Mechanisms Regulating Alternative pre-mRNA Splicing

大多数真核基因都被非编码内含子打断，这些内含子必须从前mRNA转录本中移除，才能产生功能蛋白。内含子的剪接是非常有效和快速的，考虑到从外显子中区分内含子的信号在高等真核生物中并不保守，这是一项令人惊讶的壮举。剪接在正常细胞功能中的重要性的推论是，大约15%的特征性遗传病涉及导致剪接缺陷的突变。此外，60%或更多的人类基因受到选择性剪接的影响，这要求细胞不仅能够准确识别内含子和外显子之间的差异，而且还能够识别选择性剪接的外显子之间的差异。我们一直在研究选择性剪接的调控，重点放在激活或抑制特定剪接位点的分子机制上。在这项建议的第一部分，实验旨在了解α-原肌球蛋白基因的互斥外显子2和3的调控。对控制剪接所需的顺式作用序列的剖析已经确定了外显子3两侧的元件，这些元件是抑制平滑肌细胞剪接所必需的。PTB(HnRNP I)与这些元件之一结合，但与直接相邻的保守UGC重复序列结合的因子的身份尚不清楚。生化纯化是为了确定这个因子，并确定它是如何参与PTB抑制剪接的。与抑制外显子3相反，在四个富含嘌呤的增强子元件和SR蛋白剪接因子家族的一个或多个成员的介导下，平滑肌细胞需要激活外显子2。体内和体外剪接试验将被用来确定哪些SR蛋白是外显子2激活所必需的。在这项提案的第二部分，将扩大SR蛋白对剪接的调节，以包括一种新的SR相关蛋白(SRrp86)的特征，该蛋白可以激活或抑制特定的SR家族成员。SRrp86的特定靶点尚不清楚，但初步数据表明，它通过蛋白质-蛋白质相互作用发挥作用。建议进行实验，以确定这种相互作用如何改变SR蛋白的活性，并确定特定的SR蛋白靶标。总体而言，该提案中的实验试图了解SR蛋白和hnRNP蛋白抑制和激活剪接的机制基础。

Most eukaryotic genes are interrupted by non-coding introns that must be removed from pre-mRNA transcripts for the production of functional proteins. The splicing of introns is remarkably efficient and rapid, an amazing feat considering that the signals that delineate introns from exons are not conserved in higher eukaryotes. A corollary of the importance of splicing in normal cell function is that approximately 15% of characterized genetic diseases involve mutations that cause defects in splicing. Further, 60% or more of human genes are subject to alternative splicing requiring that cells not only be able to accurately recognize the difference between introns and exons, but also between alternatively spliced exons. We have been studying the regulation of alternative splicing focusing on the molecular mechanisms by which specific splice sites are activated or repressed. In the first part of this proposal, experiments are designed to understand the regulation of the mutually exclusive exons 2 and 3 of the alpha- tropomyosin gene. Dissection of the cis-acting sequences needed to control splicing have identified elements flanking exon 3 that are needed to repress splicing in smooth muscle cells. PTB (hnRNP I) binds to one of these elements but the identity of factors that bind to a directly adjacent, conserved UGC repeat remains unknown. Biochemical purification is proposed to identify this factor and determine how it participates with PTB to repress splicing. In contrast to repression of exon 3, activation of exon 2 is needed in smooth muscle cells mediated by four purine-rich enhancer elements and one or more members of the SR protein family of splicing factors. In vivo and in vitro splicing assays will be used to identify which SR proteins are essential for exon 2 activation. In the second part of this proposal, regulation of splicing by SR proteins will be expanded to include characterization of a new SR-related protein (SRrp86) that can activate or repress specific SR family members. The specific targets of SRrp86 are unknown but preliminary data suggests that it functions by protein-protein interaction. Experiments are proposed to determine how such interaction alters SR protein activity and to identify specific SR protein targets. Overall, the experiments in this proposal seek to understand the mechanistic basis underlying the repression and activation of splicing by SR proteins and hnRNP proteins.