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，对所有细胞的生命至关重要，其破坏是癌细胞的标志。mRNA的合成需要RNA聚合酶II （pol II）转录以产生前体，该前体通过盖帽、剪接和裂解/聚腺苷酸化成熟，并与RNA结合蛋白包装成mRNP出口到细胞质。被转录的模板是由染色质构成的：DNA包裹在被称为核小体的组蛋白上。转录和前mRNA加工不是独立的事件；它们以一种依赖于RNA pol II的一个特殊区域CTD的方式相互结合，CTD作为处理蛋白质的着陆点。事实上，由pol II以外的RNA聚合酶或由缺乏CTD的pol II合成的转录本不能正确地加工成成熟的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”复合体完成的，该复合体包括pol II和通过与CTD接触而结合在一起的加工因子。这个模型是一种思考一组蛋白质的新方法的一个例子，这些蛋白质曾经被认为是彼此独立运作的，而不是作为一个完整的团队。我们使用遗传和生化方法来询问“mRNA工厂”如何有效和准确地生产出口能力的mRNP，协调合成和成熟由pol II制成的转录本。本建议的目标是：1。确定允许前mrna剪接和染色质结构修饰之间的串扰的机制。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. 确定出口能力的RNP的组装如何与mRNA的切割-聚腺苷化加工协调。

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.