CHARACTERIZATION OF microRNA-BINDING PROTEINS

描述(由申请人提供)： LIN-4和let-7编码两个小的(21-25nt)RNA，这两个RNA是协调钴线虫的发育时间所必需的。两者都是在时间上表达的，并通过与靶RNA的3‘UTRs中的互补序列结合而发挥翻译抑制物的功能。最近发现，这些小RNA代表了一类新的进化保守基因的创始成员，称为microRNAs。目前，只有两个线虫RNA结合蛋白Alg-1和Alg-2参与了L/n-4和let-7 RNA的加工和功能。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。具体地说，高通量RNAi筛选将被用于识别其他需要alg-1/alg-2才能发挥功能的线虫基因，并将确定需要alg-1/alg-2才能有效调控的其他调控/发育途径。这一遗传策略将与分子/生化技术相辅相成，以识别额外的需要ALG-1/ALG-2进行加工的microRNA和与ALG-1/ALG-2核糖核蛋白复合体相关的其他蛋白质/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.