REGULATION OF THYROID HORMONE RECEPTOR MRNA PROCESSING

在哺乳动物中，c-erbApha基因编码两个重叠的mRNAs，除了它们的3‘端外显子外，它们是相同的。其中一个mRNAs的蛋白质产物是α-甲状腺激素受体(TRalpha1)，这是一种核受体蛋白，介导细胞对甲状腺激素的反应。另一种信使核糖核酸编码一个非激素结合变异体(TRalpha2)，该变异体作为显性负抑制因子发挥作用，拮抗TRalpha1的功能。受体及其对应的mRNAs的比例因组织和阶段而异。因此，对erbApha信使核糖核酸的选择性处理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-ErbApha(Rev-ErbApha)的3‘端部分重叠，后者以相反的方向转录。在大多数细胞中，TRalpha1和TRalpha2mRNAs的比例随着混响表达水平的不同而变化。由于混响的3‘端与TRalpha2的3’外显子重叠，但不与TRalpha1重叠，因此假设Reflb和TRalpha2转录物之间的碱基配对相互作用相对于TRalpha1选择性地阻断TRalpha2的表达。进一步的证据表明，混响mRNA特异性地阻止了TRalpha2特异的3‘外显子的剪接。这里提出的研究将探讨调节TRalpha1和TRalpha2mRNA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。这些实验将重点放在两个内含子剪接增强剂的功能上，这些内含子剪接增强剂专门用于处理TRalpha2mRNA。将进一步绘制这些元素的图谱，并对与这些元素结合的蛋白质进行表征。(2)研究混响表达在改变TRalpha1/TRalpha2比值中的作用。在调控启动子的控制下，我们将构建稳定表达混响基因mRNAs的细胞，以检测混响基因对TRalpha1和TRalpha2mRNAs表达的影响。将通过在重叠区域内构建突变来探索混响和TRalpha1之间的重叠程度。(3)确定互补转录本是否形成稳定的碱基配对双链。一种敏感的基于聚合酶链式反应的策略将被用来搜索剪接和非剪接RNA之间的双链。核糖核酸酶保护试验将被用作替代方法。这项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以及体内互补转录本之间碱基配对的反义相互作用的要求是重要的。

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.