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GENETIC ANALYSIS OF TRNA SPLICING IN THE YEAST NUCLEUS

酵母细胞核中 TRNA 剪接的遗传分析

基本信息

批准号：
2616395
负责人：
MICHAEL R CULBERTSON
金额：
$ 5.94万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1999-08-31
项目状态：
已结题

项目摘要

Intervening sequences are spliced from eukaryotic precursor-tRNAs through the action of three enzymes, a heterotrimeric endonuclease, a multi-functional but monomeric ligase, and an NAD-linked phosphatase. The endonuclease and ligase form a physical complex that is localized in the nuclear periphery in the vicinity of nuclear pores, suggesting the potential for functional coupling of splicing and nuclear export. Two genes, SEN1 and SEN2, were identified previously in the yeast Saccharomyces cerevisiae that are required for endonuclease activity. SEN1 codes for a nuclear-localized, positive effector of endonuclease activity that appears not to be a catalytic subunit of the enzyme. SEN2 codes for the 42 Kd catalytic subunit of the enzyme. We have isolated mutations in six new genes that affect SEN1 function. Mutations in two of the genes, SEN3 and SEN4, affect the expression of SEN1 at a post-translational level. One temperature-sensitive mutation, sen3-l, also causes excess accumulation of tRNA splicing intermediates in vivo. In addition, four genes have been identified that suppress the temperature-sensitive senl-l mutation when present in multiple copies and may encode products that interact with the SEN1 protein. By continuing with our analysis of SEN1 and SEN2 function and by initiating analyses of SEN3, SEN4, and the high-copy suppressor genes, we hope to provide critical information on the structure, organization, function, and intracellular location of the splicing complex. It is also possible that we may learn something about the import of splicing enzymes into the nucleus and the export of spliced tRNA products from the nucleus.

干预序列是通过以下方式从真核前体-tRNA拼接而来的三种酶的作用，异源三聚体核酸内切酶，多功能但单体连接酶和NAD连接的磷酸酶。核酸内切酶和连接酶形成定位于细胞中的物理复合物。核孔附近的核周边，表明剪接和核输出的功能性偶联的可能性。两 SEN 1和SEN 2基因先前在酵母中被鉴定酿酒酵母的核酸内切酶活性所必需的。 SEN 1编码核酸内切酶的核定位阳性效应子活性似乎不是酶的催化亚基。 Sen2 编码该酶的42 Kd催化亚基。我们已经分离出六个新基因的突变影响SEN 1功能。两个突变基因SEN 3和SEN 4影响SEN 1的表达，翻译后水平。一个温度敏感突变sen 3-l 也导致体内tRNA剪接中间体的过量积累。此外，已经鉴定出四种基因，当以多个拷贝存在时，可能编码与SEN 1蛋白相互作用的产物。若继续通过我们对SEN 1和SEN 2功能的分析， SEN 3，SEN 4和高拷贝抑制基因的表达，我们希望提供关于结构、组织、职能和剪接复合物的细胞内定位。也有可能我们可能会了解到一些关于剪接酶进入细胞的信息，细胞核和剪接的tRNA产物从细胞核输出。