Mechanisms Regulating Alternative pre-mRNA Splicing

大多数真核基因被非编码内含子中断，非编码内含子必须从前mRNA转录物中去除以产生功能蛋白。内含子的剪接是非常有效和快速的，考虑到从外显子中描绘内含子的信号在高等真核生物中并不保守，这是一个惊人的壮举。 剪接在正常细胞功能中的重要性的必然结果是，约15%的特征性遗传疾病涉及导致剪接缺陷的突变。此外，60%或更多的人类基因经历选择性剪接，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，而且能够准确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节，重点是特定剪接位点被激活或抑制的分子机制。在这个建议的第一部分，实验的目的是了解相互排斥的外显子2和3的α-原肌球蛋白基因的调节。对控制剪接所需的顺式作用序列的解剖已经确定了在平滑肌细胞中抑制剪接所需的外显子3侧翼的元件。PTB（hnRNP I）结合这些元件之一，但与直接相邻的保守UGC重复序列结合的因子的身份仍然未知。建议进行生化纯化以鉴定该因子并确定其如何参与PTB抑制剪接。与外显子3的抑制相反，在平滑肌细胞中需要外显子2的激活，其由四个富含嘌呤的增强子元件和一个或多个剪接因子的SR蛋白家族成员介导。体内和体外剪接试验将用于鉴定哪些SR蛋白是外显子2激活所必需的。在本提案的第二部分，SR蛋白的剪接调控将扩大到包括一个新的SR相关蛋白（SRrp 86），可以激活或抑制特定的SR家族成员的表征。SRrp 86的具体靶点尚不清楚，但初步数据表明它通过蛋白质-蛋白质相互作用发挥作用。实验提出，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蛋白的目标。总体而言，本提案中的实验旨在了解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.