Uncovering Mechanisms of 5' Splice Site Fidelity

揭示 5 剪接位点保真度的机制

基本信息

批准号：
10532793
负责人：
ALAN M ZAHLER
金额：
$ 36.97万
依托单位：
UNIVERSITY OF CALIFORNIA SANTA CRUZ
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-15 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10532793
关键词：
5&apos Splice Site Active Sites Affect Alleles Binding Biochemical Biochemistry Biological Assay Caenorhabditis elegans Catalysis Catalytic Domain Cells Code Complex Cryoelectron Microscopy DNA Data Dinucleoside Phosphates Ensure Eukaryota Event Explosion Genes Genetic Genetic Diseases Genetic Screening Genomics Goals Hereditary Disease Homologous Gene Human Human Genetics Introns Learning Messenger RNA Microscopic Modeling Molecular Conformation Mutation Nature Nucleotides Positioning Attribute Prevalence Process Proteins RNA RNA Sequences RNA Splicing RNA, Messenger, Splicing Reaction Regulation Role Saccharomyces cerevisiae Secure Site Slide Small Nuclear RNA Spliceosome Assembly Pathway Spliceosomes Suppressor Mutations System Testing Time Translational Research Yeasts dominant genetic mutation falls human disease mRNA Precursor mutant

项目摘要

PROJECT SUMMARY/ABSTRACT Precursor messenger RNA (pre-mRNA) splicing is essential in higher eukaryotes in order to produce functional messenger RNAs to code for proteins. The splicing reaction is carried out by a large macromolecular machine called the spliceosome. The spliceosome assembles onto pre-mRNA substrates through a complex binding, rearrangement and release of 5 small nuclear RNAs and over 100 associated proteins. The exact roles of many of the spliceosome-associated factors are poorly understood. Our lab uses the microscopic worm C. elegans as a model to explore pre-mRNA splicing through integrated genetic, genomic and biochemical approaches. We have established sensitives genetic screens to identify factors important for the accurate assembly of the spliceosome at the 5' splice site. Our data suggest the hypothesis that key spliceosomal components have an important role in securing the accurate transfer of the 5' splice site from its initial recognition by U1snRNP to its loading into the active site of the spliceosome. The dominant suppressor mutations that we uncovered fall into two classes; 1) those that allow the spliceosome to remain in an open conformation, which allows the 5' splice site position to slide between two regions 23nt apart during loading into the active site and 2) those that promote the usage of an intron that begins with an unusual UU dinucleotide. We will explore the following questions: 1. How do protein factors control the accurate selection of 5' splice sites during spliceosome assembly? We hypothesize that components of the assembled spliceosome control the final determination of 5' splice site selection. We will study how alleles of PRPF8, SNRNP27 and SNRNP200 (Brr2) affect precise 5' splice site usage in worms. Because of the prevalence of human genetic disease alleles with mutations identical to the ones explored in our genetic screen, we also plan to take our studies into human cells to determine if the alleles identified in worms can be targets for translational research into splicing regulation. 2. How do KIN17 and PRCC maintain 5'ss fidelity? We have found a phenomenon in which two 5'ss separated by a single nucleotide are both used even in the presence of a wild type spliceosome. We have identified a class of dominant suppressors that promote usage of introns that begin with UU instead of the canonical GU dinucleotide. These alleles are in the worm homologs of human KIN17 and PRCC; little is known about a role for either protein in splicing other than that they transiently interact with mammalian Bact spliceosomes. We now have functional splicing assays for these factors to exploit in characterizing their function in maintaining splice site fidelity.

项目摘要/摘要前体信使RNA（前MRNA）剪接在较高的真核生物中至关重要，以生产功能性信使RNA代码蛋白质。剪接反应是由大的大分子机器称为剪接体。剪接组合到前mRNA底物上通过复杂的结合，重排和释放5个小核RNA和超过100个相关的蛋白质。许多与剪接体相关的因素的确切作用鲜为人知。我们的实验室使用微观蠕虫秀丽隐杆线虫作为模型，通过综合遗传，基因组和生化方法。我们已经建立了灵敏度遗传筛选以识别因素对于在5'剪接位点精确组装剪接体的精确组装很重要。我们的数据表明假设关键的剪接组成分在确保准确转移的准确转移方面具有重要作用从U1SNRNP的初始识别到其加载到剪接体的活跃位点的5'剪接位点。我们发现的主要抑制突变分为两个类别。 1）那些允许剪接体保持开放构象，允许5'剪接位点位置在两个之间滑动在加载到活动地点的过程中，区域分开23nt，以及2）促进内含子使用的区域从异常的UU二核苷酸开始。我们将探讨以下问题： 1。蛋白质因子如何控制剪接组装过程中5'剪接位点的准确选择？我们假设组装剪接体的组件控制5'剪接的最终测定站点选择。我们将研究PRPF8，SNRNP27和SNRNP200（BRR2）的等位基因如何影响精确的5'剪接蠕虫的现场使用。由于人类遗传疾病等位基因的流行，突变与在我们的遗传筛查中探讨的那些，我们还计划将研究带入人类细胞，以确定是否是否在蠕虫中鉴定出的等位基因可以是转化剪接调节的目标。 2. KIN17和PRCC如何保持5's忠诚？我们发现了一种现象，其中，即使在存在野生型剪接体。我们已经确定了一类主导抑制器，以促进用UU开头的内含子使用而不是规范的GU Dinucleotide。这些等位基因在蠕虫中人类Kin17和PRCC的同源物；关于任何一种蛋白质在剪接以外的蛋白质的作用知之甚少它们与哺乳动物BACT剪接体瞬时相互作用。我们现在有功能剪接测定法这些因素可以利用它们在维持剪接站点保真度中的功能方面的功能。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A forward genetic screen in C. elegans identifies conserved residues of spliceosomal proteins PRP8 and SNRNP200/BRR2 with a role in maintaining 5' splice site identity.

通过对 elegans 进行前向遗传筛选，发现剪接体蛋白 PRP8 和 SNRNP200/BRR2 的保守残基在维持 5'剪接位点一致性方面发挥作用。

DOI：
10.1093/nar/gkac991
发表时间：
2022-11-11
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Cartwright-Acar, Catiana H.;Osterhoudt, Kenneth;Suzuki, Jessie M. N. G. L.;Gomez, Destiny R.;Katzman, Sol;Zahler, Alan M.
通讯作者：
Zahler, Alan M.

A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5' splice site identity.