ALTERNATIVE SPLICING OF CONTACTILE PROTEIN GENES

碱性肌球蛋白轻链（MLC 1/3）、α-原肌球蛋白（Alpha-TM）和 肌钙蛋白T（TNT）是在肌节肌肉中表达的三种基因。 每一个 这些基因中的每一个产生多个mRNA，每个mRNA编码不同的 蛋白质异构体，通过选择性剪接的过程。 的情况 MLC 1/3基因，两个选择性剪接的mRNA（编码MLC 1和MLC 3， 分别）是由两个不同的和重叠的 启动子相隔10 kb但终止于 在相同的聚腺苷酸添加位点。 Alpha-TM基因产生最低限度的 在5'和3'端都选择性剪接的三种不同mRNA， 形接头. 初级转录本的构象可能与 在5'端、3'端或两者的差异，负责 剪接位点的选择决定了可变剪接 途径。 另一方面，肌钙蛋白T产生至少10种不同的 mRNA，具有产生多达64个的潜力，具有相同的5'和3'端， 通过选择性剪接内部外显子从一个独特的初级 成绩单。 由于这些不同的TNT mRNA亚型的表达， 发育和组织特异性，很可能是细胞特异性 扩散因子参与剪接途径的调节 这份原始记录 该提案的具体目标有三个方面：1）确定 初级转录本和假定转录本结构的相对作用 调节可变剪接产生中的反式作用因子 在肌肉细胞中的模式。 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， 细胞 3)使用体外方法开始表征这些因子 和体内剪接系统。

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.