al. 1997). Antibodies directed against the CTD and CTD peptides have been shown to inhibit splicing in vitro (Chabot et al. 1995; Yuryev et al. 1996). Like capping, splicing (and polyadenylation) of RNAs transcribed in transient transfection assays by CTD-truncated RNAP II was inefficient (McCracken et al. 1997b), and overexpression of phosphorylated CTD peptides was shown to inhibit splicing in cultured mammalian cells (Du and Warren 1997). These observations provided the initial evidence that the hyperphosphorylated CTD of elongating RNAP II may function in splicing, perhaps serving as a platform upon which processing factors bind, thus helping to promote efficient and accurate splicing by targeting necessary factors to transcription sites.

Microscopic observations in mammalian cells have provided visual support for this targeting model (Misteli and Spector 1999). Following activation of a reporter gene in cells expressing either full-length or CTD-truncated RNAP II as the only source of active enzyme, sites of accumulation of both the newly synthesized reporter transcripts and splicing factors were simultaneously visualized by immunohistochemistry techniques. Although both sites colocalized well in cells expressing wild-type RNAP II, the transcription sites did not colocalize above random levels with either SR proteins or snRNP particles in cells expressing the CTD-truncated RNAP II. Estimation of splicing levels by in situ hybridization with short exon-spanning probes suggested that truncation of the CTD prevented accumulation of spliced products despite the presence of significant amounts of unspliced pre-mRNAs. These results support the idea that the CTD is required for targeting splicing factors to transcription sites and that this can be important for efficient splicing. The same authors also showed by immunoprecipitation experiments using transiently transfected cells expressing wild-type or mutant SR proteins that the RS domain of several SR proteins was necessary and sufficient for associating with the phosphorylated RNAP II largest subunit, although it is not clear whether these associations were direct or indirect.

The above findings are consistent with the view that the CTD is required for targeting splicing factors to transcription sites to ensure efficient splicing. However, is the efficient splicing in vivo due only to the increased local concentration of processing factors? Or does the CTD participate more directly in the actual splicing reaction? Like in other processing reactions such as capping or polyadenylation (see below), recent experiments have shown that RNAP II also plays a direct and active role in splicing in vitro in the absence of transcription (Fig. 3; Hirose et al. 1999). Purified RNAP IIO was found to strongly activate the splicing of several different pre-mRNAs in reconstituted splicing assays. RNAP IIO significantly increased formation of spliceosomal complexes, and the pre-spliceosomal A complex was notably increased at very early times of the reaction. These results indicate that RNAP IIO stimulates splicing by accelerating the rate of one of the first steps in spliceosomal assembly, probably by facilitating in some way binding of U1 and/or U2 snRNP particles to the pre-mRNA 5' splice site and/or branch site, respectively. RNAP IIA, on the other hand, was capable of inhibiting splicing of some but not all of the pre-mRNA substrates tested, apparently by disrupting early pre-splicing complexes. The CTD was necessary for these effects on splicing because the CTD-less form of RNAP II (IIB) was without significant effect, but, unlike with capping and polyadenylation (see below), was not sufficient. These results provide additional support for the idea that RNAP II not only controls spatial distribution of pre-mRNA processing factors in the cell nucleus to couple transcription to processing, but also can function directly with splicing factors to enhance the efficiency of splicing. Furthermore, this study also suggested that differential CTD phosphorylation has the potential to play a significant role in splicing regulation.

The concept of a physical and functional coupling between transcription and splicing has been extended by the finding that promoter structure can contribute to splice site selection (Cramer et al. 1997, 1999). One of the exons in the fibronectin (FN) gene, called EDI, has long been known to be subject to alternative splicing (inclusion or exclusion). Inclusion of this exon depends on the presence of specific cis-acting sequences located within EDI that function by binding specific SR proteins (Lavigueur et al. 1993). Various reporter constructs containing EDI under the control of different promoters were transiently transfected into human cultured cells and the extent of EDI inclusion was measured by RT-PCR and Northern blot analysis. The authors found that varying the promoter resulted in significant changes in the ratio of EDI inclusion versus exclusion (Cramer et al. 1997). Important controls showed that the effects were not due to differences in promoter strength or to differences in the site of transcription initiation, but rather to the identity of the promoter (Cramer et al. 1999). The authors further demonstrated that overexpression of specific SR proteins markedly stimulated EDI inclusion, but the effects of these SR proteins depended on the promoter from which the transcript was produced. These results have suggested that the transcription machinery can affect the recruitment of specific SR proteins to exonic cis-acting elements on the newly transcribed RNA.

How might promoter structure modulate alternative splicing? One possible explanation is that the precise nature of the initiation complex assembled on a particular promoter may affect the extent of phosphorylation of the RNAP II-CTD, and this in turn modulates the recruitment by the CTD of specific SR proteins to cis-acting regulatory sequences in the nascent RNA. Alternatively, different degrees of phosphorylation could affect the ability of the CTD to participate directly in early spliceosome assembly, as discussed above. Another possible mechanism, not related to CTD phosphorylation, involves recruitment of promoter-specific factors that may in turn function with the CTD to influence splicing.

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