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解旋酶几乎参与真核生物RNA代谢的所有方面。尽管最近在了解DEAD-box蛋白的分子机制方面取得了进展，但尚未鉴定出细胞底物rna，并且尚不清楚细胞中哪些rna -蛋白复合物是这些重要酶的靶标。我建议使用一种新的实验方法，结合几种互补的体内交联方法、变性条件下的复杂纯化和深度测序，从酿酒酵母中鉴定必需的DEAD-box蛋白Ded1p的生理RNA靶点。此外，Ded1p在其RNA靶标上的结合位点将通过控制RNA酶切交联RNA和随后的深度测序来绘制。这项工作将首次全面定义DEAD-box蛋白的细胞RNA结合位点，从而为这些必需蛋白的生物学功能提供独特和前所未有的见解。同样重要的是，所开发的方法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.