Abstract

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.

Project-related publications

Ogorodnikov A, Danckwardt.
TRENDseq – A highly multiplexed high throughput RNA 3’ end sequencing for mapping alternative polyadenylation. Methods in Enzymology. 2021; book chapter in press

Marini F, Scherzinger D, Danckwardt S.
TREND-DB – A transcriptome-wide atlas of the dynamic landscape of alternative polyadenylation. Nucleic Acids Res. 2021; 49(D1):D243; http://shiny.imbei.uni-mainz.de:3838/trend-db/

Nourse J, Danckwardt S.
A novel rationale for targeting FXI: Insights from the hemostatic microRNA targetome for emerging anticoagulant strategies. Pharmacol Ther, 2021; 218:107676

Nourse J, Spada S, Danckwardt S.
Emerging roles of RNA 3’end cleavage and polyadenylation in pathogenesis, diagnosis and therapy of human disorders. Biomolecules, 2020; 10(6):915

Ogorodnikov A, Levin M, Tattikota S, Tokalov S, Hoque M, Scherzinger D, Marini F, Tian B, Schaefer M, Lackner KJ, Westermann F, Danckwardt S.
Transcriptome 3'end organization by PCF11 links alternative polyadenylation to formation and neuronal differentiation of neuroblastoma. Nature Comm. 2018; 9(1):5331

Kargapolova Y, Levin M, Lackner K, Danckwardt S.
sCLIP – An integrated platform to study RNA-protein interactomes in biomedical research: identification of CSTF2tau in alternative processing of small nuclear RNAs. Nucleic Acids Res. 2017;45(10):6074