REGULATION OF THYROID HORMONE RECEPTOR MRNA PROCESSING

甲状腺激素受体 mRNA 加工的调节

• 批准号: 6159760
• 负责人: STEPHEN H MUNROE
• 金额: $ 14.29万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-17 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6159760
• 关键词: RNA splicing; RNase protection assay; SDS polyacrylamide gel electrophoresis; affinity chromatography; autoradiography; gel mobility shift assay; gene mutation; genetic enhancer element; genetic mapping; genetic regulatory element; hormone receptor; immunoprecipitation; messenger RNA; northern blottings; polymerase chain reaction; posttranscriptional RNA processing; protein binding; receptor expression; thyroid hormones; ultraviolet radiation

In mammals the c-erbAalpha gene encodes two overlapping mRNAs which are identical except for their 3' terminal exons. The protein product of one of these mRNAs is the alpha-thyroid hormone receptor (TRalpha1), a nuclear receptor protein which mediates the cellular response to thyroid hormone. The other mRNA encodes a non-hormone binding variant (TRalpha2) which functions as a dominant negative repressor, antagonizing the function of TRalpha1. The ratio of receptors and their corresponding mRNAs varies in a tissue-specific and stage- specific manner. Alternative processing of the erbAalpha mRNA thus determines the cellular response to thyroid hormone. A remarkable feature of this locus is that it partially overlaps the 3' end of a gene for another receptor protein, Rev-ErbAalpha (RevErb), which is transcribed in the opposite direction. In most cells, the ratio of TRalpha1 to TRalpha2 mRNAs varies with the level of RevErb expression. Since the 3' end of RevErb overlaps the 3' exon of TRalpha2 but not TRalpha1, it has been hypothesized that base pairing interactions between RevErb and TRalpha2 transcripts selectively block expression of TRalpha2 relative to TRalpha1. Further evidence suggests that RevErb mRNA specifically blocks splicing of the TRalpha2-specific 3' exon. Research proposed here will investigate molecular mechanisms which regulate alternative processing of TRalpha1 and TRalpha2 mRNA. The specific aims of the research are as follows: (1) To characterize cis-acting elements which regulate mRNA processing. These experiments will focus on the function of two intronic splicing enhancers specific for processing TRalpha2 mRNA. These elements will be further mapped and proteins which bind to these elements will be characterized. (2) To characterize the role of RevErb expression in altering the ratio of TRalpha1 and TRalpha2. Cells stably transfected with minigenes expressing RevErb mRNA under the control of a regulated promoter will be constructed to examine the effect of RevErb on expression of TRalpha1 and TRalpha2 mRNAs. The extent of overlap between RevErb and TRalpha1 will be explored by constructing mutations within the region of overlap. (3) To determine whether the complementary transcripts form a stably base-paired duplex. A sensitive PCR- based strategy will be used to search for duplexes between spliced and unspliced RNAs. RNase protection assays will be used as an alternative approach. The proposed research is important for understanding more fully regulation of the physiological response to thyroid hormone in mammals and requirements fir for base-paired antisense interactions between complementary transcripts in vivo.

在哺乳动物中，c-erbA α基因编码两个重叠的mRNA，除了它们的3'末端外显子外，这两个mRNA是相同的。 这些mRNA之一的蛋白质产物是α-甲状腺激素受体（TRalpha 1），一种介导细胞对甲状腺激素反应的核受体蛋白。 另一种mRNA编码一种非激素结合变体（TRalpha 2），其作为显性负性阻遏物发挥作用，拮抗TRalpha 1的功能。受体及其相应mRNA的比例以组织特异性和阶段特异性方式变化。 因此，erbA α mRNA的替代加工决定了细胞对甲状腺激素的反应。 该基因座的一个显著特征是它与另一种受体蛋白Rev-ErbA α（RevErb）基因的3'端部分重叠，该受体蛋白以相反方向转录。 在大多数细胞中，TRalpha 1与TRalpha 2 mRNA的比例随RevErb表达水平而变化。 由于RevErb的3'端与TRalpha 2的3'外显子重叠，但不与TRalpha 1重叠，因此假设RevErb和TRalpha 2转录物之间的碱基配对相互作用相对于TRalpha 1选择性地阻断TRalpha 2的表达。 进一步的证据表明，RevErb mRNA特异性地阻断TR α 2特异性3'外显子的剪接。本文提出的研究将探讨调节TRalpha 1和TRalpha 2 mRNA交替加工的分子机制。 本研究的具体目的如下：（1）研究调控mRNA加工的顺式作用元件。这些实验将集中在两个内含子剪接增强子的功能，特异性加工TRalpha 2 mRNA。 这些元件将被进一步绘制，与这些元件结合的蛋白质将被表征。 (2)描述RevErb表达在改变TRalpha 1和TRalpha 2比例中的作用。将构建用在受调控启动子控制下表达RevErb mRNA的小基因稳定转染的细胞，以检查RevErb对TR α 1和TR α 2 mRNA表达的影响。 将通过在重叠区域内构建突变来探索RevErb和TR α 1之间的重叠程度。 (3)确定互补转录物是否形成稳定的碱基配对双链体。 一个敏感的PCR为基础的策略将被用来寻找剪接和未剪接的RNA之间的双链体。 RNA酶保护试验将用作替代方法。 该研究对于更全面地了解哺乳动物对甲状腺激素的生理反应的调节以及体内互补转录本之间碱基配对反义相互作用的要求具有重要意义。