Mechanisms of alternative splicing of pre-mRNA

描述(由申请人提供)： 这项建议中描述的实验旨在深入了解正常细胞过程中替代的前mRNA剪接(AS)的机制和调控，以及在不同病理条件下的错误调控。本研究拟探讨RNA/DNA结合蛋白TLS/FUS(TLS)在神经退行性疾病肌萎缩侧索硬化症(ALS)中的作用，hnRNP蛋白在癌症，特别是胶质母细胞瘤(GBM)中的作用，核心剪接因子在骨髓增生异常综合征(MDS)中突变的作用，以及AS在人类胚胎干细胞(HESCs)分化中的作用。提出了以下具体目标。1.TLS/FUS和ALS。正在进行的研究证实，编码甲基CpG结合蛋白2的MECP2基因是在ALS突变蛋白存在下解除调控的TLS靶标，将继续作为TLS如何调节AS和基因表达以及这在ALS中如何出错的模型。与N.Shneider合作，将通过检测MECP2在ALS小鼠模型和衍生的分化为运动神经元的ESC系中的表达来研究MECP2失调与疾病的相关性。最后，显示相似的其他基因 用MECP2观察到的表达中断将被识别和表征。2.hnRNPs A1/A2和PTB：在基底膜中的过表达及其调控机制。研究hnRNP A1/A2和PTB在GBM中的作用的实验将继续进行。最近确定的受PTB/A1/A2调控的剪接靶标将被研究AS如何对两个 关键的癌症表型，肿瘤抑制活性的丧失和促进增殖功能的获得。将分析PTB/A1/A2水平的变化如何影响它们与前mRNA靶标中的位点结合，以检验这些hnRNP水平的降低以不同方式影响不同位点的占用的假设。3.MDS中的SF3B1和SRSF2突变。本研究旨在探讨MDS及相关恶性肿瘤中SF3B1和SRSF2突变的分子机制和功能意义。突变蛋白可能与剪接有关的缺陷将通过体外检测进行检查。对于A.raza和S.Mukherjee，将确定突变蛋白诱导的表型。将继续分析来自MDS患者的RNA-SEQ数据以检测剪接缺陷，特别是内含子保留，并检查这涉及U11/U12内含子的可能性。突变的SF3B1和SRSF2通过诱导R环的形成和基因组重排而促进MDS的假设将得到验证。已经检测到的假定的基因融合的意义将被确定。4.AS在hESC分化中的调控作用。通过对伴随hESC分化的AS变化的RNA-SEQ分析获得的结果将被扩展到检测额外的针对hESCs的AS事件。已确定的hESC特异性AS事件的生物学意义和潜在机制，包括编码TCF3和Tra2�的转录本，以及相关蛋白亚型的功能，都将被研究。TCF3是一种先前参与维持ESCs多能性的转录因子，Tra2是一种已知调节组织特异性AS的剪接因子。

DESCRIPTION (provided by applicant): The experiments described in this proposal are designed to provide insight into the mechanism and regulation of alternative pre-mRNA splicing (AS) in normal cellular processes as well as into its misregulation in different pathological conditions. Studies are proposed to investigate the roles of the RNA/DNA binding protein TLS/FUS (TLS) in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), of hnRNP proteins in cancer, specifically glioblastoma (GBM), of core splicing factors mutated in myelodysplastic syndromes (MDS), and of AS in differentiation of human embryonic stem cells (hESCs). The following Specific Aims are proposed. 1. TLS/FUS and ALS. Ongoing studies establishing that the MECP2 gene, which encodes methyl CpG binding protein 2, is a TLS target deregulated in the presence of ALS mutant proteins will be continued, as a model for how TLS regulates AS and gene expression generally and how this goes awry in ALS. With N. Shneider, the disease relevance of MECP2 misregulation will be investigated by examining MECP2 expression in ALS mouse models and derived ESC lines differentiated into motor neurons. Finally, additional genes that display similar disruptions in expression as observed with MECP2 will be identified and characterized. 2. hnRNPs A1/A2 and PTB: overexpression in GBM and mechanism of regulation. Experiments examining the roles of hnRNP A1/A2 and PTB in GBM will be continued. PTB/A1/A2-regulated splicing targets recently identified will be investigated with respect to how AS contributes to two key cancer phenotypes, loss of tumor suppressor activity and gain of proliferation-promoting function. How changes in PTB/A1/A2 levels affect their binding to sites in pre-mRNA targets will be analyzed to test the hypothesis that reducing levels of these hnRNPs affects occupancy of different sites in distinct ways. 3. SF3B1 and SRSF2 mutations in MDS. Experiments are proposed to address both the molecular mechanisms and functional significance of SF3B1 and SRSF2 mutations in MDS and related malignancies. Possible splicing-related defects of the mutant proteins will be examined using in vitro assays. With A. Raza and S. Mukherjee, phenotypes induced by mutant proteins will be determined. Analysis of RNA-seq data from MDS patients to detect splicing defects, notably intron retention, will be continued, and the possibiliy that this involves U11/U12 introns examined. The hypothesis that mutant SF3B1 and SRSF2 contribute to MDS by inducing R-loop formation and genomic rearrangements will be tested. The significance of a putative gene fusion already detected will be determined. 4. AS regulation in hESC differentiation. Results obtained by RNA-seq analysis of AS changes accompanying hESC differentiation will be extended to detect additional AS events specific to hESCs. The biological significance and underlying mechanisms of identified hESC-specific AS events, including in transcripts encoding Tcf3, a transcription factor previously implicated in the maintenance of ESC pluripotency, and Tra2�, a splicing factor known to regulate tissue-specific AS, will be investigated, as will the functions of the relevant protein isoforms.