How are some genes that specify cell fates translationally regulated so that cells are determined at the right time and place? We propose to study this fundamental question of developmental gene regulation by analyzing the translational regulation of the C. elegan sex determination gene, tra-2. We have shown that a direct repeat, located in the 3' untranslated region (3'UTR), is necessary for repressing the translation of tra-2 mRNA. This repression is required for the proper specification of sexual fates. We have identified an RNA binding factor that binds to the direct repeat. We have also identified a gene, laf-1, that may be involved in this regulation; mutations in laf-1 disrupt the transitional control of reporter constructs by the 3'UTR. During the next five years, we propose to investigate the mechanism by which the 3'UTR controls translation of tra-2 mRNA during development. First, using reporter constructs, we will delineate the necessary cis- acting sequences required for the 3'UTR regulation. In addition, we will ask if the position or orientation of these sequences is important for control. We will study whether the direct repeat controls translation by regulating the length of the poly(A) tail. Second, using RNA gel retardation analysis and reporter constructs, we will investigate whether the binding factor may be a translational inhibitor by correlating the ability of the factor to bind mutant 3'UTRs with the capability of these 3'UTRs to regulate translation. Third, we will genetically characterize the known laf-1 alleles and determine the laf-1 loss-of-function phenotype. This analysis will elucidate the role of laf-1 in the 3'UTR control, and is necessary for the cloning of the gene. Fourth, we will clone laf-1, which will give further insight into its part in the 3'UTR control, and give us the tools to analyze the 3'UTR regulation, biochemically. Fifth, using genetics, reporter constructs, and TRA-2 antibodies, we will study how the 3'UTR regulation changes during development, and identify genes that may control these changes. The health relatedness of this work derives from its contribution to an understanding of fundamental control of gene activity by translational regulation. This class of post-transcriptional regulation is being found to be common in many metazoans, including man.
