RNA Methylation Keeps R-loops Under Control to Maintain Genome Integrity
December 1, 2021
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Gene transcription in mammalian cells can lead to the formation of hybrid structures known as “R-loops,” in which a single-stranded nascent RNA molecule interacts with double-stranded DNA, altering its typical configuration. While a wide range of studies have indicated that bubble-like R-loops play critical roles in transcriptional regulation and DNA repair (Santos-Pereira and Aguilera, 2015). RNA/DNA hybrid sequences also represent a significant source of genome instability (Allison and Wang, 2019) that can negatively impact normal cell function if left unchecked. Furthermore, diseases associated with genomic instability, including neurodegenerative diseases and certain cancers (Groh and Gromak, 2014), have been linked to the uncontrolled accumulation of R-loops. So how do cells manage to keep these potentially problematic RNA/DNA hybrid sequences in check?
Epigenetic research has previously underscored the general importance of the prevalent N6-methyladenosine (m6A) modification of RNA in regulating molecular stability/turnover and hence RNA translation (Yu et al., 2015 and Roundtree et al., 2017). Recently, two fascinating articles have described how RNA methylation may also help to maintain genome integrity by modulating R-loop persistence.
m6A-modified R-loops – The Epigenetic Guardians of Genome Stability?
Researchers from the laboratories of Arne Klungland (University of Oslo, Norway), Natalia Gromak (University of Oxford, UK), and Alexey Ruzov (University of Nottingham, UK) began their study of R-loops and RNA methylation with a different but related target in mind. Abakir and colleagues from these three labs initially sought to evaluate the general abundance of a specific form of methylated adenosine in DNA (N6-methyldeoxyadenosine; 6mA) in the human stem and cancer cell genome due to a poor understanding of the prevalence and relevance of this modification in mammalian systems.
Interestingly, preliminary immunoprecipitation and mass spectrometry results provided evidence for a general lack of 6mA-modified DNA. Instead, the authors encountered the widespread nature of m6A-modified RNA/DNA hybrid sequences in human cells, thereby suggesting an essential link between RNA methylation and R-loops.
Next, the team asked whether R-loops containing m6A-modified RNA played a role in genome integrity by analyzing the RNA methyltransferases involved and evaluating those factors recruited by the m6A-modified RNA component. Fascinatingly, the team discovered that the knockdown of the METTL3 N6-adenosine-methyltransferase (part of a larger methyltransferase complex) prompted the accumulation of RNA/DNA hybrid sequences; therefore, they hypothesized that the METTL3-mediated m6A-modification of RNA within R-loops may act as a signal for their removal/resolution to maintain genome integrity.
In addition to METTL3, the authors also discovered that YTHDF2—a known m6A-interacting protein that regulates cytoplasmic mRNA degradation (Wang et al., 2014)—interacted with RNA/DNA hybrid sequences. Fascinatingly, YTHDF2 knockdown prompted a considerable increase in the number of m6A-modified RNA/DNA hybrid sequences, the induction of DNA damage marker expression, and a correlative decrease in cell growth potential.
Overall, these findings suggest that METTL3 methylates the RNA component of R-loops to attract YTHDF2 binding to inhibit the accumulation of RNA/DNA hybrid sequences and protect against genome instability. But the question arises; just how does METTL3 make its way to R-loops in the first place and what mechanism supports the resolution of R-loops?
You Can Lead a Horse to Water – TonEBP Shows METTL3 the Way
These vexing questions were soon answered by a team of researchers from the laboratories of Kyungjae Myung, Ja Yil Lee, and Hyug Moo Kwon (Ulsan National Institute of Science and Technology/Institute for Basic Science, Ulsan, South Korea) in a subsequent R-loop and RNA methylation study. Kang, Cheon, and colleagues had sought to build upon the previous study´s findings and define the mechanisms involved in the recognition and resolution of R-loops, which they induced by exposing human cell lines to DNA damaging agents (ultraviolet light and topoisomerase inhibitors). Their fascinating findings indicate that the transcriptional regulator tonicity-responsive enhancer-binding protein (TonEBP, also called NFAT5) recognizes R-loops, recruits METTL3, and helps an RNA cleaving enzyme to resolve R-loops, thereby maintaining genome integrity (Choi et al., 2020).
Previous research underlined the ability of TonEBP to sense DNA damage and orchestrate signaling events via interactions with multiple enzymes (Kang et al., 2019), and initial studies combining immunoprecipitation and mass spectrometry provided robust evidence of an interaction between METTL3 and TonEBP. The authors discovered that TonEBP rapidly and efficiently recognized DNA damage-induced R-loops through an unusual “3D collision and 1D diffusion” dual search mechanism, allowing TonEBP to recruit METTL3 to R-loops methylate the RNA component of the RNA/DNA hybrid sequence. The Rel homology domain, present in the NF-B proteins and several other transcription factors, facilitates the formation of DNA-bound protein complexes, mediated both the recognition of R-loops by TonEBP and the interaction between TonEBP and METTL3. Regarding the role of YTHDF2, the authors discovered that TonEBP also controlled the previously noted YTHDF2-R-loop interaction.
Finally, the authors showed that TonEBP also recruited RNaseH1 (a non-specific endonuclease that hydrolytically cleaves RNA) to R-loops through an interaction with METTL3. This prompted the removal of RNA to allow the reformation of the double-stranded DNA into its typical conformation. In confirmation of these findings, the loss of TonEBP expression prompted a decrease in m6A-modified RNA levels, an increase in the number of R-loops, and a reduction in cell proliferation and survival following exposure to the DNA damaging agents.
These data provide evidence for a significant role of a TonEBP-METTL3-RNA methylation pathway in resolving DNA damage-induced R-loops via the recruitment of RNaseH1. The authors strongly suggest exploring the full range of activity of TonEBP as a critical next task given the importance of R-loop homeostasis to cell physiology and the pathogenesis of various diseases.
The Future of RNA Methylation and R-loops
Overall, these exciting findings support the development of future studies into the potential role of RNA methylation. Furthermore, the mechanistic insight into R-loop resolution may also foster the development of therapeutic strategies that protect against genome instability in specific cancer types and neurodegenerative diseases.
About the author
Stuart P. Atkinson, Ph.D.
Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!
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