Accurate detection of m6A RNA modifications in native RNA sequences

Liu, Huanle; Begik, Oguzhan; Lucas, Morghan C.; Ramírez, José Miguel; Mason, Christopher E.; Wiener, David; Schwartz, Schraga; Mattick, John S.; Smith, Martin A.; Novoa, Eva María

doi:10.1038/s41467-019-11713-9

Cited by 373 publications

(495 citation statements)

References 44 publications

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“…So far, comparison to this data suggests that antibody-based approaches may underestimate the number of m 6 A sites 45 . Alternative methods to detect m 6 A based using single-molecule sequencing (including direct RNA sequencing and real-time cDNA synthesis) are under development and may offer ways to detect, quantify, and phase m 6 A sites, but these have not yet been shown to accurately detect m 6 A across a cellular transcriptome 46,82,83 . For now, site-specific SCARLET is the only option to biochemically validate proposed changes in m 6 A at most motifs.…”

Section: Discussionmentioning

confidence: 99%

“…We use our results to propose approaches to MeRIP-seq experimental design and analysis to improve reproducibility and more accurately measure differential regulation of m 6 A (m) in response to stimuli. These data and analyses emphasize the need for further research and alternative assays, for example recently developed endoribonuclease-based sequencing methods 44,45 or direct RNA nanopore sequencing 46 , to resolve the extent to which m 6 A changes in response to specific conditions.…”

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confidence: 96%

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Limits in the detection of m6A changes using MeRIP/m6A-seq

McIntyre

Gokhale

Cerchietti

et al. 2020

Sci Rep

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155

122

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Many cellular mRNAs contain the modified base m 6 A, and recent studies have suggested that various stimuli can lead to changes in m 6 A. the most common method to map m 6 A and to predict changes in m 6 A between conditions is methylated RNA immunoprecipitation sequencing (MeRIP-seq), through which methylated regions are detected as peaks in transcript coverage from immunoprecipitated RNA relative to input RNA. Here, we generated replicate controls and reanalyzed published MeRIP-seq data to estimate reproducibility across experiments. We found that m 6 A peak overlap in mRNAs varies from ~30 to 60% between studies, even in the same cell type. We then assessed statistical methods to detect changes in m 6 A peaks as distinct from changes in gene expression. However, from these published data sets, we detected few changes under most conditions and were unable to detect consistent changes across studies of similar stimuli. Overall, our work identifies limits to MeRIP-seq reproducibility in the detection both of peaks and of peak changes and proposes improved approaches for analysis of peak changes. Methylation at the N6 position in adenosine (m 6 A) is the most common internal modification in eukaryotic mRNA. A methyltransferase complex composed of METTL3, METTL14, WTAP, VIRMA, and other cofactors catalyzes methylation at DRACH/DRAC motifs, primarily in the last exon 1,2. Most m 6 A methylation occurs during transcription 3. The modification then affects mRNA metabolism through recognition by RNA-binding proteins that regulate processes including translation and mRNA degradation 4-9. However, whether m 6 A is lost and gained in response to various cellular changes remains contentious 3,10-15. To assess the evidence for proposed dynamic changes in m 6 A, a reliable and reproducible method to detect changes in methylation as distinct from changes in gene expression is necessary. The first and most widely-used method to enable transcriptome-wide studies of m 6 A, MeRIP-seq or m 6 A-seq, involves the immunoprecipitation of m 6 A-modified RNA fragments followed by peak detection through comparison to background gene coverage 16,17. A second method was developed in 2015, miCLIP or m 6 A-CLIP, which involves crosslinking at the site of antibody binding to induce mutations during reverse transcription for single-nucleotide detection of methylated bases 2,18. MeRIP-seq is still more often used than miCLIP, despite less precise localization of m 6 A to peak regions of approximately 50-200 base pairs that can contain multiple DRAC motifs, since it follows a simpler protocol, requires less starting material, and generally produces higher coverage of more transcripts. Antibodies for m 6 A can also detect a second base modification, N 6 ,2′-O-dimethyladenosine (m 6 A m), found at a lower abundance than m 6 A and located at the 5′ ends of select transcripts 15,18. We thus refer to the base modifications detected through MeRIP-seq collectively as m 6 A (m) , although most are likely m 6 A. As of late 2018, over fifty studies used M...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 96%

Limits in the detection of m6A changes using MeRIP/m6A-seq

McIntyre

Gokhale

Cerchietti

et al. 2020

Sci Rep

Self Cite

155

122

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“…Unlike commonly used NGS-based methods that typically necessitate RNA fragmentation, reverse transcription and extensive PCR amplification, nanopore methodology enables direct sequencing of very long RNA molecules without amplification. This sequencing method has been recently applied to measure poly(A) tails (Krause et al, 2019;Workman et al, 2019), as well as to detect epi-transcriptomic modifications (Liu et al, 2019a;Kim et al, 2020;Lorenz et al, 2020). Despite the current limitations, this technology is under constant development, which makes it highly likely that in the near future, it will become a critical tool enabling the scientific community to address multiple open questions in the field of RNA biology, particularly of epitranscriptomics.…”

Section: Discussionmentioning

confidence: 99%

So close, no matter how far: multiple paths connecting transcription to mRNA translation in eukaryotes

Slobodin

Dikstein

2020

EMBO Reports

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Transcription of DNA into mRNA and translation of mRNA into proteins are two major processes underlying gene expression. Due to the distinct molecular mechanisms, timings, and locales of action, these processes are mainly considered to be independent. During the last two decades, however, multiple factors and elements were shown to coordinate transcription and translation, suggesting an intricate level of synchronization. This review discusses the molecular mechanisms that impact both processes in eukaryotic cells of different origins. The emerging global picture suggests evolutionarily conserved regulation and coordination between transcription and mRNA translation, indicating the importance of this phenomenon for the fine‐tuning of gene expression and the adjustment to constantly changing conditions.

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“…More recent work has determined that m 6 A can be detected through an analysis of increased systematic errors and decreased base‐calling qualities. This method can detect m 6 A with approximately 90 per cent accuracy in synthetic sequences and with 87 per cent accuracy in vivo [187].…”

Section: Epigenetic Modificationsmentioning

confidence: 99%

Invited Review: Epigenetics in neurodevelopment

Salinas

Connolly

Song

2020

Neuropathology Appl Neurobio

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Neural development requires the orchestration of dynamic changes in gene expression to regulate cell fate decisions. This regulation is heavily influenced by epigenetics, heritable changes in gene expression not directly explained by genomic information alone. An understanding of the complexity of epigenetic regulation is rapidly emerging through the development of novel technologies that can assay various features of epigenetics and gene regulation. Here, we provide a broad overview of several commonly investigated modes of epigenetic regulation, including DNA methylation, histone modifications, noncoding RNAs, as well as epitranscriptomics that describe modifications of RNA, in neurodevelopment and diseases. Rather than functioning in isolation, it is being increasingly appreciated that these various modes of gene regulation are dynamically interactive and coordinate the complex nature of neurodevelopment along multiple axes. Future work investigating these interactions will likely utilize ‘multi‐omic’ strategies that assay cell fate dynamics in a high‐dimensional and high‐throughput fashion. Novel human neurodevelopmental models including iPSC and cerebral organoid systems may provide further insight into human‐specific features of neurodevelopment and diseases.

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Accurate detection of m6A RNA modifications in native RNA sequences

Cited by 373 publications

References 44 publications

Limits in the detection of m6A changes using MeRIP/m6A-seq

Limits in the detection of m6A changes using MeRIP/m6A-seq

So close, no matter how far: multiple paths connecting transcription to mRNA translation in eukaryotes

Invited Review: Epigenetics in neurodevelopment

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