Regulation of translation initiation in eukaryotes

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DESCRIPTION (provided by applicant): This proposal describes experiments to investigate the scope and importance of base-pairing between messenger RNA (mRNA) and ribosomal RNA (rRNA) as a mechanism for regulating translation in eukaryotes. Until recently, this mechanism was thought to be restricted to bacteria. However, by using the same rigorous criteria that were used to establish the Shine-Dalgarno base-pairing interaction in bacteria, the applicant has shown that this mechanism also affects translation in mammalian cells. This demonstration used a 9-nt translational enhancer element from the 5' leader of the Gtx homeodomain mRNA. The rRNA binding site for the Gfx-element is in the platform of the 40S ribosomal subunit, close to the predicted mRNA binding tract. The applicant has identified numerous other translational enhancer elements with potential binding sites in the 18S rRNA. These sites are primarily in two discrete regions in the intersubunit face of the 40S subunit: the platform and the right foot. A major goal of the proposed studies is to use both biochemical and functional approaches to determine if these sequences bind to rRNA and to determine if this binding affects translation. UV cross-linking together with RNase H localization and toeprinting methods will be used to evaluate and define binding sites. Functional evaluation of base-pairing will involve targeting candidate sites in the 18S rRNA with complementary oligoribonucleotides and by mutation of both mRNA and rRNA sequences to determine whether the activities of individual mRNA-elements require intact complementary matches to the rRNA. For these studies, the applicant has developed a novel yeast system with ribosomes containing a mouse-yeast hybrid 18S rRNA. The second major goal of the proposed studies is to assess the physiological relevance of mRNA:rRNA interactions. Natural mRNAs that contain ribosome binding sites will be tested in yeast and in mammalian cells to determine whether base-pairing occurs in this context and whether these interactions affect protein synthesis. In addition, the extent to which such base-pairing interactions affect the proteome will be assessed by blocking and mutating binding sites in 40S subunits and measuring the effects of these mutations on global and specific protein synthesis both in yeast and in mammalian cells. These studies should provide valuable insights into how the translation of mRNAs are regulated, allowing novel means for controlling translation, e.g. for the production of therapeutic proteins, and new avenues for analyzing the potential contributions of mis-regulation to disease.