PCF11-dependent regulation of transcriptome 3’end dynamics during neuronal development
Alternative polyadenylation (APA) is an evolutionary conserved mechanism of post-transcriptional gene regulation. It affects >70% of all genes and thereby profoundly contributes to transcriptome 3’end diversification (TREND). APA controls a variety of cellular processes including differentiation and dedifferentiation.
Using a large scale RNAi screening, we recently profiled the dynamic landscape of TREND and characterized underlying mechanisms. Among various drivers directing APA, we discovered PCF11 as pervasive component for proximal polyadenylation. We showed that PCF11 is down-regulated postnatally, and that the re-organization of the transcriptome 3’end architecture by PCF11 is crucial for neurodifferentiation. 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 want to investigate how PCF11 affects the fate of mRNA molecules undergoing APA. We use PCF11 depletion and resulting global 3’UTR lengthening of the transcriptome as a prototype for a naturally occurring change during development, regulating the RNP dynamics of hundreds of 3’UTRs within a single cell. To this end, we will use 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 the RNA binding properties of selected RBPs in a dynamic model of neurodifferentiation using state of the art crosslinking immunoprecipitation (CLIP) and RNA sequencing. In combination with functional studies, we ultimately want to decipher the principles of how isoform changes at the transcriptome 3’end influence the RNP dynamics and how this determines the mRNA fate and downstream function in a model of neurodevelopment.
Ogorodnikov A, Danckwardt.
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