m6A modification in R-loop homeostasis: a potential target for cancer therapeutics

Abstract

R-loops or DNA-RNA hybrids are prominent nucleic acid structures that commonly arise during transcription. These structures play important biological functions, such as regulating gene expression and DNA repair. However, when unresolved by nucleic acid processing factors, pathological R-loops can be harmful and lead to genome instability. N ⁶-Methyladenosine (m6A), the most prevalent modification in messenger RNA, has been recently identified to be crucial for regulating R-loop balance and maintaining genome stability. Strikingly, m6A-modified R-loop formation can have opposing consequences, either stabilization or resolution, depending on the biological context. In this review, we discuss the current knowledge of the regulatory roles of m6A on R-loops across various processes, including gene transcription, DNA repair, and centromere and telomere stability. Additionally, we explore other m6A-mediated processes, such as nascent transcription and chromatin landscape, that potentially affect R-loop dynamics. Finally, we discuss the current limitations and future directions of studying the m6A-R-loop axis, as well as the opportunities to target this pathway as a potential therapeutic strategy.

Graphical Abstract

Figure 1.

Overview of m6A regulators on mRNA and R-loops/DNA–RNA hybrids. The methyltransferase writer complex includes the core components (METTL3, METTL14, and WTAP) and the regulatory proteins (VIRMA, HAKAI, RBM15, and ZC3H13). The writer complex methylates adenosine at the N⁶ position of an mRNA transcript and a DNA–RNA hybrid transcribed by RNA polymerase II (RNAPII). The m6A erasers include ALKBH5 and FTO, which remove the methylation of adenosine from target mRNAs. Modified mRNAs are recognized by m6A readers, with each reader playing a different role in RNA metabolism. Modified DNA–RNA hybrids or R-loops are also stabilized or resolved by different m6A readers. The endonuclease RNase H1 and the histone H3 variant CENPA are also implicated as potential m6A readers (in question mark).

Figure 2.

Molecular and potential functions of m6A on different classes of R-loops and DNA–RNA hybrids. (A) m6A deposition occurs co-transcriptionally at different stages of transcription. At TSSs, m6A-modified R-loops from paused RNAPII might be recognized by m6A readers (such as YTHDC1) that recruit helicases or nucleases (such as RNase H1) for R-loop resolution to promote elongation. m6A might regulate co-transcriptional splicing, which can influence transcription processing and R-loop levels. At TTSs, R-loops are unwound by DDX21 for m6A deposition, which is recognized by the reader YTHDC1, potentially recruiting XRN2 exonuclease for RNAPII termination. The m6A writers and readers also regulate active (green) or repressive (red) histone marks, contributing to R-loop metabolism. (B) Upon DSBs, ARID1A as well as TonEBP helps the recruitment of METTL3 to methylate RNA-associated DSBs. YTHDC1 stabilizes m6A-modified hybrids, which attracts RAD51 to coat single-strand DNA overhangs and BRCA1 to promote HR repair. RNase H1 is also recruited, either directly or indirectly by m6A, to resolve the hybrids for efficient repair. (C) In centromeres, m6A-modified centromeric RNAs (cenRNA), potentially forming R-loops, are recognized and bound by CENPA to ensure centromeric integrity during cell division. In telomeres, telomeric-repeat-containing RNA (TERRA) is m6A marked and stabilized by YTHDC1 or hnRNPA2B1 readers to form R-loops, promoting HR to maintain telomere length in alternative lengthening of telomere (ALT) pathway.