Project 19. R-loop epitranscriptomics: Do RNA modifications determine R-loop fate?
R-loops are three-stranded nucleic acid structures consisting of an RNA:DNA hybrid and a displaced single DNA strand. They perform important functions in gene regulation, chromatin organisation, RNA processing, telomere maintenance and DNA repair. However, persistent or misregulated R-loops can also cause replication stress, DNA damage and genome instability. What determines whether an R-loop acts as a beneficial regulatory structure or becomes a pathological threat to the genome remains largely unknown.
This project investigates the hypothesis that chemical modifications of the RNA within an R-loop contribute to determining its functional fate. RNA modifications can alter RNA structure, base-pairing behaviour, molecular stability and interactions with RNA-binding proteins. Modifications such as m6A, pseudouridine, inosine and 2′-O-methylation may therefore influence how readily an RNA hybridises with DNA, how long the resulting R-loop persists and how efficiently it is recognised and resolved by cellular factors.
To investigate this previously unexplored regulatory layer, we will combine genome-wide R-loop maps generated within the 4R consortium with direct RNA nanopore sequencing. In contrast to conventional RNA sequencing, nanopore sequencing retains information about chemical RNA modifications and simultaneously identifies the transcript on which they occur. Building on the experimental resources and machine-learning methods developed in our laboratory, we will determine whether R-loop-forming transcripts carry characteristic epitranscriptomic signatures.
The project will address three central questions:
- Do transcripts associated with regulatory and pathological R-loops carry distinct RNA modification patterns?
- Can these patterns, together with RNA sequence, transcript architecture and structural features, predict R-loop formation, persistence and functional outcome?
- Are the identified modification pathways functionally linked to R-loop stability, DNA damage and genome instability?
Figure 1. Epitranscriptomic regulation of R-loop fate.
Interpretable machine-learning approaches will be used to identify combinations of molecular features that distinguish different R-loop states and to formulate experimentally testable hypotheses. Candidate modification pathways will subsequently be investigated using perturbation systems and complementary R-loop, chromatin and genome-instability data available within the 4R consortium. These studies will connect the computational predictions to the biological consequences of altered R-loop homeostasis.
The project builds on close collaborations with 4R laboratories studying R-loop mapping, RNA processing, chromatin organisation and R-loop-associated DNA damage. It thereby establishes a bridge between epitranscriptomics and the central research areas of the 4R programme.
Ultimately, we aim to determine whether the epitranscriptome represents a previously unrecognised control layer of R-loop biology. This could explain why some RNA:DNA hybrids support genome function, whereas others persist and become drivers of genome instability and disease.
