Cross-regulation between transcription and pre-mRNA splicing

 描述（由申请人提供）：前体mRNA剪接-内含子去除和外显子连接的过程-通常共转录发生，即在RNA聚合酶II（Pol II）转录期间。转录和剪接之间的交叉调节代表了重要的基因调控途径，其影响基因的高度表达、将产生何种选择性剪接异构体以及转录延伸的效率。然而，剪接和转录机制是如何交流的还不清楚。我们的目标是了解剪接和转录是如何协调的。因此，我们将测试的假设，暂停Pol II延长基因内可能是由剪接，特定的基因特征，或不完全剪接的方式检查点。我们的总体策略是从染色质中生物化学纯化新生RNA，并使用下一代测序作为工具来精确确定转录过程中何时发生剪接。我们对少数基因的初步数据表明，剪接发生后不久，3'剪接位点出现从Pol II，比预测的间接测定快得多。我们将研究数百个内源性基因，以探索基因特异性特征在共转录剪接中的作用，例如序列，外显子-内含子结构，启动子身份，核小体定位和组蛋白或Pol II的翻译后修饰。我们将修改内源性基因，并采用剪接和转录机制的突变体来探测机制。S.酿酒酵母，具有约300个单内含子基因，使我们能够研究和实验改变转录，剪接和基因结构。S.粟酒裂殖酵母含有约1000个含有多个内含子的基因，使我们能够确定剪接和转录如何影响内含子去除的顺序，这必须在选择性剪接过程中进行调节。我们的发现将有助于解释细胞如何通过剪接控制mRNA丰度和mRNA亚型。由于转录和剪接的错误调节经常与人类疾病（如癌症）相关，因此对其交叉调节的分子理解对人类健康具有重要意义。

 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.