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Alternative processing and antisense regulation of thyroid hormone receptor mRNA

甲状腺激素受体 mRNA 的选择性加工和反义调控

基本信息

批准号：
7253718
负责人：
STEPHEN H MUNROE
金额：
$ 22.35万
依托单位：
MARQUETTE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-10 至 2011-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7253718
关键词：
Address Alternative Splicing Antisense RNA Binding Cell physiology Cells Chromosomes Circadian Rhythms Cis-Acting Sequence Code Developmental Process Dominant-Negative Mutation Enhancers Equilibrium Exons Genes Genetic Genetic Enhancer Element Genetic Transcription Goals Health Hereditary Disease Heterogeneous Nuclear RNA Hormones Human In Vitro Introns Link Mammalian Cell Mammals Maps Mediating Mediator of activation protein Messenger RNA Molecular Nature Neuraxis Nuclear Receptors Orphan Overlapping Genes Physiological Physiological Processes Physiology Play Polyadenylation Post-Transcriptional Regulation Process Protein Isoforms Proteins RNA Splicing Reaction Regulation Relative (related person)Research Ribonucleoproteins Role Signal Transduction Staging System Thyroid Hormone Receptor Thyroid Hormones Tissues Transcript Transcription Process Triiodothyronine Variant Work cis acting element in vivo mRNA Precursor mammalian genome receptor research study response rev Protein transcription factor

项目摘要

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.

描述(由申请人提供)：拟议的项目将研究两个重叠基因的转录后调控机制，一个编码α-甲状腺激素受体(Tra)的异构体，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mRNAs，编码两个功能上相互拮抗的转录因子。一种被命名为TRA1，它介导细胞对甲状腺激素(T3)的反应。另一种是TRa2，是一种非激素结合变异体，作为T3诱导基因的显性负抑制因子，从而拮抗TRa1的生理作用。虽然TRa1和TRa2在大多数细胞和组织中都有表达，但这些蛋白及其mRNAs的相对水平存在很大差异。TrA的一个不寻常的特征是，它与另一个编码结构相关的核受体蛋白Rev-erba的基因在染色体的相反链上有互补重叠，使得Rev-erba mRNA与TRa2重叠，而不与TRa1 mRNA重叠。有几条证据表明，这种反义重叠具有调节作用。第一个具体目标是鉴定调节TRa2mRNA选择性剪接的顺式作用元件和反式作用蛋白。实验将集中在先前映射的两个剪接增强子元件上。第二个具体目标是鉴定TRa1、TRa2和Rev-erba mRNAs的3‘加工要求。这项工作将集中在TRa2剪接和竞争多腺苷基化反应要求之间的平衡。还将研究与REV-ERBA 3‘末端形成相关的非常规上游多聚腺苷信号的作用。第三个具体目标是开发一种系统，在该系统中，这三个内源mRNAs的表达在生理上各有不同。REV-ERBA、Tra1和TRa2的这种阶段特异性变异将使调控和反义相互作用的分析与特定目标1和2中进行的替代处理机制的分析相结合。这些实验利用体内和体外方法来分析替代处理的mRNAs。该项目的长期目标是充分表征这些基因的转录后调控，并开发有助于研究其他双向表达基因的表达的方法。最近的研究表明，选择性剪接和反义表达在哺乳动物基因组中广泛存在，并且选择性剪接在人类遗传病中的重要性。因此，这项研究对于理解广泛影响人类健康和生理的基因调控的基本方面是重要的。它还将为本科生大量参与研究提供更多的机会。这项研究对于理解三种核受体蛋白TRa1、TRa2和Rev-erba的表达具有重要意义，这三种核受体蛋白介导了包括人类在内的所有哺乳动物的重要生理过程。TRA1是对甲状腺激素反应的主要介质，甲状腺激素是一种主要激素，调节包括与人类中枢神经系统更高水平功能相关的发育过程和动态平衡过程。TRA2是调节甲状腺激素反应的主要非激素结合变异体。Rev-erba是另一种重要的受体蛋白，最近被确定为调节哺乳动物昼夜节律的分子时钟的核心组成部分。除了了解这三个紧密相连的调控因子的调控外，这项研究还将有助于我们从根本上理解替代的mRNA加工以及反义RNA在哺乳动物细胞中作为生理调节因子的作用。