The role of non-canonical intronic motifs in splicing

前体mRNA剪接是真核生物基因表达的重要组成部分，了解剪接位点是如何被识别的对于解释这一基本过程的调控和机制至关重要。很明显，选择性前体mRNA剪接在增加高等真核生物蛋白质组多样性中起着重要作用。大多数选择性剪接是在内含子识别水平上调节的。内含子由多个RNA元件定义，其中一些位于内含子中，另一些位于侧翼外显子中。已知的内含子元件是5'剪接位点、分支点序列、多聚嘧啶（PY）区和3'剪接位点;后三个元件通常存在于内含子的最后30-40个核苷酸中。该项目的目标是双重的，（1）确定缺乏典型PY束的内含子被识别和剪接的机制，以及（2）PY束结合蛋白U2 AF 65在识别和剪接这些缺乏典型PY束的内含子中的作用。生物信息学工作揭示了三种类型的内含子基序，它们可能充当内含子剪接增强子（ISE），可以补偿缺乏典型PY区的内含子的剪接。体内和体外剪接试验被用来测试的假设，这些推定的ISE功能，以弥补非经典的PY道。初步数据表明，三个ISE中的两个作为剪接增强子和反式作用因子的功能将使用交联和质谱法进行鉴定。突变的U2 AF 65分子已被创建，保留了必要的蛋白质-蛋白质相互作用的能力，为内含子识别，但削弱了RNA结合，以测试的假设，即RNA结合的U2 AF 65非典型的PY tracts是不必要的内含子recognition和splicing.Broader的影响：大多数人类基因更多的内含子比外显子，但大多数内含子序列服务于未知的目的。一个有趣的可能性是，许多内含子含有多种调控元件，这些元件决定何时发生剪接以及使用哪些潜在的剪接位点。本研究的重点是新的内含子序列基序，并将提供信息的相对未开发的内含子序列多样性。生物信息学工作已经导致创建了一个名为Intron Motif的网络工具（introns.uoregon.edu），该工具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的基序的内含子。这个工具也被用在课堂上教本科生如何开始进行生物信息学分析。例如，一个45人的生物化学学生班阅读了一篇描述剪接因子NOVA如何通过内含子UCAY基序调节剪接的论文，然后学生们继续使用内含子基序识别NOVA可能通过搜索外显子下游和上游具有多个UCAY基序的内含子来调节的潜在新外显子。

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.