RNA metabolism, RNA processing: This proposal aims to define cellular RNA targets of the essential, highly conserved DEAD-box helicase Ded1p.

DEAD-box蛋白家族的RNA解旋酶几乎参与了真核细胞RNA代谢的各个方面。尽管最近在了解DEAD-box蛋白的分子机制方面取得了进展，但细胞底物RNA还没有被识别出来，也不知道细胞中的RNA-蛋白质复合体是这些重要酶的靶标。我建议使用一种新的实验方法来鉴定酿酒酵母必需的DEAD-box蛋白Ded1p的生理RNA靶标，该方法结合了几种互补的体内交联法、变性条件下的复杂纯化和深度测序。此外，Ded1p在其RNA靶标上的结合位点将通过受控的RNase对交联RNA的消化和随后的深度测序来定位。这项拟议的工作将为DEAD-box蛋白提供第一个全面的细胞RNA结合位点的定义，从而为这些基本蛋白的生物学功能提供独特的和前所未有的见解。同样重要的是，所开发的方法学将能够定义其他细胞RNA解旋酶的RNA靶标，这对于理解这些关键酶的细胞作用至关重要。

RNA helicases of the DEAD-box protein family are involved in virtually all aspects of eukaryotic RNA metabolism. Despite recent progress towards understanding the molecular mechanisms of DEAD-box proteins, cellular substrate RNAs have not been identified, and it is unknown which RNA-protein complexes in the cell are targeted by these important enzymes. I propose to identify physiological RNA targets of the essential DEAD-box protein Ded1p from Saccharomyces cerevisiae using a novel experimental approach that combines several complimentary in vivo crosslinking methods, complex purification under denaturing conditions, and deep sequencing. In addition, the binding sites of Ded1p on its RNA targets will be mapped by controlled RNase digestion of crosslinked RNAs and subsequent deep sequencing. The proposed work will provide the first comprehensive definition of cellular RNA binding sites for a DEAD-box protein and thus offer unique and unprecedented insights into the biological function of these essential proteins. Equally important, the developed methodology will enable the definition of RNA targets for other cellular RNA helicases, which is pivotal for understanding the cellular roles of these critical enzymes.