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

选择性RNA 3 '端切割和多聚腺苷化（APA）是一种进化保守的转录后基因调控机制。它影响约70%的基因，因此对转录组3′端多样化有着深远的影响。APA控制多种细胞过程2,3,4，包括分化和去分化3,5-13。在大规模的RNAi筛选中，我们最近描绘了转录组3 ‘端多样化的动态景观并探索了影响转录组3 ’端多样化的机制。在指导APA的各种驱动因素中，我们发现PCF11是近端聚腺苷化的普遍成分。我们证明了该成分在出生后下调，并且转录组3 '端结构的pcf11依赖性组织对神经分化至关重要。相反，持续高水平的PCF11表达抑制未分化状态的神经元前体，并由于具有致癌功能的转录异构体缩短而导致神经母细胞肿瘤。在这里，我们打算阐明PCF11如何影响经历APA的mRNA分子的整体和/或特定命运。我们将使用PCF11耗竭和转录组整体3'UTR延长作为发育过程中自然发生的变化的原型，该变化可能在mRNP形成中具有重要功能，从而调节单个细胞内数百个3'UTR的RNP动态。为此，我们将采用具有不同水平PCF11表达的细胞系模型，研究其对基因连续表达层（如mRNA输出、定位、周转和翻译）的影响，并候选出可能具有选择性apa -转录物亚型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，我们想要破译转录组3 ‘端的同工异构体变化如何影响mRNA 3 ’端的RNP动力学，以及这如何决定mRNA的命运和神经发育模型中的下游功能。

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.