This proposal outlines a plan to investigate three RNA-protein complexes involved in regulation of tra-2 mRNA in C. elegans. Post-transcriptional regulation of TRA-2 protein expression is a key step in both sex determination and germline development. A nuclear retention complex is formed by binding of proteins NXF- 2 and ALY-1 to an RNA element in the 3'-untranslated region of tra-2 mRNA. A nuclear export complex is formed by binding of proteins TRA-1 and ALY-1 to the same RNA element. A translational repression complex is formed by binding of proteins GLD-1 and FOG-2 to an adjacent RNA element. Quantatitave analysis of the thermodynamics of binding and the sequence requirements for binding will be carried out for these three complexes, with a focus on the cooperative nature of assembly of the RNA-protein complexes. The biochemical studies will serve as a prelude to structural biology investigations of the molecular basis for the RNA-protein and protein-protein interactions involved in these complexes. Finally, proteomic and microarray experiments will be conducted to identify other proteins involved in these complexes, as well as additional RNA targets regulated by these complexes. These studies will provide mechanistic and structural insights into common mechanisms for post-transcriptional gene regulation.
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