Cross-regulation between transcription and pre-mRNA splicing

转录和前 mRNA 剪接之间的交叉调节

• 批准号: 9133424
• 负责人: Karla M Neugebauer
• 金额: $ 38.6万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9133424
• 关键词: 3' Splice Site; Address; Alternative Splicing; Architecture; Biogenesis; Biological Assay; Biological Models; Cells; Characteristics; Chromatin; Communication; Complex; DNA; Data; Dependence; Disease; Environment; Eukaryota; Eukaryotic Cell; Excision; Exons; Fission Yeast; Gene Expression; Genes; Genetic Transcription; Health; Histones; Human; Individual; Introns; Kinetics; Knowledge; Ligation; Malignant Neoplasms; Messenger RNA; Methods; Molecular; Nucleosomes; Polymerase; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Protein Isoforms; RNA; RNA Polymerase II; RNA Processing; RNA Sequences; RNA Splicing; Regulation; Regulatory Pathway; Resolution; Resources; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Speed; Spliced Genes; Spliceosome Assembly Pathway; Spliceosomes; Structure; System; Testing; Time; Transcription Elongation; Transcription Process; Transcriptional Regulation; Untranslated RNA; cell type; human disease; in vivo; mRNA Precursor; models and simulation; mutant; next generation sequencing; programs; promoter; single molecule; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): Pre-mRNA splicing - the process of intron removal and exon ligation -often occurs co-transcriptionally, i.e. during transcription by RNA polymerase II (Pol II). Cross-regulation between transcription and splicing represents an important gene regulatory pathway that influences how highly expressed a gene is, which alternative splice isoforms will be produced, and the efficiency of transcription elongation. Yet how the splicing and transcription machineries communicate is unknown. Our objective is to understand how splicing and transcription are coordinated. Accordingly, we will test the hypotheses that pausing of Pol II elongation within genes may be caused by splicing, specific gene characteristics, or incomplete splicing in the manner of a checkpoint. Our general strategy is to biochemically purify nascent RNA from chromatin and use next generation sequencing as a tool to precisely determine when during transcription splicing occurs. Our preliminary data on a small number of genes indicate that splicing occurs soon after the 3' splice site emerges from Pol II, much faster than predicted from indirect assays. We will investigate hundreds of endogenous genes in order to explore roles in co-transcriptional splicing for gene-specific features, such as sequence, exon-intron structure, promoter identity, nucleosome positioning and post-translational modifications on histones or Pol II. We will modify endogenous genes and employ mutants of the splicing and transcriptional machinery to probe mechanism. The simplicity of S. cerevisiae, with ~300 single-intron genes, allows us to investigate and experimentally alter transcription, splicing, and the architecture of genes. The relative complexity of S. pombe, with ~1000 genes harboring multiple introns, allows us to determine how splicing and transcription impact the order of intron removal, which must be regulated during alternative splicing. Our findings will help explain how cells control mRNA abundance and mRNA isoforms through splicing. Because mis-regulation of transcription and splicing are frequently associated with human diseases, such as cancer, a molecular understanding of their cross-regulation is significant for human health.

 描述（由应用程序提供）：前MRNA剪接 - 内含子去除和外显子连接的过程 - 通常是共转录的，即在通过RNA聚合酶II（POL II）转录过程中。转录和剪接之间的交叉调节代表了一个重要的基因调节途径，它影响了一个基因的高度表达，将产生哪种替代剪接同工型，以及转录伸长的效率。然而，剪接和转录机的通信方式尚不清楚。我们的目标是了解剪接和转录的协调方式。彼此之间，我们将检验以下假设，即基因内的pol II伸长可能是由剪接，特定基因特征或以检查点的方式引起的。我们的一般策略是从染色质中从生化纯化的新生RNA，并使用下一代测序作为一种工具，以精确确定在转录剪接期间发生何时发生。我们关于少数基因的初步数据表明，剪​​接发生在3'剪接位点从POL II出现后不久，比间接测定的预测快得多。我们将研究数百个内源基因，以探索基因特异性特征的共转录剪接中的作用，例如序列，外显子内结构，启动子身份，核小体定位和对组蛋白或POL II的转录后修饰。我们将修改剪接和转录机械的内源基因和雇员突变体，以探测机制。酿酒酵母的简单性具有〜300个单间基因的基因，使我们能够研究并实验改变基因的转录，剪接和结构。 S. pombe的相对复杂性具有约1000个带有多个内含子的基因的基因，使我们能够确定剪接和转录如何影响内含子删除的顺序，这必须在替代剪接过程中调节。我们的发现将有助于解释细胞如何通过剪接来控制mRNA抽象和mRNA同工型。由于转录和剪接的错误调节通常与人类疾病（例如癌症）有关，因此对它们的交叉调节的分子理解对人类健康很重要。