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

神经元发育过程中 PCF11 依赖的转录组 3âend mRNP 动态调节

• 批准号: 427452638
• 负责人: Professor Dr. Sven Danckwardt
• 金额: --
• 依托单位: Centrum für Thrombose und Hämostase (CTH)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/427452638?language=en
• 关键词: PCF11; dependent; regulation; transcriptome; end

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.

替代RNA 3'End裂解和聚腺苷酸化（APA）是转录后基因调控的进化配置的关键机制。它影响了所有基因的70％，从而对转录组3的多样化产生了深远的影响。 APA控制各种细胞过程2,3,4，包括分化和去分化3,5-13。在大规模的RNAi筛选中，我们最近描述了影响转录组3的多样化的机制的动态景观。在指挥APA的各种驱动因素中，我们发现PCF11是用于近端聚腺苷酸化的普遍成分。我们证明了该组件在产后下调，并且转录组3'End体系结构的PCF11依赖性组织对于神经分化至关重要。相比之下，持续的高级PCF11表达会阻碍未分化状态的神经元前体，并导致由于转录本缩短了具有致癌功能的转录本相结合而导致的神经母细胞肿瘤。我们打算阐明PCF11如何影响未经APA的MRNA分子的全球和/或特定的特定率。我们将使用PCF11部署并导致转录组的全局3'UTR延长作为在开发过程中自然发生变化的原型，并在MRNP形成中可能重要的功能，因此控制单个单元格中数百个3'UTR的RNP动力学。为此，我们将采用具有不同水平的PCF11表达的细胞系模型，研究对基因表达的连续层的影响（例如mRNA输出，定位，周转和翻译）以及候选名单潜在的潜在相关的RBP，可能具有可能选择性的APA-转录同类药特异性特异性结合亲密关系。我们将通过最先进的交联免疫沉淀（夹子）和RNA测序来定义它们的RNA结合特性。最终，与功能研究结合，我们希望解释原理如何在转录组3'End上的同工型在mRNA 3'End处影响RNP动力学，以及这如何决定神经发育模型中的mRNA命运和下游功能。