Messenger RNA Control by Pumilio

Abstract

Pumilio (Pum) is a sequence-specific RNA-binding protein (RBP) that recognizes Pum Response Elements (PREs) in the 3'UTR of target mRNAs. Pum represses an extensive network of mRNAs to control embryogenesis, stem cell maintenance, fertility, and neurological function in Drosophila. Moreover, Pum orthologs have roles in cancer, neurodegeneration, ataxia, and epilepsy. Given these crucial functions, the primary goal of my thesis research has been to identify the mechanisms of mRNA regulation by Pum. Pum accelerates degradation of target mRNAs, and this activity is primarily caused by three repression domains (RDs) in the protein's N terminus. The RDs are unique to Pum and its orthologs, and can function autonomously when directed to a reporter mRNA. Each Pum RD causes repression and mRNA degradation, and I found that their activity requires the Ccr4-Not (CNOT) deadenylase complex. The Not1 scaffold and Pop2 deadenylase subunits of CNOT are crucial for Pum RD-mediated repression. Moreover, Pop2's catalytic activity is necessary to rescue RD function, indicating that Pum RDs require the deadenylation activity of CNOT. Our biochemical data reveal that multiple regions of Pum recruit CNOT to target mRNAs, including the three N-terminal RDs, and the C-terminal RNA-binding domain. Consistent with this model, the ability of Pum to accelerate decay of target mRNAs requires CNOT. I also observed that decapping factors participate in repression by the Pum N terminus. Together, the data reveal that Pum utilizes multiple mRNA decay pathways to repress target mRNAs. Ongoing work includes studying the relevance of the RDs in vivo during embryogenesis using transgenic flies with deletions in one or more RD. These mechanistic insights into Pum repression may shed light on its combinatorial regulation with other RBP partners, such as Nanos (Nos). We previously showed that Pum and Nos bind cooperatively to RNA, thereby strengthening repression of mRNAs. In collaboration with Traci Hall at NIEHS and Zachary Campbell at UT Dallas, we reported the structure and specificity of the Pum-Nos-RNA complex. Nos binds Pum and nucleotides upstream of the PRE, strengthening its RNA binding and repression activities. I contributed to the validation of Nos-Pum contacts through mutagenesis, and cell-based experiments demonstrating that Nos can expand Pum’s target repertoire and confer repression to canonically weak PREs. To expand on our molecular studies of Pum and Nos, we also integrated transcriptome-wide analyses and bioinformatics predictions to assess their global impacts on gene regulation. Taken together, these findings support the potential for pervasive, dynamic post-transcriptional control by these RBPs both individually and combinatorially.