PCF11-dependent regulation of transcriptome 3’end mRNP dynamics during neuronal development

RNA 3 '端切割和多聚腺苷酸化（阿帕）是一种进化上保守的转录后基因调控机制。它影响>70%的所有基因，从而深刻地促进转录组3 '端多样化。阿帕控制各种细胞过程2，3，4包括分化和去分化3，5 -13。在大规模RNAi筛选中，我们最近描绘了影响转录组3 '端多样化的动态景观并探索了其机制。在指导阿帕的各种驱动程序中，我们发现PCF 11是近端聚腺苷酸化的普遍组分。我们证明了该成分在出生后下调，并且转录组3 '端结构的PCF 11依赖性组织对于神经分化至关重要。相反，持续高水平的PCF 11表达逮捕神经元前体细胞在未分化状态，并导致神经母细胞肿瘤由于缩短转录异构体与致癌function. There我们打算阐明PCF 11如何影响全球和/或特定的命运的mRNA分子进行阿帕。我们将使用PCF 11耗竭和由此产生的转录组的全局3 'UTR延长作为发育期间自然发生的变化的原型，其在mRNP形成中可能具有重要功能，因此调节单个细胞内数百个3' UTR的RNP动力学。为此，我们将采用不同水平的PCF 11表达的细胞系模型，研究对基因表达的连续层（如mRNA输出，定位，周转和翻译）的影响，并筛选出可能具有选择性APA转录异构体特异性结合亲和力的潜在相关RBP。我们将通过最先进的交联免疫沉淀（CLIP）和RNA测序在神经分化的动态模型中定义它们的RNA结合特性。最后，结合功能研究，我们希望破译的原则，异构体的变化在转录组3 '端的影响RNP动力学在mRNA的3'端，这如何决定mRNA的命运和下游功能的神经发育模型。

Alternative RNA 3’end cleavage and polyadenylation (APA) is an evolutionary conserved key mechanism of post-transcriptional gene regulation. It affects >70% of all genes and thereby profoundly contributes to transcriptome 3’end diversification. APA controls a variety of cellular processes2,3,4 including differentiation and dedifferentiation 3,5-13. In a large scale RNAi screening, we recently delineated the dynamic landscape of and explored mechanisms influencing transcriptome 3’end diversification. Among various drivers directing APA, we discovered PCF11 as pervasive component for proximal polyadenylation. We demonstrated that this component is down-regulated postnatally, and that PCF11-dependent organization of the transcriptome 3’end architecture is critical for neurodifferentiation. In contrast, sustained high-level PCF11 expression arrests neuronal precursors in an undifferentiated state and leads to neuroblastic tumors due to shortened transcript isoforms with oncogenic function.Here we intend to shed light onto how PCF11 influences the global and/or specific fate of mRNA molecules undergoing APA. We will use PCF11 depletion and resulting global 3’UTR lengthening of the transcriptome as a prototype for a naturally occurring change during development with likely important function in mRNP formation, hence regulating the RNP dynamics of hundreds of 3’UTRs within a single cell. To this end, we will employ cell line models with varying levels of PCF11 expression, study the impact on consecutive layers of gene expression (such as mRNA export, localization, turnover and translation) and shortlist potentially relevant RBPs with likely selective APA-transcript isoform-specific binding affinities. We will define their RNA binding properties in a dynamic model of neurodifferentiation by state of the art crosslinking immunoprecipitation (CLIP) and RNA sequencing. Ultimately, combined with functional studies we want to decipher principles how isoform changes at the transcriptome 3’end influence the RNP dynamics at the mRNA 3’end and how this determines the mRNA fate and downstream function in a model of neurodevelopment.