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UPF GENE PRODUCT FUNCTION IN TURNOVER OF NATURAL MRNAS

天然 MRNAS 周转中的 UPF 基因产物功能

基本信息

批准号：
2518828
负责人：
JEFFREY N DAHLSEID
金额：
$ 2.86万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-09-01 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/2518828
关键词：
Saccharomyces cerevisiae cell cycle centromere chromosome movement fungal genetics gene expression genetic regulation messenger RNA

项目摘要

UPF genes accelerate nonsense mRNA decay in Saccharomyces cerevisiae. Recent evidence indicates UPF genes directly modulate the decay of the natural CTF13 mRNA, which encodes an essential kinetochore protein. This proposal will focus on understanding the UPF-mediated decay of natural mRNA targets that code for kinetochore proteins. We aim to determine the nature of the CTF13 mRNA decay pathway, to determine to what extent UPF genes affect kinetochore mRNA levels, and to investigate the molecular mechanism of UPF-mediated decay of natural mRNAs. We will analyze the intermediates in the decay of CTF13 mRNA to determine if it is degraded by a deadenylation-dependent or -independent pathway. We will determine whether CTF13 mRNA is decapped and degraded exonucleolyticly 5'-->3' or whether it decays by another pathway. We will examine the steady-state levels and half-lives of two other kinetochore mRNAs encoded by CTF14 and CBF3b to determine if they are directly modulated by UPF function. If two or more kinetochore mRNAs are affected, we will address the possibility that UPF genes regulate the expression of kinetochore mRNAs during the cell cycle, by monitoring CTF13 mRNA accumulation in synchronized cells carrying UPF loss-of-function alleles. CTF13 and potentially other direct targets of UPF function will be studied further to define the sequences or structures in the mRNAs that allow them to be recognized as substrates of the UPF-mediated decay pathway. This will be accomplished through the analysis of target mRNAs either containing deletions or as part of lacZ gene fusions. Gel mobility shift assays will be used to address what protein(s) bind the recognition sequence(s) identified above. The long- range goal is to understand what cellular purpose is served by UPF- mediated decay of specific, natural mRNAs. As UPF-mediated mRNA decay modulates kinetochore gene expression, its study has health-related importance for cancer.

UPF基因加速酿酒酵母中无意义的mRNA衰退。最近的证据表明，UPF基因直接调节细胞的腐烂天然CTF13信使核糖核酸，编码一种重要的动粒蛋白。这提案将侧重于了解UPF介导的自然腐烂编码动粒蛋白的信使核糖核酸。我们的目标是确定 CTF13mRNA衰变途径的性质，以确定UPF在多大程度上影响动粒mRNA水平的基因，并研究其分子水平 UPF介导的天然mRNAs衰变机制。我们将分析 CTF13信使核糖核酸降解过程中的中间体一种依赖于或非依赖于去烯基化的途径。我们将决定 CTF13基因是否被5‘--&gt；3’或不管它是否通过另一条途径腐烂。我们将研究稳态 CTF14和CTF14编码的另外两个动粒mRNAs的水平和半衰期 CBF3b以确定它们是否直接受到UPF功能的调制。如果是两个或者更多的动粒mRNAs受到影响，我们将解决这种可能性 UPF基因调控着动粒mRNAs的表达细胞周期，通过监测同步化细胞中CTF13基因的积累携带UPF功能丧失等位基因。CTF13和可能的其他直接将进一步研究UPF函数的目标，以定义序列或 MRNAs中的结构，允许它们被识别为 UPF介导的衰变途径。这将通过含有缺失或作为LacZ一部分的靶基因的分析基因融合。凝胶迁移率变化分析将用于解决蛋白质(S)与上面确定的识别序列(S)结合。长的- 射程目标是了解UPF的蜂窝目的是什么- 介导特定的、自然的mRNAs的衰变。作为UPF介导的mRNA衰变对动粒基因表达的调控，其研究与健康有关对癌症的重要性。