Comprehensive determination of the human proteins that define the splicing code

全面测定定义剪接代码的人类蛋白质

• 批准号: 8513387
• 负责人: RUSS Paul CARSTENS
• 金额: $ 20万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-18 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513387
• 关键词: Accounting; Affinity; Alternative Splicing; Area; Binding; Binding Sites; Bioinformatics; Biological Assay; Cells; Chimeric Proteins; Code; Collection; Complementary DNA; Complex; Couples; Databases; Destinations; Development; Disease; Enhancers; Equipment and supply inventories; Evolution; Exons; Gene Expression Profile; Gene Expression Regulation; Genes; Goals; Health; Human; Human Genome; Indium; Lead; Libraries; Ligands; Luciferases; Malignant Neoplasms; Maps; Mediating; Messenger RNA; Methods; Minority; Molecular; Outcome; Pattern; Peptides; Positioning Attribute; Process; Production; Protein Binding; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Resources; Site; Specificity; Staging; Tissue-Specific Splicing; Tissues; Transcript; Validation; Variant; base; cDNA Library; cell type; combinatorial; disorder risk; genome-wide; high throughput screening; human disease; improved; innovation; insight; novel; preference; programs; screening; transcriptome sequencing; vector

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.

描述（由申请人提供）：替代剪接大大扩展了人类转录组的复杂性，几乎所有人类基因都会产生编码不同蛋白质的多个mRNA。 剪接的调节主要是由RNA结合蛋白（RBP）介导的，这些蛋白（RBP）与外显子和内含子序列结合，并作为剪接增强子或附近剪接位点或外显子的剪接增强子或消音器的作用。 在组合内部或附近绑定的许多RBP的净活动在组合中决定了剪接结果。 对几个良好特征的剪接因子的研究表明，结合的“ RNA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图被预测确定全局剪接模式。 因此，这样的地图将定义一个不同序列基序和特征的“剪接代码”，这些图案可以确保预测剪接中组织特异性的差异。 该领域的最新进展表明，可以根据RNA序列特征预测组织特异性剪接，并揭示了大量RNA序列基序和包含该代码的特征。 但是，我们对完整剪接代码的理解中存在许多差距。 首先，仅定义了总剪接因子的少数。其次，与调节剪接的大多数序列基序结合的蛋白质调节剂是未知的。 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的剪接上更复杂的差异，需要大量的新输入。 解决这些问题的第一步将是 完成所有直接调节剪接并确定其同源结合序列基序的所有人类蛋白质的完整清单。 我们将通过以下特定目的进行此步骤：1）使用人RNA结合蛋白的综合库进行高吞吐量筛选（HTS）剪接测定。 我们将使用RNA绑定测定法与先前验证的基于荧光素酶的剪接记者结合使用，以筛选几乎所有人类RBP的库，以从不同的内含子位置增强或沉默外显子剪接的能力。这种筛选管道和随后的验证步骤将大大扩展人类基因组中的一组已知替代剪接调节剂。 2）通过多重RNA SELEX-SEQ测定新剪接调节剂的RNA结合特异性。 使用创新的新方法，将使用基于细胞的方法来确定所有已知和新型剪接调节剂的高亲和力结合基序，以通过高指数富集（SELEX）和高吞吐量测序（RNA-SEQ）通过指数富集（SELEX）进行系统演化。 这些研究的结果将为定义剪接代码的替代剪接的顺式和反式调节剂提供全面的看法。 当与现有的实验和生物信息学数据库的夫妻夫妻夫妇将基本上改善我们对剪接代码的定义，并为导致组织特异性剪接的分子机制提供新的见解。