MECHANISMS OF ALTERNATIVE SPLICING OF PRE MRNA

选择性剪接在细胞的生命周期中扮演着至关重要的角色 DNA肿瘤病毒SV40以及其他病毒，以及在 大量细胞基因的表达。例如，SV40 早期的转录本可以被拼接以产生编码的mRNAs 两种病毒所需的大肿瘤抗原 复制和致癌细胞转化，以及小细胞 肿瘤抗原，其功能也与细胞生长有关。 尽管我们对将军的理解有了很大进步 Pre-mRNA剪接的机制，人们对其知之甚少 控制备选剪接的因素。这个问题是关于 特别重要，因为越来越多的证据表明 受调控的剪接不仅在 致癌病毒的复制，也在基因的控制下 在不同的发育阶段或在 不同的组织。这里提出的实验旨在 提供对控制可选剪接的因素的见解， 它们是如何运作的，它们本身又是如何被控制的。这个 将解决以下具体问题：在体外使用 突变技术，对受控至关重要的序列 SV40前mRNA的替代剪接将通过以下两种方式识别 不同类型培养的哺乳动物细胞的转基因，以及 通过显微注射莱氏X.laevis卵母细胞。这些小路和 SV40早期剪接参数的研究将使用 用于在体外处理前mRNAs的哺乳动物细胞的提取物。也是 利用体外处理系统，反式作用的身份 影响选择性剪接途径选择的因素 将会被确定。反动行为的性质可能发生变化 开发过程中的因素将通过引入 将SV40RNA(或DNA)导入乳突圆线虫卵母细胞、卵子和胚胎中。 最后，对编码剪接因子的基因的调控， 人类的U1和U2基因将被研究，并且有可能 SV40T抗原或细胞同系物可以影响其表达 这些基因将被研究。

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.