Alternative splicing is the major contributor to protein diversity. Recent findings justify a renewed interest in alternative splicing. Estimated to affect nearly 60 percent of human genes, alternative splicing is more the rule than the exception, and mutations that affect alternative splicing–regulatory sequences are a widespread source of human disease. Indeed, many genetic disorders and cancers are caused by mutations in coding sequences that were previously overlooked owing to the fact they do not affect amino acid sequences but instead alter the function of alternative splicing–regulatory sequences. In addition, alternative splicing is particularly important in the development of the nervous system. Finally, alternative splicing regulation not only depends on the interaction of splicing factors with their pre-mRNA target sequences but, like other pre-mRNA processing reactions, is coupled to RNA pol II transcription.
Our laboratory investigates the mechanisms of coupling between transcription and alternative splicing and the coupling's regulatory roles in human cells. Both transcription and splicing are highly complex processes, involving thousands of protein factors, RNA molecules, and DNA sequences. Control of alternative splicing by a promoter was originally discovered in our laboratory and later confirmed by others. Two non-exclusive mechanisms have been proposed to explain the promoter effect. On the one hand, the promoter might affect the rate of pol II elongation, influencing in turn the timing of cotranscriptional splicing. On the other hand, the promoter might affect the recruitment to the transcribing gene of splicing factors or factors acting simultaneously on both transcription and splicing.
Pol II Elongation Affects Alternative Splicing
Low rates of pol II elongation or internal pauses for elongation would favor the inclusion of alternative cassette exons, whereas a highly elongating pol II, or the absence of internal pauses, would favor exclusion of these types of exons. We are using the fibronectin EDI cassette exon as a model to study alternative splicing mechanisms. The manner in which the elongation rate affects alternative splicing of EDI is a consequence of EDI's pre-mRNA sequence. EDI exon skipping occurs because the 3′ splice site of the upstream intron is suboptimal compared with the 3′ splice site of the downstream intron. If the polymerase pauses anywhere between these two sites, only elimination of the upstream intron can take place. Once the pause is passed or the polymerase proceeds, the only option for the splicing machinery is to eliminate the downstream intron, which leads to exon inclusion. A highly elongating pol II, or the absence of internal pauses, would favor the simultaneous presentation of both introns to the splicing machinery; in such a situation, the stronger 3′ splice site of the downstream intron outcompetes the weaker 3′ splice site of the upstream intron, resulting in exon skipping. A direct proof for the elongation mechanism in the transcriptional control of alternative splicing in human cells was provided when we used a mutant form of pol II (called C4) with a lower elongation rate. This slow polymerase mutant stimulates the inclusion of the fibronectin EDI exon four-fold, confirming the hypothesis of an inverse correlation between elongation rate and inclusion of this alternative exon.
Factor Recruitment to Pol II and Alternative Splicing
The recruitment mechanism may be in part due to the ability of the carboxy-terminal domain (CTD) of RNA polymerase II (pol II) to bind and “piggyback” some of the processing factors in a complex known as the “mRNA factory.” Previous studies have linked CTD with co-transcriptional pre-mRNA processing such as capping and 3′ end formation, but little is known about CTD function in alternative splicing. We used various pol II CTD mutants and fibronectin reporter minigenes to study the role of the CTD in the regulation of alternative splicing. We found that the CTD is required for the inhibitory action of the serine-arginine-rich protein SRp20 on the inclusion of the EDI cassette exon into the mature mRNA in an elongation-independent manner. Our results suggest that the CTD promotes exon skipping by recruiting SRp20 and that elongation and factor recruitment contribute independently to the transcriptional control of alternative splicing.
Conclusions and Perspectives
Elongation and factor recruitment may contribute independently or in a concerted way to the transcriptional control of alternative splicing. We are currently investigating three different mechanisms that control alternative splicing through the kinetic coupling.
1. UV radiation affects alternative splicing of many genes, including the upregulation of the pro-apoptotic isoform of Bcl-x, an expected physiological response to DNA damage. The UV effect does not require p53, is not caused by damage of the DNA template in cis and only affects co-transcriptional splicing. UV light causes hyperphosphorylation of the carboxyterminal domain of RNA polymerase II, which is responsible for alteration in alternative splicing through the inhibition of transcriptional elongation. Pol II mutants engineered to mimic the hyperphosphorylated state duplicate the effects of UV light on alternative splicing.
2. The chromatin context affects Pol II elongation rates and, in turn, alternative splicing. We discovered the mechanism by which membrane depolarization in nerve cells affects alternative splicing of the NCAM pre-mRNA. Depolarization promotes local heterochromatinization around the alternative exon on the NCAM gene, which creates roadblocks to Pol II elongation.
3. In our search for new tools to control alternative splicing at the chromatin level, we found that small interfering RNAs (siRNAs) targeted at the intron located downstream of an alternative exon affect alternative splicing through a mechanism known as transcriptional gene silencing (TGS). The intronic siRNAs trigger heterochromatinization on DNA target sequences by causing histone H3 Lys9 dimethylation. The effects of intronic siRNAs on alternative splicing are not caused by conventional post-transcriptional gene silencing (PTGS), are abolished by inhibitors of histone deacetylation and methylation, and depend on the presence of the protein Ago2, known to be necessary for TGS.
Last updated August 2010
