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

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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.