Investigating the cotranscriptional regulation of pre-mRNA splicing and 3'-end processing.
Published: 2004
Language: eng
Number of pages: 250
Description: Transcriptional activators play an important role in the assembly of the transcriptional apparatus at the promoter regions of genes. We examined whether activators participate in the coupling of transcription with pre-mRNA processing, as well. Strong activation domains resulted in higher levels of splicing and cleavage compared to weak activation domains when targeted to the promoter of reporter genes. Truncation of the CTD abrogated this effect indicating that the CTD is involved in mediating the effect of strong activators on efficient processing. Further exploration of this mechanism revealed that splicing factor PSF binds preferentially to strong activation domains, and stimulates splicing and cleavage in vivo, in a CTD dependent manner. Therefore, PSF likely mediates the effect of a strong activator on efficient processing, whereby strong activators facilitate the association of PSF with the elongation apparatus. Our findings therefore implicate both transcriptional activators and PSF in cotranscriptional splicing and 3 '-end formation.The production of a messenger RNA (mRNA) is a complex process that involves many concerted steps, including the processing of the primary transcript, or precursor mRNA (pre-mRNA). Processing involves capping, 3' -end cleavage and polyadenylation, and splicing of introns from within the pre-mRNA. Pre-mRNAs are transcribed by RNA polymerase II (pol II), and it has been found that pre-mRNA processing is coupled to transcription by pol II, facilitating efficient and coordinated production of mature mRNA. Here I report the results of investigations of cotranscriptional splicing and 3'-end formation of pre-mRNAs.The carboxyl-terminal domain (CTD) of pol II is a highly-repetitive sequence unique to pol II that plays key roles in coupling gene expression events leading to the production of mRNA. We examined the CTD requirement for processing of different pre-mRNAs by exploring the effect of CTD mutations on splicing and cleavage of reporter genes in mammalian cells. We found that the length, rather than the type of CTD repeats, can be the major determinant in the efficient processing of pre-mRNA substrates. Furthermore, our results suggest that the requirement for the CTD in pre-mRNA processing is dependent on sequences within the gene itself. The degree of CTD-dependence therefore appears to be pre-mRNA specific.
Notes: Adviser:  Benjamin J. Blencowe.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2218.