Alternative processing and antisense regulation of thyroid hormone receptor mRNA

Munroe, Stephen

Abstract

The proposed project will investigate post-transcriptional mechanisms regulating expression of two overlapping genes, one encoding isoforms of the a-thyroid hormone receptor (TRa) and the other encoding the orphan receptor, Rev-erba. In mammals the TRa gene produces two alternatively spliced mRNAs that encode two functionally antagonistic transcription factors. One, designated TRa1, mediates the cellular response to thyroid hormone (T3). The other, TRa2, is a non-hormone-binding variant that acts as a dominant negative repressor of T3-induced genes, thereby antagonizing the physiological action of TRa1. Although TRa1 and TRa2 are expressed in most cells and tissues, the relative levels of these proteins and their mRNAs vary substantially. An unusual feature of TRa is that it shares a complementary overlap with another gene encoding a structurally related nuclear receptor protein, Rev- erba, on the opposite strand of the chromosome, such that Rev-erba mRNA overlaps TRa2 but not TRa1 mRNA. Several lines of evidence suggest a regulatory role for this antisense overlap. The first specific aim is to characterize both cis-acting elements and trans-acting proteins that regulate alternative splicing specific to TRa2 mRNA. Experiments will focus on two splicing enhancer elements that have previously been mapped. The second specific aim is directed at characterizing requirements for 3' processing of TRa1, TRa2, and Rev-erba mRNAs. This work will focus on the the balance between TRa2 splicing and requirements for competing polyadenylation reactions. The role of unconventional upstream polyadenylation signals associated with Rev-erba 3' end formation will also be examined. The third specific aim is to develop a system in which the expression of each of these three endogenous mRNAs varies physiologically. Such stage-specific variation in Rev-erba, Tra1 and TRa2 will permit analysis of regulation and antisense interactions to be integrated with the analysis of alternative processing mechanisms carried out in Specific Aims 1 and 2. These experiments utilize both in vivo and in vitro approaches for the analysis of alternatively processed mRNAs. The long-term goal of this project is to fully characterize post-transcriptional regulation of these genes and to develop approaches useful for investigating the expression of other bidirectionally expressed genes. Recent studies have demonstrated the widespread occurrence of alternative splicing and antisense expression in mammalian genomes and the importance of alternative splicing in human genetic diseases. Thus, this research is important for understanding fundamental aspects of genetic regulation that broadly impact human health and physiology. It will also provide expanded opportunities for significant undergraduate research participation. The proposed research is important for understanding the expression of three nuclear receptor proteins, TRa1, TRa2 and Rev-erba which mediate important physiological processes in all mammals, including humans. TRa1 is the principal mediator of the response to thyroid hormone, a master hormone that mediates both developmental processes, including those associated with higher level functions of the human central nervous system, and homeostatic processes. TRa2 is the major non-hormone binding variant that modulates the response to thyroid hormone. Rev-erba is another important receptor protein that has recently been identified as a core component of the molecular clock that regulates circadian rhythms in mammals. In addition to understanding the regulation of these three tightly linked regulatory factors this research will contribute to our fundamental understanding of alternative mRNA processing and of the role of antisense RNA as a physiological regulator in mammalian cells. ? ? ?