Coupling of transcription with pre-mRNA metabolism

描述（由申请人提供）：制造信使RNA（mRNA）是蛋白质编码基因表达的主要事件，是所有细胞生命的核心，其腐败是癌细胞的标志。mRNA合成需要通过RNA聚合酶II（pol II）转录以产生前体，所述前体通过加帽、剪接和切割/聚腺苷酸化而成熟，并与RNA结合蛋白一起包装成mRNP以输出到细胞质中。被转录的模板是由染色质组成的：DNA包裹在组蛋白周围，形成一个叫做核小体的集合体。转录和前mRNA加工不是独立的事件;它们以依赖于RNA pol II的称为CTD的专门结构域的方式彼此整合，该结构域充当加工蛋白质的着陆垫。事实上，由pol II以外的RNA聚合酶或缺乏CTD的pol II所产生的转录物不能正确地加工成成熟mRNA。我们的工作假设是，mRNA前体的合成和加工是由一个“mRNA工厂”的复合物，其中包括pol II和加工因子保持在一起的CTD接触。这个模型是一种新的思维方式的一个例子，一组蛋白质曾经被认为是独立运作的，而不是作为一个完整的团队。我们使用遗传和生物化学的方法来询问“mRNA工厂”如何工作，以实现有效和准确的生产出口主管mRNP的协调合成和成熟的转录由pol II。该提案的目标是1。确定允许前体mRNA剪接和染色质结构修饰之间串扰的机制。2.以高分辨率绘制pol II CTD磷酸化模式，并确定它们如何与正常乳腺细胞和乳腺癌细胞中剪接和3'端加工复合物的共转录组装相关。3.确定输出感受态RNP的组装如何通过切割-聚腺苷酸化与mRNA的加工协调。 公共卫生相关性： 癌细胞中基因错误表达的主要成分可以追溯到前体mRNA剪接和通过切割/多聚腺苷酸化的3'加工的异常模式。这项工作可能有助于阐明mRNA的转录和加工在正常条件下是如何调节的，以及它们在癌细胞中是如何被错误调节的。我们将使用ChIP-seq的全基因组分析来比较pol II CTD磷酸化和加工因子与正常乳腺细胞和乳腺癌细胞系中基因的募集模式。这项研究将有助于确定“mRNA工厂”功能的缺陷，这些缺陷可能在破坏癌细胞中的剪接和切割/多聚腺苷酸化中起主要作用。

DESCRIPTION (provided by applicant): Making messenger RNA (mRNA), the primary event in expression of protein coding genes is central to the life of all cells and its corruption is a hallmark of cancer cells. mRNA synthesis requires transcription by RNA polymerase II (pol II) to produce a precursor that is matured by capping, splicing and cleavage/polyadenylation and packaged with RNA binding proteins into mRNP's for export to the cytoplasm. The template that is transcribed is made of chromatin: DNA wrapped around histone proteins in assemblies called nucleosomes. Transcription and pre- mRNA processing are not independent events; they are integrated with one another in a manner that depends on a specialized domain of RNA pol II called the CTD that acts as a landing pad for processing proteins. In fact transcripts made by RNA polymerases other than pol II or those made by pol II lacking the CTD are not processed correctly into mature mRNA. Our working hypothesis is that synthesis and processing of mRNA precursors is performed by an 'mRNA factory' complex which comprises pol II and processing factors held together by contacts with the CTD. This model is an example of a new way of thinking about a set proteins that were once thought to operate independently of one another rather than as a integrated team. We use genetic and biochemical approaches to ask how the 'mRNA factory' works to achieve efficient and accurate production of export-competent mRNP's coordinated synthesis and maturation of transcripts made by pol II. The objectives of this proposal are to 1. To determine the mechanism that permits cross-talk between pre-mRNA splicing and modification of chromatin structure. 2. To map patterns of pol II CTD phosphorylation at high resolution and determine how they related to co-transcriptional assembly of splicing and 3' end processing complexes in normal breast cells and breast cancer cells. 3. To determine how assembly of export competent RNP's is coordinated with processing of the mRNA by cleavage-polyadenylation. PUBLIC HEALTH RELEVANCE: A major component of gene misexpression in cancer cells can be traced to abnormal patterns of pre-mRNA splicing and 3' processing by cleavage/polyadenylation. This work may help elucidate how the transcription and processing of mRNAs are regulated under normal conditions and how they become mis-regulated in cancer cells. We will use genome-wide analysis by ChIP-seq to compare patterns of pol II CTD phosphorylation and recruitment of processing factors to genes in normal breast cells and breast cancer cell lines. This study will help identify defects in the function of the 'mRNA factory" that may play a major role in corrupting splicing and cleavage/polyadenylation in cancer cells.