The long term goal of our research is to determine how gene expression is regulated at the post- transcriptional level. Control of messenger RNA (mRNA) translation and degradation underlies important biological processes including development, fertility and neurological functions. The proposed work focuses on the archetypal mRNA regulator, Drosophila Pumilio (Pum), which belongs to a family of RNA-binding proteins that are conserved throughout eukarya. Pum binds an extensive group of mRNAs including those that encode key developmental morphogens. Upon binding to an mRNA, Pum represses expression of the encoded protein. We find that Pum accelerates decay of the target mRNA and the proposed research seeks to discover the mechanism of Pum-mediated mRNA degradation. We developed novel assays to measure Pum activity in Drosophila cells and discovered multiple unique Repression Domains that potently repress target mRNAs. In preliminary work, we identified key co-repressors that are necessary for Pum repression. In the first aim, we measure the impact of the Repression Domains on protein expression and mRNA degradation and interrogate the functional roles of two classes of mRNA decay factors in repression by each Pum Repression Domain. We interrogate the physical interactions of the co-repressors with each Pum Repression Domain and measure their recruitment to Pum-regulated target mRNAs. In the second aim, we investigate the role of each Pum Repression Domain in embryonic development and proper spatial and temporal control of gene expression during early development. This research will reveal novel mechanisms of Pum repression. The resulting discoveries are expected to broadly enhance our understanding of post-transcriptional control and specifically improve knowledge of gene regulation in development, fertility, stem cell proliferation, and the nervous system.
This research is relevant to human biology and health because Pumilio proteins are conserved in humans where, like their Drosophila counterpart, they are implicated in control of cell proliferation, fertility and neurological functions. The RNA-binding and Repression Domains of Pumilio are functionally conserved in human Pum orthologs; therefore, our discoveries are expected to be translatable to post-transcriptional regulation of human cancer biology, developmental defects, neurological disorders and infertility.
|T Abshire, Elizabeth; Chasseur, Jennifer; Bohn, Jennifer A et al. (2018) The structure of human Nocturnin reveals a conserved ribonuclease domain that represses target transcript translation and abundance in cells. Nucleic Acids Res 46:6257-6270|
|Wolfe, Michael B; Goldstrohm, Aaron C; Freddolino, Peter L (2018) Global analysis of RNA metabolism using bio-orthogonal labeling coupled with next-generation RNA sequencing. Methods :|
|Bohn, Jennifer A; Van Etten, Jamie L; Schagat, Trista L et al. (2018) Identification of diverse target RNAs that are functionally regulated by human Pumilio proteins. Nucleic Acids Res 46:362-386|
|Hughes, Kelsey L; Abshire, Elizabeth T; Goldstrohm, Aaron C (2018) Regulatory roles of vertebrate Nocturnin: insights and remaining mysteries. RNA Biol 15:1255-1267|
|Goldstrohm, Aaron C; Hall, Traci M Tanaka; McKenney, Katherine M (2018) Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins. Trends Genet 34:972-990|
|Arvola, René M; Weidmann, Chase A; Tanaka Hall, Traci M et al. (2017) Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins. RNA Biol 14:1445-1456|
|Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan et al. (2017) Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS). Methods 118-119:171-181|
|Weidmann, Chase A; Qiu, Chen; Arvola, René M et al. (2016) Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio. Elife 5:|
|Weidmann, Chase A; Raynard, Nathan A; Blewett, Nathan H et al. (2014) The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation. RNA 20:1298-319|
|Hrit, Joel; Raynard, Nathan; Van Etten, Jamie et al. (2014) In vitro analysis of RNA degradation catalyzed by deadenylase enzymes. Methods Mol Biol 1125:325-39|