This proposal is primarily aimed at understanding mechanisms that regulate the expression of a key molecule, TCR-beta, on the surface of T lymphocytes. A central aim is to elucidate the mechanisms that dictate the steady state levels of TCR-beta MRNA during the ontogeny of T cells. This regulation may be achieved by a combination of both positive and negative regulatory mechanisms acting at different levels. We plan to focus our attention on the role of negative controls, which we have defined, that act at the post-transcriptional level. We shall examine the underlying molecular basis for the down-regulation of out-of-frame TCR-beta transcripts derived from non-productively rearranged genes. We will assess if RNA splicing is a developmentally regulated process that controls TCR-beta transcript levels during thymic maturation and/or maintains lineage fidelity. We will also characterize cis-acting elements and candidate RNA-binding proteins that dictate the post-transcriptional regulation of TCR-beta transcripts. Particular emphasis will be placed on studies deliniating the cis- and trans-acting factors that participate in RNA splicing regulation. The regulation of TCR-beta expression is of interest for several reasons: First, it is possible that elucidation of mechanisms that regulate TCR- beta gene expression will help advance future investigations on the precise molecular defects responsible for some autoimmune and immune deficiency diseases. Second, the expression of TCR-beta transcripts is developmentally regulated in a lineage and stage specific fashion. Since TCR-beta is specifically expressed in a cell type whose developmental program is well understood, there is a great potential for ultimately understanding the connections between developmental status and gene expression. Third, TCR-beta is of interest since it is one of a very few mammalian genes that undergo programmed rearrangements in vivo. An important consequence of gene rearrangement events in the TCR-beta gene is a high rate of potentially deleterious mutations. It is not known what potential effect these """"""""mutant' TCR-beta genes have on T cells, but it is clear that the expression of transcripts derived from these genes is down-regulated by a post-transcriptional mechanism (see progress report). Finally, our results indicate that TCR-beta expression is regulated at the level of RNA splicing. The potential consequences of this form of regulation include control of T cell maturation status and lineage fidelity. Extensive experiments are aimed at studying RNA splicing regulation not only because of what it may tell us about T cell biology, but also because these studies may reveal important new information about control of gene expression, in general.
