Comprehensive determination of the human proteins that define the splicing code

描述(申请人提供)：选择性剪接极大地扩展了人类转录组的复杂性，几乎所有人类基因都产生多个mRNAs，编码不同的蛋白质。剪接调控主要由RNA结合蛋白(RNAbindingProteins，RBPs)介导，它与外显子和内含子序列结合，作为邻近剪接位点或外显子的剪接增强剂或沉默因子。结合在受调控外显子内或附近的许多限制性商业惯例的净活性组合决定了剪接结果。对几种具有良好特性的剪接因子的研究揭示了结合的“RNA图谱”，这些结合图定义了它们是促进还是抑制外显子剪接。因此，预测在给定组织或细胞中表达的剪接因子集合的RNA图谱，以确定全局剪接模式。 因此，这样的图谱将定义不同序列基序和特征的“剪接代码”，可以自信地预测剪接过程中组织特异性的差异。该领域的最新进展表明，组织特异性剪接可以根据RNA序列特征进行预测，并揭示了组成该密码的大量RNA序列基序和特征。然而，在我们对完整剪接代码的理解中仍然存在许多差距。首先，只定义了整个剪接因子集合中的一小部分。其次，与大多数调控剪接的序列基序结合的蛋白质调节器是未知的。第三，一个更完整的剪接代码也可以预测所有细胞类型和条件下更复杂的剪接差异，需要大量新的输入。解决这些问题的第一步将是 完成所有直接调节剪接的人类蛋白质的完整清单，并确定它们的同源结合序列基序。我们将通过以下具体目标进行这一步骤：1)使用全面的人RNA结合蛋白文库进行高通量筛选(HTS)剪接分析。我们将结合先前验证的基于荧光素酶的剪接报告，使用RNA拴系分析来筛选几乎所有人类限制性商业惯例的文库，以增强或抑制不同内含子位置的外显子剪接。这一筛选流程和随后的验证步骤将极大地扩大人类基因组中已知的替代剪接调控因子的集合。2)用多重RNA SELEX-Seq法测定新型剪接调节剂的RNA结合特异性。使用一种创新的新的高通量方法，所有已知和新的剪接调节子的高亲和力结合基序将使用基于细胞的方法来确定，该方法将通过指数富集法(SELEX)和高通量测序(RNA-SEQ)相结合。这些研究的结果将为定义剪接密码的选择性剪接的顺式和反式调控因子提供一个全面的视角。当与现有的实验和生物信息学数据库相结合时，将大大提高我们对剪接密码的定义，并为导致组织特异性剪接的分子机制提供新的见解。

DESCRIPTION (provided by applicant): Alternative splicing massively expands the complexity of the human transcriptome and nearly all human genes produce multiple mRNAs that encode distinct proteins. Regulation of splicing is primarily mediated by RNA binding proteins (RBPs) that bind to exonic and intronic sequences and function as splicing enhancers or silencers of nearby splice sites or exons. The net activities of a number of RBPs that bind within or near regulated exons combinatorially determine the splicing outcome. Studies of several well characterized splicing factors have revealed "RNA maps" of binding that define whether they promote or repress exon splicing. The RNA maps of a collection of splicing factors expressed in a given tissue or cell are therefore predicted to determine global splicing patterns. Such maps will thus define a "splicing code" of diverse sequence motifs and features that can confidently predict tissue-specific differences in splicing. Recent progress in this area has shown that tissue specific splicing can be predicted on the basis of RNA sequence features and revealed a vast collection of RNA sequence motifs and features that comprise this code. However, many gaps in our understanding of the complete splicing code remain. First, only a minority of the total set of splicing factors are defined. Second, the protein regulators that bindto the most of the sequence motifs that regulate splicing are unknown. Third, a more complete splicing code that can also predict more complex differences in splicing in all cell types and conditions requires substantial new inputs. A first step towards resolving these issues will be to complete the full inventory of all human proteins that directly regulate splicing and determine their cognate binding sequence motifs. We will undertake this step through the following specific aims: 1) Perform high throughput screening (HTS) splicing assays using a comprehensive library of human RNA binding proteins. We will use RNA tethering assays in conjunction with previously validated luciferase-based splicing reporters to screen a library of nearly all human RBPs for the ability to enhance or silence exon splicing from different intronic positions. This screening pipeline and subsequent validation steps will vastly expand the set of known alternative splicing regulators in the human genome. 2) Determination of RNA binding specificities for novel splicing regulators by multiplexed RNA SELEX-Seq. Using an innovative new high throughout method the high affinity binding motifs for all known and novel splicing regulators will be determined using a cell-based method to that couples systematic evolution of ligands by exponential enrichment (SELEX) with high throughput sequencing (RNA-Seq). The results from these studies will provide a comprehensive view of the cis- and trans-regulators of alternative splicing that define the splicing code. When couples with existing experimental and bioinformatics databases will substantially improve our definition of the splicing code and provide new insights into the molecular mechanisms that lead to tissue-specific splicing.