MECHANISMS OF ALTERNATIVE SPLICING OF PRE MRNA

PRE mRNA 的选择性剪接机制

基本信息

批准号：
2185763
负责人：
James L. Manley
金额：
$ 36.97万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-07-01 至 1996-06-30
项目状态：
已结题

项目摘要

Alternative splicing plays a crucial role in the life cycle of the DNA tumor virus SV40, as well as other viruses, and in the expression of numerous cellular genes. For example, the SV40 early transcript can be spliced to produce the mRNAs encoding both the large tumor antigen, which is required for both viral replication and oncogenic cell transformation, and the small tumor antigen, whose function is also related to cell growth. Despite significant advances in our understanding of the general mechanism of pre mRNA splicing, little is known about the factors that control alternative splicing. This question is of particular significance because of the increasing evidence that regulated splicing plays an important role not only in the replication of oncogenic viruses, but also in the control of genes that are expressed at different developmental stages or in different tissues. The experiments proposed here are designed to provide insights into the factors that control alternative splicing, how they function and how they themselves are controlled. The following specific issues will be addressed: Using in vitro mutagenesis techniques, sequences crucial for controlled alternative splicing of SV40 pre mRNAs will be identified both by transfection of different types of cultured mammalian cells, and by micro-injection of X. laevis oocytes. The pathways and parameters of SV40 early splicing will be investigated by using extracts of mammalian cells to process pre mRNAs in vitro. Also using in vitro processing systems, the identities of trans-acting factors that influence the choice of alternative splicing pathways will be determined. Possible changes in the nature of trans-acting factors during development will be investigated by introducing SV40 RNA (or DNA) into X. laevis oocytes, ova and embryos. Finally, regulation of genes that encode splicing factors, the human U1 and U2 genes, will be studied, and the possibility that SV40 T antigen, or a cellular homologue, can affect expression of these genes will be investigated.

选择性剪接在植物的生命周期中起着至关重要的作用 DNA肿瘤病毒SV40以及其他病毒，并且在许多细胞基因的表达。例如，SV40 早期转录本可以被剪接以产生编码的mRNA 两种病毒都需要大肿瘤抗原复制和致癌细胞转化，以及小肿瘤抗原，其功能也与细胞生长有关。尽管我们对一般性的理解取得了重大进展对于pre mRNA剪接的机制，人们知之甚少。控制选择性剪接的因素。这个问题是关于特别重要的是因为越来越多的证据表明调控剪接不仅在致癌病毒的复制，还在于基因的控制表现在不同的发育阶段或不同的组织。这里提出的实验旨在提供对控制选择性剪接的因素的见解，它们如何运作以及它们本身如何受到控制。这将解决以下具体问题：使用体外诱变技术，对于控制至关重要的序列 SV40 pre mRNA 的选择性剪接将通过以下方式鉴定转染不同类型的培养哺乳动物细胞，以及通过显微注射 X. laevis 卵母细胞。途径和 SV40早期剪接的参数将通过使用进行研究哺乳动物细胞提取物在体外加工前 mRNA。还使用体外处理系统，反式作用的身份影响选择性剪接途径选择的因素将被确定。交易性质可能发生的变化将通过引入来调查开发过程中的因素 SV40 RNA（或 DNA）进入非洲爪蟾卵母细胞、卵子和胚胎。最后，编码剪接因子的基因的调控，人类 U1 和 U2 基因将被研究，并且可能性 SV40 T 抗原或细胞同源物可以影响这些基因将被研究。