Eukaryotic mRNAs can be controlled at many steps. In the nucleus, transcription and mRNA processing are needed to generate mRNAs that can be translated. In the cytoplasm, mature mRNAs can be regulated at the levels of stability, translation and localization. The objective of the proposed work is to understand the molecular mechanisms that regulate mature mRNAs in animal cells. We focus on controls mediated by sequences that beyond the termination codon -- in the 3' untranslated region (3'UTR) and the poly(A) tail. Regulated changes in poly(A) length occur throughout development and affect translation and stability of many mRNAs. Here,we focus on a novel form of cytoplasmic poly(A) polymerase in C. elegans, consisting of a catalytic subunit, GLD-2, with distinct RNA-binding protein partners. One partner binds to and antagonizes FBF, a founder of a family of 3'UTR repressors, the PUF proteins. Each protein we focus on -- GLD-2, its partners, and FBF -- controls key events in development. Our ultimate goals are to understand, in molecular terms, how these proteins control the fate and function of mature mRNAs. The approach taken is first to elucidate how the novel polyadenylation system functions. Using molecular genetics and biochemistry, we identify regions of GLD-2 that promote catalysis and bind partners. Through grafting and mutagenesis, we test the model that GLD-2 lacks RNA-binding activity, but gains it via distinct protein partners. This work is consummated by determining structures of the key components. We elucidate the evolutionary breadth of the polyadenylation system, focusing on vertebrates and in vivo assays, and elucidate how it antagonizes PUF-mediated repression. Finally, combining the yeast three-hybrid system and other methods, we identify mRNA targets of the system. Throughout, we combine the use of Xenopus oocytes, yeast molecular genetics, and in vitro systems to elucidate how these proteins function. We exploit novel methods we developed that have broader utility. The rich genetics and biology of C. elegans development provides a critical biological foundation. In focusing sharply on a few specific examples, we hope to illuminate the broad molecular questions of how 3'UTR controls function, evolve, and coordinate exoression of multiole mRNAs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM031892-21
Application #
7149154
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Rhoades, Marcus M
Project Start
1983-04-01
Project End
2007-12-16
Budget Start
2006-12-01
Budget End
2007-12-16
Support Year
21
Fiscal Year
2007
Total Cost
$360,515
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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Ellery, Paul E R; Maroney, Susan A; Martinez, Nicholas D et al. (2014) Translation of human tissue factor pathway inhibitor-? mRNA is controlled by alternative splicing within the 5' untranslated region. Arterioscler Thromb Vasc Biol 34:187-95
Lapointe, Christopher P; Wickens, Marvin (2013) The nucleic acid-binding domain and translational repression activity of a Xenopus terminal uridylyl transferase. J Biol Chem 288:20723-33
Menichelli, Elena; Wu, Joann; Campbell, Zachary T et al. (2013) Biochemical characterization of the Caenorhabditis elegans FBF.CPB-1 translational regulation complex identifies conserved protein interaction hotspots. J Mol Biol 425:725-37
Wu, Joann; Campbell, Zachary T; Menichelli, Elena et al. (2013) A protein.protein interaction platform involved in recruitment of GLD-3 to the FBF.fem-3 mRNA complex. J Mol Biol 425:738-54
Zhang, Yan; Cooke, Amy; Park, Sookhee et al. (2013) Bicaudal-C spatially controls translation of vertebrate maternal mRNAs. RNA 19:1575-82
LeGendre, Jacqueline Baca; Campbell, Zachary T; Kroll-Conner, Peggy et al. (2013) RNA targets and specificity of Staufen, a double-stranded RNA-binding protein in Caenorhabditis elegans. J Biol Chem 288:2532-45
Campbell, Zachary T; Menichelli, Elena; Friend, Kyle et al. (2012) Identification of a conserved interface between PUF and CPEB proteins. J Biol Chem 287:18854-62
Gerstner, Jason R; Vanderheyden, William M; LaVaute, Timothy et al. (2012) Time of day regulates subcellular trafficking, tripartite synaptic localization, and polyadenylation of the astrocytic Fabp7 mRNA. J Neurosci 32:1383-94
Friend, Kyle; Campbell, Zachary T; Cooke, Amy et al. (2012) A conserved PUF-Ago-eEF1A complex attenuates translation elongation. Nat Struct Mol Biol 19:176-83

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