MECHANISMS OF ALTERNATIVE SPLICING OF PRE MRNA

选择性剪接在细胞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 大的肿瘤抗原，这是两种病毒所必需的， 复制和致癌细胞转化，以及小的 肿瘤抗原，其功能也与细胞生长有关。 尽管我们对宇宙的理解有了很大的进步， 前mRNA剪接的机制，很少有人知道， 控制可变剪接的因素。 这个问题是 特别重要的是，越来越多的证据表明， 调控剪接不仅在细胞内起重要作用， 复制致癌病毒，而且在控制基因 在不同的发育阶段表达， 不同的组织 这里提出的实验旨在 提供控制可变剪接的因素的见解， 它们是如何运作的以及它们自身是如何被控制的。 的 将解决以下具体问题： 诱变技术，控制基因突变的关键序列 SV40前体mRNA的选择性剪接将通过以下两种方法进行鉴定： 转染不同类型的培养的哺乳动物细胞，和 通过微量注射X.卵母细胞 道路和 SV40早期剪接的参数将通过使用 哺乳动物细胞提取物在体外加工前mRNA。 也 使用体外处理系统，反式作用的身份 影响选择性剪接途径的因素 将被确定。 交易行为性质的可能变化 在开发过程中的因素将通过引入 SV40 RNA（或DNA）插入X. 卵母细胞、卵子和胚胎。 最后，编码剪接因子的基因的调节， 人类U1和U2基因，将被研究， SV40 T抗原或细胞同源物可影响 将对这些基因进行研究。

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.