The role of non-canonical intronic motifs in splicing

非规范内含子基序在剪接中的作用

• 批准号: 0616264
• 负责人: Andy Berglund
• 金额: --
• 依托单位: University of Oregon Eugene
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0616264&HistoricalAwards=false
• 关键词: role; non; canonical; intronic; motifs

Pre-mRNA splicing is a critical component of gene expression in eukaryotic organisms and understanding how splice sites are recognized is vital to deciphering the regulation and mechanism of this fundamental process. It has become apparent that alternative pre-mRNA splicing plays an important role in increasing the diversity of the proteome in higher eukaryotes. The majority of alternative splicing is regulated at the level of intron recognition. Introns are defined by multiple RNA elements, some of which reside in the intron and some of which reside in flanking exons. The known intronic elements are the 5' splice site, the branchpoint sequence, polypyrimidine (PY) tract and 3' splice site; the latter three elements are usually found in the last 30-40 nucleotides of the intron. The goals of this project are two-fold, (1) determine the mechanisms through which introns that lack a canonical PY tract are recognized and spliced and (2) what is the role of U2AF65, the PY tract binding protein, in the recognition and splicing of these introns lacking a canonical PY tract. Bioinformatics work has revealed three classes of intronic motifs that may function as intronic splicing enhancers (ISEs) that could compensate in the splicing of introns lacking the canonical PY tract. In vivo and in vitro splicing assays are being used to test the hypothesis that these putative ISEs function to compensate for the noncanonical PY tract. Preliminary data indicates that two of the three ISEs function as splicing enhancers and trans-acting factors will be identified using cross-linking and mass spectrometry. Mutant U2AF65 molecules have been created that retain the ability to make the necessary protein-protein interactions for intron recognition but have weakened RNA binding to test the hypothesis that RNA binding of U2AF65 to non-canonical PY tracts is not necessary for intron recognition and splicing.Broader Impacts:Most human genes are more intron than exon, yet the majority of intron sequences serve no known purpose. An interesting possibility is that many introns harbor a variety of regulatory elements that dictate when splicing will take place and which potential splice sites will be used. This study focuses on novel intronic sequence motifs, and will provide information about the relatively unexplored sequence diversity of introns. The bioinformatics work has lead to the creation of a webtool termed the Intron Motif Finder (introns.uoregon.edu), which allows researchers without programming skills to manipulate large sequence databases to easily identify introns containing motifs of interest. This tool is also being used in the classroom to teach undergraduate students how to begin performing bioinformatics analysis. For example, a class of 45 biochemistry students read a paper describing how the splicing factor NOVA regulates splicing through intronic UCAY motifs, the students then went on to use the Intron Motif Finder to identify potentially new exons that NOVA might regulate by searching for introns with multiple UCAY motifs downstream and upstream of exons.

mRNA 前体剪接是真核生物基因表达的关键组成部分，了解剪接位点的识别方式对于破译这一基本过程的调节和机制至关重要。很明显，可变的前 mRNA 剪接在增加高等真核生物中蛋白质组的多样性方面发挥着重要作用。大多数选择性剪接在内含子识别水平上受到调节。内含子由多个 RNA 元件定义，其中一些位于内含子中，一些位于侧翼外显子中。已知的内含子元件有5'剪接位点、分支点序列、多聚嘧啶(PY)束和3'剪接位点；后三个元件通常在内含子的最后30-40个核苷酸中发现。该项目的目标有两个，(1) 确定缺乏规范 PY 束的内含子被识别和剪接的机制；(2) PY 束结合蛋白 U2AF65 在这些缺乏规范 PY 束的内含子的识别和剪接中的作用是什么。生物信息学工作揭示了三类内含子基序，它们可能充当内含子剪接增强子（ISE），可以补偿缺乏规范 PY 束的内含子剪接。体内和体外剪接测定被用来检验这些假定的 ISE 具有补偿非规范 PY 束功能的假设。初步数据表明，三个 ISE 中的两个充当剪接增强子，并且将使用交联和质谱法来鉴定反式作用因子。突变的 U2AF65 分子已被创建，保留了内含子识别所需的蛋白质-蛋白质相互作用的能力，但削弱了 RNA 结合，以测试以下假设：U2AF65 与非规范 PY 束的 RNA 结合对于内含子识别和剪接不是必需的。更广泛的影响：大多数人类基因的内含子数量多于外显子，但大多数内含子序列没有已知的用途。一个有趣的可能性是，许多内含子含有多种调控元件，这些元件决定剪接何时发生以及将使用哪些潜在剪接位点。这项研究重点关注新的内含子序列基序，并将提供有关相对未探索的内含子序列多样性的信息。生物信息学工作导致创建了一个名为“内含子基序查找器”(introns.uoregon.edu) 的网络工具，它允许没有编程技能的研究人员操作大型序列数据库，以轻松识别包含感兴趣基序的内含子。该工具还在课堂上使用，教本科生如何开始进行生物信息学分析。例如，一个由 45 名生物化学学生组成的班级阅读了一篇论文，描述了剪接因子 NOVA 如何通过内含子 UCAY 基序调节剪接，然后学生们继续使用内含子基序查找器，通过搜索外显子下游和上游具有多个 UCAY 基序的内含子来识别 NOVA 可能调节的潜在新外显子。