Cross-regulation between transcription and pre-mRNA splicing

 描述(申请人提供)：前mRNA剪接-内含子移除和外显子连接的过程-通常发生在共转录过程中，即在RNA聚合酶II(POL II)转录过程中。转录和剪接之间的交叉调控是一条重要的基因调控途径，它影响基因的高表达程度，产生哪些剪接异构体，以及转录延伸的效率。然而，剪接和转录机器是如何沟通的尚不清楚。我们的目标是了解剪接和转录是如何协调的。因此，我们将测试假设，即基因内POL II延伸的暂停可能是由剪接、特定的基因特征或检查点方式的不完全剪接引起的。我们的总体策略是从染色质中生物化学地纯化新生RNA，并使用下一代测序作为一种工具来精确确定转录剪接发生的时间。我们对少数基因的初步数据表明，剪接在Pol II的3‘剪接点出现后不久就发生了，比间接分析预测的要快得多。我们将研究数百个内源基因，以探索基因特异性特征在共转录剪接中的作用，如序列、外显子-内含子结构、启动子同源性、核小体定位以及组蛋白或POL II的翻译后修饰。我们将修改内源基因，并利用剪接和转录机制的突变来探索机制。酿酒酵母具有约300个单内含子基因，其简单性使我们能够研究和实验改变基因的转录、剪接和结构。庞氏链霉菌具有约1000个含有多个内含子的基因，其相对复杂性使我们能够确定剪接和转录如何影响内含子去除的顺序，内含子去除的顺序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信使核糖核酸丰度和信使核糖核酸异构体。由于转录和剪接的错误调控经常与癌症等人类疾病有关，因此了解它们的交叉调控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.