Dynamics of transcriptome 3’end diversity in development and disease

2 PhD projects offered in the IPP summer call 2021

Scientific background

Next generation RNA sequencing has led to the discovery of a perplexingly complex metazoan transcriptome architecture arising from the alternative use of transcription start sites, exons and introns and polyadenylation sites. The combinatorial use, and incorporation, of such elements into mature transcript isoforms considerably expands genomic information and is subject to dynamic spatial and temporal modulation during development and adaptation. Recently, diversification of the transcriptome at the 3’end through alternative polyadenylation (APA) evolved as an important and evolutionarily conserved layer of gene regulation. APA results in transcript isoforms varying at the RNA 3’ end, which can encode proteins with profoundly distinct functions or regulatory properties. Furthermore APA can affect the mRNA fate via the inclusion or exclusion of regulatory elements. In 2018 we unraveled the universe of drivers affecting transcriptome 3’end diversification (TREND; Ogorodnikov et al, 2018; Marini et al, 2021). We showed that dysregulation of TREND can impair normal neuronal development and result in tumor formation (Ogorodnikov et al, 2018). The goal of our projects is to further analyze the translational implications of perturbations altering TREND.

PhD project 1: The role of alternative polyadenylation in the maintenance of genome stability

Alternative polyadenylation (APA) plays an important role in various developmental and adaptive programs (Nourse et al, 2020). This involves the modulation of central cell signaling pathways including WNT and others (Ogorodnikov et al, 2018). We recently obtained evidence that APA is likely involved in mechanisms that confer genome stability by directing pathways both sensing and repairing double strand breaks and single nucleotide mismatches (Marini et al, 2021).

Here we want to dissect the role of APA in the maintenance of genome stability. To this end, we will use genetically engineered cell lines and mouse models in combination with a deep sequencing approach suitable for the detection of transcriptome 3’end diversity (TRENDseq; Ogorodnikov et al, in press). First, we will characterize TREND signatures in response to genotoxic stress. From these signatures we can infer potentially involved APA drivers based on our recently released TREND-DB atlas shiny.imbei.uni-mainz.de/trend-db/. The role of APA in genotoxic stress will then be examined using select KO cell lines and animals of APA drivers, followed by an in-depth functional characterization of how APA-affected target RNAs govern genome stability. These examinations will ultimately be complemented by studies of human material to uncover similarities and differences in these mechanism(s) between species.

PhD Project 2: Generation of a molecular imaging reporter mouse model to interrogate alternative polyadenylation non-invasively in vivo

Alternative polyadenylation (APA) plays an important role in various developmental and adaptive programs (Nourse et al, 2020). Recently we showed that dysregulation of APA impairs normal neuronal development and results in tumor formation (Ogorodnikov et al. 2018).

Here we want to generate a molecular imaging reporter mouse model to interrogate APA dynamics non-invasively in a living organism. To this end, we will make use of our recently released atlas covering the transcriptome-wide landscape of APA in response to 170 different experimental conditions (Marini et al, 2021). Based on this catalog, and our expertise in non-invasive molecular imaging (Schott et al. 2020), we will first generate rationalizable multiplexed optical imaging APA reporters in cell line models. Upon validation, these APA reporters will serve to generate a genetically engineered non-invasive imaging reporter mouse model. By crossing this with animals in which we can selectively deplete potent APA regulators (Ogorodnikov et al. 2018) we aim to retrieve established APA signatures and to cross-correlate this with our multiplexed optical imaging APA reporters. This model will then serve to chart APA dynamics during development and aging, and in dedicated disease model(s) non-invasively in vivo. It thereby complements mechanistic studies in the lab to uncover disease-eliciting mechanisms and to identify novel therapeutic avenues.

Publications relevant to the project

Kargapolova Y, Levin M, Lackner K, Danckwardt S (2017) 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, 45(10):6074

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

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

Marini F, Scherzinger D, Danckwardt S (2021) TREND-DB – A Transcriptome-wide Atlas of the Dynamic Landscape of Alternative Polyadenylation. Nucleic Acids Res, 49(D1):D243

Ogorodnikov A, Danckwardt S. TRENDseq - A highly multiplexed high throughput 3’end RNA sequencing for mapping alternative polyadenylation. Methods in Enzymology, in press.

Schott LK, Pruschinski L, Khokar S, Eder L, Muhammad K, Tokalov S, Satrapa J, Klein M, Lackner K, Danckwardt S (2020) Non-invasive optical in vivo imaging reveals tumor-derived prothrombin with a functional role in hemostasis and tumor progression. Res Pract Thromb Haemost, 4 (Suppl 1)