Wednesday, December 4, 2024
12/4/2024
PROJECT SUMMARY The mechanisms by which eukaryotes regulate gene expression are important for understanding many complex biological phenomena including human diseases. Prevention and treatment of such diseases have been and will continue to be improved by basic knowledge of gene regulation, especially because molecular mechanisms of transcriptional initiation are highly conserved in eukaryotic organisms ranging from human to yeast. This proposal will continue to investigate basic issues concerning molecular mechanisms of cleavage/ polyadenylation (3’ end formation), and mRNA stability in yeast, by combining molecular genetic, and functional genomic approaches. It will take advantage of our novel methodologies for measuring half-lives, structure (DREADS via chemical probes), protein binding (CLIP-READS), and poly(A) length (A-READS) of individual mRNA 3’ isoforms. Our previous work identified strong and unexpected mechanistic links between transcriptional elongation, cleavage/polyadenylation, and mRNA decay. Novel findings include a nucleotide- level link between elongation and cleavage/polyadenylation, condition-specific 3’ isoform half-lives and mRNA stability elements, and a compensatory mechanism in which the rate of cleavage/polyadenylation in the nucleus is linked to 3’ isoform stability in the cytoplasm. The proposed experiments represent a highly integrated approach to study the generation, structure, stability, and regulation of 3’ isoforms on a transcriptome scale. In addition to our previous experiments that involved relatively stable cytoplasmic mRNAs, the new studies will use a metabolically labeling, pulse-chase approach to analyze newly-synthesized RNAs that are generated in the nucleus prior to transport to the cytoplasm. First, for polyadenylation, we will (A) define the sequence requirements for 3’UTRs by generating large libraries of yeast cells each of which contains a genetically modified 3’UTR, B) use the 3’UTR cell libraries to address the mechanism(s) by which polyadenylation is restricted to the 3' UTR. C) identify factors that are responsible for the wild-type poly(A) pattern, D) determine the mechanistic basis for how individual proteins involved in transcriptional elongation, cleavage/polyadenylation, and mRNA decay affect poly(A) profiles of some genes but not others, and E) elucidate 3'-isoform variation and regulation of poly(A) tail length. Second, for studies of mRNA stabilization/destabilization elements and half-lives of 3' isoforms, we will A) perform RNA structural analysis during the degradation process, B) identify proteins that affect/mediate the large differences in mRNA isoform stabilities C) perform directed genetic experiments to address how secondary structure affects mRNA stability, D) elucidate our recently discovered compensatory mechanism between cleavage/polyadenylation and mRNA decay, E) identify isoform-selective mRNA-binding proteins, and F) investigate the mechanism of condition- specific isoform half-lives and stability elements that we recently identified. Overall, the proposal will answer fundamental questions about the interlinked processes of transcription, polyadenylation, and mRNA stability.
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