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 lie 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'UTRs control function, evolve, and coordinate expression of multiple mRNAs.
Showing the most recent 10 out of 54 publications
