The nucleic acid specificity and endogenous substrate of Rei1

University Of Utah, Salt Lake City UT

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): RNA binding proteins play key roles in important cellular pathways. A new class of zinc finger proteins that bind double-stranded RNA has recently been discovered. My broad aim is to characterize zinc finger proteins that bind double-stranded RNA to determine what cellular roles they play. I will first investigate the Saccharomyces cerevisiae protein Reilp, a zinc finger protein that I have shown binds to double-stranded RNA. Reilp has been shown to be involved in the maturation of the large ribosomal subunit, perhaps by regulating the nucleocytoplasmic shuttling of other ribosome maturation factors. It is my hypothesis that Rei1 p binds to double-stranded or highly structured RNA regions on the 60S subunit, and that this interaction is crucial for its cellular function. Therefore, my specific aims are 1) To identify the endogenous RNA substrate of the S. cerevisiae protein Rei1 p; and 2) To determine the regions of Rei1 p that contribute to RNA binding in vitro and ribosome biogenesis in vivo. [unreadable] [unreadable] To identify the endogenous substrate of Reilp, I will utilize hydroxyl radical probing to determine ReMp's large subunit RNA "footprint". Having established the region of RNA to which Reilp binds, I will more precisely determine, through in vitro binding experiments, the specific RNA sequence/structure that is bound by Reilp. Finally, I will confirm the Rei1p-RNA interaction in vivo by mutating key residues of the ribosomal RNA and demonstrating an accompanying loss in Reilp-ribosome association. [unreadable] [unreadable] To determine the regions of Reilp that contribute to RNA binding and ribosome biogenesis, I will create a series of truncation, deletion and point mutants, all of which will be probed for in vitro RNA binding as well as in vivo function. This will allow me to determine the regions of Reilp that affect its RNA binding, as well as to determine the extent to which RNA binding by Reilp correlates with in vivo function. In addition, these studies will allow me to identify regions that, while not important for RNA binding, are important for Rei1 p's cellular function-such as regions involved in interactions with other cellular factors. [unreadable] [unreadable] Reilp appears to play an important regulatory role in creating ribosomes, and thus in protein synthesis. It is increasingly clear that control and coordination of protein synthesis is a critical issue in cell growth control, and defects in the biogenesis of ribosomes lead to certain types of cancer. [unreadable] [unreadable] [unreadable]