The long term goal of this research is to reveal 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, Pumilio (Pum). Pum is a member of the PUF family of eukaryotic RNA binding proteins. Pum binds an extensive group of messenger RNAs including those that encode cell cycle proteins and key developmental morphogens. Upon binding to an mRNA, Pum represses expression of the encoded protein. The proposed research seeks to discover the mechanism of Pum repression and determine how Pum activity is modulated. We developed novel assays to measure Pum activity in Drosophila cells and discovered multiple new domains that potently repress protein expression. The repressive activity of the Pum repression domains do not rely on previously identified cofactors, indicating novel repression mechanism(s). In the first aim, we measure the impact of the repression domains on translation and mRNA degradation. We seek to discover cofactors necessary for their activity. We discovered an autoregulatory switch that controls Pum activity and may be modulated by phosphorylation. In the second aim, we dissect the mechanism of this autoregulatory switch using functional and protein interaction assays. In the third aim, we explore how the Zn finger protein Nanos regulates repression by Pum. Nanos interacts with Pum and enhances repression. We propose that Nanos enhances interaction of Pum with mRNA and, in effect, changes the collection of mRNAs repressed by Pum. This research will determine novel mechanisms of Pum repression and illuminate how that activity is regulated in response to protein modifications and combinatorial action with Nanos. 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.

Public Health Relevance

This research is relevant to human biology and health because Pumilio and Nanos are conserved in humans where, like their Drosophila counterpart, they are implicated in control of cell proliferation, fertility and neurological functions. The RNA binding, repression and autoregulatory domains of Pumilio are homologous to human Pums;therefore, our discoveries are expected to be translatable to post-transcriptional regulation of human cancer biology, developmental defects, neurological disorders and infertility.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM105707-01
Application #
8476706
Study Section
Special Emphasis Panel (ZRG1-GGG-E (91))
Program Officer
Bender, Michael T
Project Start
2013-09-20
Project End
2018-07-31
Budget Start
2013-09-20
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$286,915
Indirect Cost
$96,915
Name
University of Michigan Ann Arbor
Department
Biochemistry
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Goldstrohm, Aaron C; Hall, Traci M Tanaka; McKenney, Katherine M (2018) Post-transcriptional Regulatory Functions of Mammalian Pumilio Proteins. Trends Genet 34:972-990
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
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