ALTERNATIVE SPLICING OF CONTRACTILE PROTEIN GENES

碱性肌球蛋白轻链(MLC1/3)、α-原肌球蛋白(Alpha-TM)和 肌钙蛋白T(TnT)是在肌肉中表达的三个基因。每一个都是 这些基因产生多个mRNAs，每个mRNAs编码一个不同的 蛋白质异构体，通过选择性剪接过程。在这种情况下 MLC1/3基因，两个交替剪接的mRNAs(编码MLC1和MLC3， 分别)是由两个不同且重叠的 启动子相距10kb但终止的转录单位 在相同的聚A加成部位。Alpha-TM基因产生最低限度的 在5‘和3’端交替剪接的三个差异mRNA 结束了。初级转录产物的构象可能与 在5‘端、3’端或两者之间的差异是导致 确定替代拼接的拼接位置选择 小路。另一方面，肌钙蛋白T至少产生10种不同的 有潜力产生高达的mRNA，具有相同的5‘和3’ 末端由来自唯一的初级基因的内部外显子的选择性拼接而成 文字记录。由于这些不同的TNT mRNA亚型的表达是 发育和组织特异性，很可能是细胞特异性 可扩散因子参与剪接通路的调节 这份主要的成绩单。 这项建议的具体目标有三个方面：1)确定 初级转录本的结构和推测的相对作用 调控选择性剪接产生中的反式作用因素 肌肉细胞中的模式。2)使用特定的基因结构，以确定 参与的反式作用因子的细胞和/或基因特异性 通过研究它们在肌肉和非肌肉中的表达来实现选择性剪接 细胞。3)启动这些因子的体外表征 以及体内的剪接系统。

Alkali myosin light chain (MLC1/3), Alpha-tropomyosin (Alpha-TM) and troponin T (TNT) are three genes expressed in sarcomeric muscles. Each one of these genes generates multiple mRNAs, each coding for a different protein isoform, by a process of alternative splicing. In the case of the MLC1/3 gene, two alternatively spliced mRNAs (coding for MLC1 and MLC3, respectively) are produced from two different and overlapping transcriptional units whose promoters are located 10kb apart but terminate at the same poly A addition site. The Alpha-TM gene produces a minimum of three difference mRNAs that are alternatively spliced at both the 5' and 3' ends. It is possible that the conformation of primary transcript with differences at either the 5' end, the 3' end or both, is responsible for the splice site selection that determines the alternative splicing pathways. Troponin T on the other hand, produces a minimum of 10 different mRNAs, with the potential to generate up to 64, with identical 5' and 3' ends by alternative splicing of internal exons from a unique primary transcript. Since the expression of these different TNT mRNA isoforms is developmental and tissue-specific, it is likely that cell-specific diffusable factors are involved in the regulation of the splicing pathways of this single primary transcript. The specific aims of this proposal are three fold: 1) To determine the relative role of the structure of primary transcripts and putative trans-acting factors in the production of regulated alternative splicing pattern in muscle cells. 2) Using specific gene constructs, to determine the cell and/or gene-specificity of the trans-acting factors involved in alternative splicing by studying their expression in muscle and non-muscle cells. 3) To initiate the characterization of these factors using in vitro and in vivo splicing systems.