Project 9. Crosstalk between R-loop formation and alternative polyadenylation.

Alternative polyadenylation (APA) is a widespread and highly dynamic mechanism of gene regulation. It affects more than 70% of all genes, resulting in transcript isoforms with distinct 3’end termini. APA thereby considerably expands the diversity of the transcriptome 3′ end, leading to mRNA isoforms with profoundly different physiological effects, by affecting protein output, production of distinct protein isoforms, or modulating protein localization. APA is globally regulated in various conditions, including developmental and adaptive programs. Perturbations of APA can disrupt biological processes, ultimately resulting in devastating disorders including cancer. Although substantial evidence for an interplay between RNA cleavage and polyadenylation (CPA) and the maintenance of genome stability exists, little is known about the role APA and CPA have in R-loop formation, and vice versa. In a systematic large-scale RNAi screening, we recently mapped the dynamic landscape of APA after depletion of >170 proteins involved in various facets of transcriptional, co- and post-transcriptional gene regulation, epigenetic modifications and further processes. We observed that key components pervasively regulating CPA and APA (including RNA processing factors involved in the coupling of transcription termination and CPA such as PCF11) control processing of various components involved in the formation and resolution of R-loops. This includes established components directly involved in the resolution of R-loops such as RNAse H1, the RNA-DNA helicase DDX5/Ddp2 or AQR, but also exosome components with a similar role (EXOSC3/hRrp40, EXOCS4/hRrp41 or EXOCS6/hMtr3). Conversely, loss of function of components involved in R-loop resolution (such as the R-loop associated helicase senataxin, SETX) also affects APA. This suggests that CPA and R-loops bidirectionally affect each other. This corresponds to the two-sided nature of R-loops, where physiological (“scheduled”) R-loops tune gene expression (including transcription termination and 3’end processing), while pathological (“unscheduled”) R-loops impair genome integrity, which is typically followed by an inhibition of CPA to limit the ‘release’ of emerging faulty transcripts and to allow for repair of the genomic lesion.

Figure 1. Crosstalk between R-loop formation and APA.

R-loop formation, location and timely removal must be tightly regulated to ensure proper gene regulation (including CPA) and to prevent deleterious effects. Here we want to study the crosstalk between R-loop formation and alternative polyadenylation, and how this contributes to R-loop homeostasis and modulation of CPA and APA, respectively. Briefly, by using various genetic models (cells/animals) available in the lab (including PCF11 loss of function) we want to explore how CPA/APA regulation affects the protein abundance of components involved in formation and removal of R-loops (including components such as RNAse H1 and DHX5) and how this in turn affects R-loop formation (by R-loop profiling together with Niehrs and Beli). We then want to study the functional effects of scheduled R-loops on transcription termination by the depletion/overexpression of RNAse H1 and DDX5 in a PCF high/low background followed by transcription termination/APA profiling and R-loop position profiling. To interrogate the consequences of unscheduled R-loop formation on genome integrity, we intend to perform permutations of depletion/addition of RNAse H1 (and DDX5, respectively) in a PCF11 low and high background with and without exposure to genotoxic stress. As a primary screening readout, we intend to perform R-loop profiling to identify the differential effect on R-loop formation, location and removal. This will be followed by profiling of downstream consequences including double-strand breaks to localize genotoxic effects and to dissect regulatory (“scheduled”) from deleterious (“unscheduled”) R-loop effects upon modulation of APA. This will be complemented by APA profiling studies in response to genotoxic stress with and without depletion/addition of RNAse H1 and DDX5.

With these exploratory studies, we first aim to resolve:

  1. The effect of APA on critical components of R-loop regulation.
  2. Whether and where R-loops are enriched under conditions of APA modulation.
  3. Whether and how this affects the regulatory function of scheduled R-loops and/or the downstream effect of unscheduled pathological R-loops on genome integrity (including the resulting effect on the compensatory modulation of CPA under genotoxic stress).

These findings will serve as a foundation for further characterization on a molecular level. They will be complemented by studies in living creatures (mice) to understand the impact of global APA changes during development and aging on the function and fidelity of R-loop-mediated regulatory processes under these conditions.