ALTERNATIVE SPLICING OF CONTRACTILE PROTEIN GENES

收缩蛋白基因的选择性剪接

• 批准号: 3157783
• 负责人: Bernardo Nadal Ginard
• 金额: $ 15.29万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1990-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157783
• 关键词: RNA splicing; developmental genetics; gene expression; genetic promoter element; genetic transcription; messenger RNA; myosins; nucleic acid probes; nucleic acid sequence; sarcomeres; striated muscles; tropomyosin; troponin

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.

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) 是肌节肌肉中表达的三种基因。 每一个 这些基因中的一些会产生多个 mRNA，每个 mRNA 编码不同的 蛋白质亚型，通过选择性剪接过程。 在这种情况下 MLC1/3 基因，两个选择性剪​​接的 mRNA（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 在相同的多聚A添加位点。 Alpha-TM 基因至少产生 三个不同的 mRNA 在 5' 和 3' 处交替剪接 结束。 初级转录本的构象可能与 5'端、3'端或两者的差异是造成 决定选择性剪接的剪接位点选择 途径。 另一方面，肌钙蛋白 T 至少产生 10 种不同的 mRNA，有可能生成多达 64 个具有相同 5' 和 3' 的 mRNA 以来自独特初级的内部外显子的选择性剪接结束 成绩单。 由于这些不同 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 扩散因子参与剪接途径的调节 这个单一的初级转录本。 该提案的具体目标有三个： 1) 确定 初级转录本结构和假定的相对作用 调控选择性剪接生产中的反式作用因子 肌肉细胞中的模式。 2) 使用特定的基因构建体，确定 涉及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) 开始使用体外表征这些因素 和体内剪接系统。