Developmental function of Rb family proteins

描述(申请人提供)：在控制细胞增殖和分化的许多途径中，视网膜母细胞瘤蛋白(RB)调控网络的基因在肿瘤发生过程中经常成为突变或放松调控的目标。虽然生化和组织培养研究表明Rb家族成员参与了广泛的细胞活动，但Rb在体内和正常发育过程中的真正功能尚不清楚。我们的长期目标是在分子水平上了解Rb家族蛋白的细胞和发育功能。为此，我们设计了一种遗传策略，使我们能够识别与线虫RB同源物LIN-35协调发挥功能的基因和途径，以控制必要的发育过程。利用这个系统，我们已经证明了LIN-35的典型细胞周期功能以及该蛋白在器官形态发生中的新作用。我们还发现了一条互补的途径，通过UBC-18/UbcH7控制器官的形态发生，Ubc-18/UbcH7是一种保守的泛素结合酶，参与蛋白质降解的靶向。拟议的实验旨在揭示LIN-35与一个或多个平行途径共同作用，调节线虫器官形态发生的潜在机制。 我们的主要目标分为两类。一个广泛的目标是确定与LIN-35和UBC-18协同作用的其他因素来控制器官发生。这些研究将包括SIR-9的克隆和鉴定，该基因与UBC-18一样，与LIN-35具有冗余的功能，以控制器官的形态发生；进行双杂交筛选以确定与UBC-18相互作用的蛋白；以及使用已知或推测的泛素途径成分进行定向RNAi馈送筛选。我们的第二个目标是确定LIN-35和UBC-18监管的功能相关的下游目标。这些研究将包括：分离抑制LIN-35致命性的突变的基因选择；UBC-18双突变体；鉴定LIN-35调控转录本的完整谱的微阵列分析；以及使用通过早期双杂交或RNAi馈送筛选确定的UBC-18的辅助因子的额外双杂交筛选。这些研究的成功完成将极大地提高我们对Rb家族功能的总体理解，并将为Rb蛋白在形态发生中的这一新的作用提供详细的机制知识。

DESCRIPTION (provided by applicant): Among the many pathways controlling cell proliferation and differentiation, genes of the retinoblastoma protein (Rb) regulatory network stand out as frequent if not obligatory targets for mutation or deregulation during tumorigenesis. Although biochemical and tissue culture studies have implicated Rb family members in a wide range of cellular activities, the bona fide functions of Rb in vivo and during normal development are not well understood. Our long-term objective is to understand the cellular, and developmental functions of Rb family proteins at the molecular level. To this end, we have devised a genetic strategy that has allowed us to identify genes and pathways that function coordinately with the C. elegans Rb homolog, lin-35, to control essential developmental processes. Using this system, we have demonstrated canonical cell cycle functions for LIN-35 as well as a novel role for this protein in organ morphogenesis. We have also uncovered a complementary pathway that acts to control organ morphogenesis through UBC-18/UbcH7, a conserved ubiquitin-conjugating enzyme involved in the targeting of proteins for degradation. The proposed experiments are designed to uncover the underlying mechanism by which LIN-35, acting in conjunction with one or more parallel pathways, regulates organ morphogenesis in C. elegans. Our main objectives fall into two categories. One broad aim is to identify additional factors that function cooperatively with LIN-35 and UBC-18 to control organogenesis. These studies will include the cloning and characterization of sir-9, a gene that, like ubc-18, functions redundantly with lin-35 to control organ morphogenesis; the execution of a two-hybrid screen to identify UBC-18-interacting proteins; and a directed RNAi feeding screen using known or putative ubiquitin pathway components. Our second objective is to identify functionally relevant downstream targets for regulation by LIN-35 and UBC-18. These studies will include genetic selections to isolate mutations that suppress the lethality of lin-35; ubc-18 double mutants; microarray analyses to identify the complete spectrum of LIN-35-regulated transcripts; and additional two-hybrid screens using co-factors of UBC-18 identified through earlier two-hybrid or RNAi-feeding screens. The successful completion of these studies will greatly enhance our general understanding of Rb family functions and will provide detailed mechanistic knowledge of this novel role for Rb proteins in morphogenesis.