Search

Maps of the RNA modification 5-methylcytosine (m 5 C) often diverge markedly not only because of differences in detection methods, data depand analysis pipelines but also biological factors. We re-analysed bisulfite RNA sequencing datasets from five human cell lines and seven tissues using a coherent m 5 C site calling pipeline. With the resulting union list of 6,393 m 5 C sites, we studied site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed tRNA:m 5 C methyltransferases NSUN2 and NSUN6 as the main mRNA m 5 C "writers," but further showed that the rRNA:m 5 C methyltransferase NSUN5 can also modify mRNA. Each enzyme recognises mRNA features that strongly resemble their canonical substrates. By analysing proximity between mRNA m 5 C sites and footprints of RNA-binding proteins, we identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. We found that lack of NSUN2 in HeLa cells affected both steady-state levels of, and UPF1-binding to, target mRNAs. Our studies emphasise the emerging diversity of m 5 C writers and readers and their effect on mRNA function., (© 2024 Guarnacci et al.)

The potential presence of 5-methylcytosine as a sparse internal modification of mRNA was first raised in 1975, and a first map of the modification was also part of the epitranscriptomics "big bang" in 2012. Since then, the evidence for its presence in mRNA has firmed up, and initial insights have been gained into the molecular function and broader biological relevance of 5-methylcytosine when present in mRNA. Here, we summarize the status quo of the field, outline some of its current challenges, and suggest how to address them in future work., (© 2024 Guarnacci and Preiss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Background: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors., Methods: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity., Results: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors., Conclusion: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability., (© 2023. The Author(s).)

Mapping the position and quantifying the level of 5-methylcytosine (m 5 C) as a modification in different types of cellular RNA is an important objective in the field of epitranscriptomics. Bisulfite conversion has long been the gold standard for the detection of m 5 C in DNA, but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification, and detection of candidate sites by sequencing on the Illumina MiSeq platform., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Circular RNAs (circRNAs), a class of covalently closed RNAs formed by a back-splicing reaction, have been involved in the regulation of diverse oncogenic processes. In this article we describe circVAMP3, a novel circular RNA overexpressed in RH4, a representative cell line of alveolar rhabdomyosarcoma. We demonstrated that circVAMP3 has a differential m 6 A pattern opposed to its linear counterpart, suggesting that the two isoforms can be differently regulated by such RNA modification. Moreover, we show how circVAMP3 depletion in alveolar rhabdomyosarcoma cells can impair cell cycle progression, through the alteration of the AKT-related pathways, pointing to this non-coding RNA as a novel regulator of the alveolar rhabdomyosarcoma progression and as a putative future therapeutic target.

N 6 -methyladenosine (m 6 A) is an RNA modification well-known for its contribution to different processes controlling RNA metabolism, including splicing, stability, and translation of mRNA. Conversely, the role of m 6 A on the biogenesis and function of circular RNAs (circRNAs) has yet to be addressed. circRNAs belong to a class of covalently closed transcripts produced via a back-splicing reaction whereby a downstream 5' splice donor site fuses to an upstream 3' splice acceptor site. Starting from circ-ZNF609 as a study case, we discover that specific m 6 As control its accumulation and that METTL3 and YTHDC1 are required to direct the back-splicing reaction. This feature is shared with other circRNAs because we find a significant direct correlation among METTL3 requirement, YTHDC1 binding, and the ability of m 6 A exons to undergo back-splicing. Finally, because circ-ZNF609 displays the ability to be translated, we show that m 6 A modifications, through recognition by YTHDF3 and eIF4G2, modulate its translation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)