The role of non-canonical intronic motifs in splicing

Pre-mRNA剪接是真核生物基因表达的一个重要组成部分，了解剪接位点如何被识别对于破译这一基本过程的调控和机制至关重要。很明显，替代前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，另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、聚嘧啶（PY）链和3′剪接位点；后三种元素通常位于内含子的最后30-40个核苷酸中。这个项目的目标有两个方面，(1)确定缺乏典型PY束的内含子被识别和剪接的机制，(2)PY束结合蛋白U2AF65在这些缺乏典型PY束的内含子的识别和剪接中起什么作用。生物信息学工作揭示了三种可能作为内含子剪接增强子（ise）的内含子基序，它们可以补偿缺乏典型PY通道的内含子剪接。体内和体外剪接试验被用来测试这些假定的ise功能来补偿非规范的PY束的假设。初步数据表明，三种ise中的两种具有剪接增强子的功能，并且将使用交联和质谱法确定反式作用因子。突变的U2AF65分子保留了识别内含子所需的蛋白质-蛋白质相互作用的能力，但削弱了RNA结合，以验证U2AF65与非规范PY束的RNA结合对内含子识别和剪接不是必需的假设。更广泛的影响：大多数人类基因是内含子多于外显子，然而大多数内含子序列没有已知的目的。一个有趣的可能性是，许多内含子含有多种调控元件，这些元件决定了剪接何时发生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，并将提供有关相对未开发的内含子序列多样性的信息。生物信息学工作导致了一个名为内含子Motif Finder的网络工具的创建（introns.uoregon.edu），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的内含子。这个工具也在课堂上用于教授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Motif Finder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。

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.