Alternative processing and antisense regulation of thyroid hormone receptor mRNA

描述（由申请人提供）：拟议的项目将研究调节两个重叠基因表达的转录后机制，一个编码α-甲状腺激素受体（TRa）的亚型，另一个编码孤儿受体Rev-erba。在哺乳动物中，TRa基因产生两个选择性剪接的mRNA，其编码两个功能拮抗性转录因子。一个称为TRa 1，介导细胞对甲状腺激素（T3）的反应。另一个，TRa 2，是一种非激素结合的变体，作为T3诱导基因的显性负阻遏物，从而拮抗TRa 1的生理作用。虽然TRa 1和TRa 2在大多数细胞和组织中表达，但这些蛋白质及其mRNA的相对水平差异很大。TRa的一个不寻常的特征是它与另一个编码结构上相关的核受体蛋白的基因在染色体的相对链上共享互补重叠，使得Rev-erba mRNA与TRa 2重叠，而不是TRa 1 mRNA。一些证据表明，这种反义重叠的调节作用。第一个具体的目的是表征顺式作用元件和反式作用蛋白，调节选择性剪接特异性的TRa 2 mRNA。实验将集中在两个剪接增强子元件，以前已被映射。第二个具体目的是针对表征TRa 1、TRa 2和Rev-erba mRNA的3'加工的要求。这项工作将集中在TRa 2剪接和竞争性聚腺苷酸化反应的要求之间的平衡。还将检查与Rev-erba 3'末端形成相关的非常规上游多聚腺苷酸化信号的作用。第三个具体目标是开发一种系统，其中这三种内源性mRNA中的每一种的表达在生理上变化。Rev-erba、Tra 1和TRa 2中的这种阶段特异性变异将允许将调节和反义相互作用的分析与特定目的1和2中进行的替代加工机制的分析整合。这些实验利用体内和体外方法来分析替代加工的mRNA。该项目的长期目标是充分表征这些基因的转录后调控，并开发用于研究其他双向表达基因表达的方法。近年来的研究表明，选择性剪接和反义表达在哺乳动物基因组中广泛存在，并在人类遗传性疾病中发挥重要作用。因此，这项研究对于理解广泛影响人类健康和生理的遗传调控的基本方面非常重要。它还将为重要的本科生研究参与提供更多的机会。这项研究对于理解三种核受体蛋白TRa 1、TRa 2和Rev-erba的表达非常重要，这三种蛋白在包括人类在内的所有哺乳动物中介导重要的生理过程。TRa 1是对甲状腺激素的反应的主要介质，甲状腺激素是介导发育过程（包括与人类中枢神经系统的更高水平功能相关的那些）和稳态过程的主激素。TRa 2是调节对甲状腺激素的反应的主要非激素结合变体。Rev-erba是另一种重要的受体蛋白，最近被鉴定为调节哺乳动物昼夜节律的分子钟的核心组分。除了了解这三个紧密相连的调节因子的调节，这项研究将有助于我们对替代mRNA加工和反义RNA在哺乳动物细胞中作为生理调节剂的作用的基本理解。

DESCRIPTION (provided by applicant): 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.