The nucleic acid specificity and endogenous substrate of Rei1

描述（由申请人提供）：RNA结合蛋白在重要的细胞通路中发挥关键作用。最近发现了一类新的结合双链RNA的锌指蛋白。我的主要目标是表征结合双链RNA的锌指蛋白，以确定它们在细胞中扮演的角色。我将首先研究酿酒酵母蛋白Reilp，这是一种锌指蛋白，我已经证明它与双链RNA结合。Reilp已被证明参与大核糖体亚基的成熟，可能是通过调节其他核糖体成熟因子的核质穿梭。我的假设是rei1p与60S亚基上的双链或高结构RNA区域结合，这种相互作用对其细胞功能至关重要。因此，我的具体目标是：1)鉴定酿酒酵母蛋白rei1p的内源性RNA底物；2)确定rei1p在体外参与RNA结合和体内核糖体生物发生的区域。

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. 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. 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. 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.