Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Shawntel Okonkwo - University of California, Los Angeles
The production of mature and functional RNA transcripts in all eukaryotes is largely in part dependent upon catalytic intron removal by the macromolecular spliceosome complex—a dynamic process more commonly referred to as splicing. In yeast, splicing of pre-mRNA molecules is co-transcriptional—a phenomenon by which intron removal of nascent RNA molecules occurs simultaneously with the process of transcription by RNA Polymerase II (RNAPII) and the entire host of associated transcription machinery. Co-transcriptional splicing is highly subject to spatiotemporal control (such as transcription elongation rate) as well as modulation by the RNAPII C-terminal domain (CTD). The CTD consists of a unique 26-tandem heptapepetide repeat sequence of amino acid residues that are subject to biochemical post-translational modifications such as phosphorylation, isomerization and dephosphorylation. It is hypothesized that these modifications may combinatorially function as a indicator or director of polymerase activity by collectively operating as a dynamic code for the regulation of transcription events such as initiation, elongation and polymerase pausing—as well as the aforementioned co-transcriptional splicing. Unfortunately, there is an insufficient amount of literature elucidating the role of the kinases that mediate these modifications in the co-transcriptional processes themselves—particularly in the context of pre-mRNA splicing. Recently, our lab has demonstrated a role for Bur1/2, a major CTD kinase responsible for transcription elongation and the bulk of Ser2 phosphorylation, in pre-mRNA splicing via spliceosome formation and snRNA interactions. Based on this previous evidence, we hypothesize that the Bur1/2 complex plays dual roles in co-transcriptional and transcription independent gene regulation. To these aims, we plan to employ RNA-seq on WT and Bur mutant yeast strains to determine if Bur association with intron-containing genes is important for their coupled splicing. We predict to see greater amount of retained introns in the mutant relative to the wildtype condition. This result can be followed up by ChIP-seq on Bur proteins and other CTD kinases to ascertain specific and differential abundance of Bur proteins on these intron-containing genes. We also plan to perform in vivo studies with RT-PCR on intron containing genes in Bur mutant backgrounds to visualize splicing defects. In conclusion, Bur1/2 may be playing a role in co-transcriptional regulation.Not Submitted
Funder Acknowledgement(s): This research is supported by the NSF LSAMP Bridge to the Doctorate Fellowship awarded to Shawntel Okonkwo.
Faculty Advisor: Tracy Johnson, email@example.com