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Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Shawntel Okonkwo - University of California, Los Angeles
Co-Author(s): Stephen Douglass, University of California Los Angeles, Los Angeles, CA; Felizza Gunderson, University of California San Diego, La Jolla, CA; Tracy Johnson, University of California Los Angeles, Los Angeles, CA;
The step-wise assembly of the spliceosome onto pre-mRNA occurs co-transcriptionally, while the nascent transcript is synthesized from RNA Polymerase II. Although the co- transcriptional nature of splicing has been well established, the mechanisms underlying the coupling and coordination of these reactions have not been thoroughly elucidated— particularly in the context of the dynamic chromatin environment. We previously discovered that the major yeast histone acetyltransferase (HAT), GCN5 of the SAGA complex, demonstrates genetic interactions with MSL1 and LEA1—two U2 small nuclear ribonucleoprotein particle (snRNP) components of the spliceosome. Additionally, we observed that Gcn5 and its acetylation activity are required for proper co-transcriptional recruitment of the spliceosome in yeast. Specifically, U2 snRNP association with the branch-point of the pre-mRNA substrate and subsequent spliceosomal rearrangements are sensitive to Gcn5-dependent acetylation. Intriguingly, Gcn5-dependent acetylation is enriched at the promoter, but the HAT is found throughout the intron-containing genes, DBP2 and ECM33. Deletion of histone deacetylases (HDACs) HOS2 and HOS3 reveals H3K9 and H3K14 acetylation throughout the gene bodies and abnormal persistence of U2 snRNP factors near the branch-point region of the pre-mRNA—with aberrant recruitment of downstream splicing factors. While Gcn5-dependent acetylation is important for the fate of spliceosomal rearrangements, the mechanism underlying this result is not yet clear. We employed RNA-seq in S.cerevisiae to uncover putative mechanistic insights into how Gcn5-dependent acetylation can affect pre-mRNA splicing. We prepared libraries from the following yeast strains: wildtype, gcn5 and H39-16 (residues 9-16 deleted from histone H3 N-terminal tail). We predicted that genes for which splicing is affected in both gcn5 and H39-16 will represent a defect specific to absence of Gcn5-dependent histone acetylation. Surprisingly, our results support an unexpected role for the histone acetyltransferase in amending the splicing efficiency of poorly spliced genes in a manner that is regulated by the competition of ribosomal protein gene pre-mRNA transcripts for the limited spliceosomes. The orchestration between HAT and HDAC activity may also be important for recruitment of factors that bind to chromatin and recruit the spliceosome or for tuning transcription elongation rates for proper stepwise recruitment of the spliceosome on the pre-mRNA substrate. Our future experiments will explore the precise mechanism for how Gcn5 is affecting the co-transcriptional recruitment of the spliceosome to the affected genes.
Abstract.docxFunder Acknowledgement(s): NSF GRFP
Faculty Advisor: Tracy Johnson, tljohnson@ucla.edu
Role: RNA isolation and library preparation for RNA-seq; Analysis and visualization of RNA-seq data; Validation of RNA-seq data by RT-PCR
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