Coupling of transcription with pre-mRNA metabolism

描述(申请人提供)：制造信使RNA(信使RNA)，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(PolII)的转录，产生一种前体，通过封端、剪接和切割/多聚腺苷作用成熟，并与RNA结合蛋白包装成mRNP，输出到细胞质。转录的模板是由染色质组成的：DNA包裹在称为核小体的组装中的组蛋白周围。转录和前mRNA加工不是独立的事件；它们以一种依赖于RNA polII的特殊结构域的方式相互整合，该结构域被称为CTD，它充当处理蛋白质的着陆台。事实上，由PolII以外的RNA聚合酶或缺少CTD的PolII产生的转录物不能正确地加工成成熟的mRNA。我们的工作假设是，信使核糖核酸前体的合成和加工是由一个‘信使核糖核酸工厂’复合体完成的，该复合体包括PolII和通过与CTD接触而结合在一起的加工因子。这个模型是思考一组蛋白质的新方式的一个例子，这些蛋白质曾经被认为是相互独立运作的，而不是作为一个完整的团队。我们使用遗传学和生物化学的方法来研究‘mRNA工厂’是如何工作的，以实现高效和准确的生产出口竞争力的mRNP的协调合成和成熟的转录本由PolII。这项建议的目标是：1.确定允许前mRNA剪接和染色质结构修改之间的串扰的机制。2.高分辨率绘制PolII CTD磷酸化图谱，并确定它们与正常乳腺细胞和乳腺癌细胞中剪接和3‘端加工复合体的共转录组装的关系。3.确定具有出口活性的RNP的组装如何与通过切割-聚腺苷酸化的mRNA的加工相协调。 公共卫生相关性： 癌细胞中基因错误表达的一个主要成分可以追溯到前mRNA剪接和3‘端切割/多聚腺苷酸化处理的异常模式。这项工作可能有助于阐明正常情况下mRNAs的转录和处理是如何调节的，以及它们是如何在癌细胞中变得错误调节的。我们将使用CHIP-SEQ进行全基因组分析，以比较正常乳腺细胞和乳腺癌细胞系中PolII、CTD的磷酸化和加工因子与基因的募集模式。这项研究将有助于确定‘信使核糖核酸工厂’的功能缺陷，该缺陷可能在破坏癌细胞的剪接和切割/多聚腺苷化方面发挥主要作用。

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.