CHARACTERIZATION OF microRNA-BINDING PROTEINS

描述（由申请人提供）： lin-4和let-7编码两个小的（21- 25 nt）RNA，它们是协调秀丽隐杆线虫发育时间所必需的。两者都是暂时表达的，并通过与靶RNA的3'UTR中发现的互补序列结合而作为翻译阻遏物发挥作用。最近发现，这些小RNA代表了一类新的进化保守基因（称为microRNA）的创始成员。 目前，只有两个C。线虫RNA结合蛋白ALG-1和ALG-2与l/n-4和let-7 RNA的加工和功能有关。本提案中概述的实验将阐明ALG-1/ALG-2与lin-4、let-7和其他microRNA之间的关系。具体而言，将采用高通量RNAi筛选来鉴定其他C. elegans基因需要alg-1/alg-2来发挥功能，并将决定需要alg-1/alg-2来有效调节的其他调节/发育途径。这种遗传策略将与分子/生物化学技术相补充，以鉴定需要alg-1/alg-2进行加工的其他microRNA以及与ALG-1/ALG-2核糖核蛋白复合物相关的其他蛋白质/RNA。

DESCRIPTION (provided by applicant): lin-4 and let-7 encode two small (21-25nt) RNAs that are required for the coordination of developmental timing in Co elegans. Both are temporally expressed and function as translational repressors by binding to complementary sequences found in the 3' UTRs of target RNAs. It has recently been discovered that these small RNAs represent the founding members of a new class of evolutionarily-conserved genes, termed microRNAs. At the present time, only two C. elegans RNA-binding proteins, ALG-1 and ALG-2, have been implicated in the processing and function of l/n-4 and let-7 RNAs. Experiments outlined in this proposal will elucidate the relationship between ALG-1/ALG-2 and lin-4, let-7 and other microRNAs. Specifically, a high-throughput RNAi screen will be employed to identify other C. elegans genes that require alg-1/alg-2 for function and will determine other regulatory/developmental pathways that require alg-1/alg-2 for efficient regulation. This genetic strategy will be complemented with molecular/biochemical techniques to identify additional microRNAs that require alg-1/alg-2 for processing and other proteins/RNAs that associate with the ALG-1/ALG-2 ribonucleoprotein complex.