Analysis of the Pumilio Repression Mechanism and its Impact on the Transcriptome

University of Minnesota Ph.D. dissertation. May 2021. Major: Biochemistry, Molecular Bio, and Biophysics. Advisors: Aaron Goldstrohm, Anja Bielinsky. 1 computer file (PDF); xiii, 198 pages.The expression of genes in an organism is controlled by many methods. One of the most important means of regulation is post-transcriptional control of messenger RNA (mRNA). RNA binding proteins (RBPs) are crucial for this process. Here we describe the characterization of a functional domain within the Drosophila RBP Pumilio (Pum) and examine the effects of Pum on the transcriptome.Pum proteins are a highly conserved family of RBPs that control the expression of mRNA by binding to specific motifs in the 3’-UTR of transcripts. Through associations with other proteins, Pum proteins regulate the fate of an RNA, primarily causing decay of the target. Drosophila Pum contains autonomous domains capable of enacting decay independently from its RNA binding domain. The most conserved domain is repression domain 3 (RD3) which we characterize here. Using conservation analysis, we define conserved regions within RD3, and show their importance for the repressive activity of RD3. We identify two specific amino acids, F1033 and F1040, which are necessary for RD3’s function. Using yeast two-hybrid assays, we show that RD3 contacts several members of the CNOT deadenylase complex, the central scaffold unit Not1, and subunits Not2 and Not3. In Drosophila cells, we show that RD3 can interact with the CNOT complex. We further explore the endogenous targets of Drosophila Pum proteins by performing RNA sequencing experiments in which we deplete Pum and members of the CNOT complex. This is the first global analysis of the functional regulation of Pum targets in Drosophila. From this analysis we find that thousands of genes rely upon the regulatory framework provided by Pum. We demonstrate specific regulation of several targets of Pum using reporter assays and identify functional Pum binding sites within target 3’-UTRs. Finally, we show that regulation by Pum also affects the levels of expressed protein for two important targets, Pde11 and Raf. The work shown here enhances our understanding of Pum proteins for all organisms and the field of post-transcriptional control