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Counting the Cuts: MAZTER-Seq Quantifies m6A Levels Using a Methylation-Sensitive Ribonuclease

2019; Cell Press; Volume: 178; Issue: 3 Linguagem: Inglês

10.1016/j.cell.2019.07.006

1097-4172

Radha Raman Pandey, Ramesh S. Pillai,

Peptidase Inhibition and Analysis

Garcia-Campos et al. describe MAZTER-seq, which deploys a sequence-specific, methylation-sensitive bacterial single-stranded ribonuclease MazF to provide nucleotide-resolution quantification of m6A methylation sites. The study reveals many new sites and supports the idea of a predictable "m6A code," where methylation levels are dictated primarily by local sequence at the site of methylation. Garcia-Campos et al. describe MAZTER-seq, which deploys a sequence-specific, methylation-sensitive bacterial single-stranded ribonuclease MazF to provide nucleotide-resolution quantification of m6A methylation sites. The study reveals many new sites and supports the idea of a predictable "m6A code," where methylation levels are dictated primarily by local sequence at the site of methylation. RNA modifications form a new layer of gene expression regulation in organisms ranging from yeast to human and plants. Of these, N6-methyladenosine (m6A) is the most abundant internal RNA modification found on messenger RNAs (mRNAs). Discovered almost 50 years ago, the m6A mark sprang back into research focus with the ability to globally profile the marks on RNAs using an antibody-based approach (Dominissini et al., 2012Dominissini D. Moshitch-Moshkovitz S. Schwartz S. Salmon-Divon M. Ungar L. Osenberg S. Cesarkas K. Jacob-Hirsch J. Amariglio N. Kupiec M. et al.Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.Nature. 2012; 485: 201-206Crossref PubMed Scopus (2656) Google Scholar, Meyer et al., 2012Meyer K.D. Saletore Y. Zumbo P. Elemento O. Mason C.E. Jaffrey S.R. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons.Cell. 2012; 149: 1635-1646Abstract Full Text Full Text PDF PubMed Scopus (2321) Google Scholar). Recognition of the modification by YTH "reader" proteins mediate functions such as modulation of RNA splicing, choice of polyadenylation site, RNA decay, and translation (Fu et al., 2014Fu Y. Dominissini D. Rechavi G. He C. Gene expression regulation mediated through reversible m6A RNA methylation.Nat. Rev. Genet. 2014; 15: 293-306Crossref PubMed Scopus (1017) Google Scholar). RNA m6A modification is deposited co-transcriptionally by writer proteins; in particular, the METTL3/METTL14 heterodimer is the major RNA m6A methyltransferase responsible for most marks on messenger RNAs. However, the level of m6A modification on different transcripts and its impact on RNA metabolism is poorly understood mainly due to the lack of a method to accurately quantify the marks across the transcriptome. The antibody-based method relies on fragmentation of RNA molecules to a desired size range (∼30–40 nt), followed by immunoprecipitation with anti-m6A antibody and deep sequencing of associated reads. Mapping the reads to transcripts identifies loci where there is an enrichment, the so-called m6A peaks. The m6A peaks usually contain a METTL3/METTL14 recognition motif (represented as DRACH [D = A/G/U, R = A/G, H = A/C/U]) somewhere in the vicinity. However, this method suffers from lack of nucleotide-resolution information on the location of the methylation mark. Variation of the method (miCLIP) using crosslinking of the antibody to the m6A mark in the RNA improves resolution (Linder et al., 2015Linder B. Grozhik A.V. Olarerin-George A.O. Meydan C. Mason C.E. Jaffrey S.R. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.Nat. Methods. 2015; 12: 767-772Crossref PubMed Scopus (827) Google Scholar), but quantification remains a problem. Finally, a quantitative measure of m6A methylation at single-nucleotide resolution can be obtained with a ligation-based method called SCARLET, but this requires working with single sites, one at a time (Liu et al., 2013Liu N. Parisien M. Dai Q. Zheng G. He C. Pan T. Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.RNA. 2013; 19: 1848-1856Crossref PubMed Scopus (326) Google Scholar). In this issue of Cell, the Schwartz lab set out to develop an antibody-independent method to quantify m6A methylation at single-nucleotide resolution (Garcia-Campos et al., 2019Garcia-Campos M.A. Edelheit S. Toth U. Safra M. Shachar R. Viukov S. Winkler R. Nir R. Lasman L. Brandis A. et al.Deciphering the "m6A Code" via Antibody-Independent Quantitative Profiling.Cell. 2019; 178 (this issue): 731-747Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). The hero of this method, which they call MAZTER-seq, is the bacterial ribonuclease MazF, which was previously shown to cleave single-stranded RNAs immediately upstream of an unmodified ACA motif but not a methylated m6A-CA (Imanishi et al., 2017Imanishi M. Tsuji S. Suda A. Futaki S. Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease.Chem. Commun. (Camb.). 2017; 53: 12930-12933Crossref PubMed Google Scholar). Thus, if there are multiple ACA motifs on an RNA molecule, all unmethylated sites will be cleaved (Figure 1). Deep sequencing of the fragments captures 5ʹ ends beginning with an ACA and 3ʹ ends ending just before a downstream ACA. Methylated ACA sites that are spared by MazF are indicated by presence of sequencing reads spanning them. Counting the number of reads beginning at and/or ending before a particular ACA site (indicating unmethylated state), versus those spanning it (methylated state), provides a quantified measure of the methylation at that particular site. As MazF is a ribonuclease specific for single-stranded RNAs, its activity can be influenced by sequence context and secondary structure at the methylation site. Thus, absolute estimation of methylation levels requires normalization in the methyltransferase mutant background. The authors use MAZTER-seq to accurately capture m6A levels on endogenous mRNAs from yeast undergoing meiosis, fluxing from nonexistent levels in the vegetative stage to a peak methylation at meiotic prophase. A striking display of the power of MAZTER-seq is its ability to uncover in yeast many more sites than previously identified by antibody-based approaches. This points to a lower sensitivity of antibody-based approaches and partially explains the low overlap of m6A sites identified by antibody-based methods in different publications. This also indicates that the currently known number of methylation sites in most systems is a huge underestimation. One important caveat of MAZTER-seq is the requirement for an ACA motif in the methylation site, but this works well for the yeast system as the motif is present in ∼50% of all methylated sites. However, given the need for relatively well-spaced ACA motifs for detection by sequencing, only 50% of all such sequences can be captured by the method, in effect allowing quantification of 25% of all m6A sites in yeast. When applied to the human transcriptome, the method can quantify a decrease in m6A levels in response to overexpression of RNA demethylases. However, given the requirement of an ACA motif, the method captures only ∼16% of the m6A (ACA-containing) sites in the mammalian system. Surveying the sites identified by MAZTER-seq, the authors define a simple sequence code consisting of the obligate ACA motif with favored flanking +4 and −4 residues (relative to the methylated adenosine). The presence of these sequences explained most of the methylation differences across the sites, although local secondary structure and, more crucially, proximity of the sites toward the 3′ end played a role in methylation efficiency. The authors were able to use this primary sequence information to predict methylation sites de novo across the genome, with many of them confirmed to be methylated. Moreover, they tested the importance of this sequence specificity by measuring in vivo methylation levels in a library of yeast mutants with hundreds of designed sequence variants of the m6A consensus site. These in vivo measures correlate well with that expected from predictions. The predictive power of their model was limited for mammalian sites, probably due to complex regulation of methylase recruitment. When MAZTER-seq was applied to quantify m6A levels in wild-type and Mettl3 knockout mouse embryonic stem cells (ESCs) the authors were able to deduce a simple m6A sequence code, very similar to yeast, which explained 35% of the variability in methylation levels among different m6A sites. This suggests that m6A methylation sites are encoded in the genome and are evolutionarily conserved from yeast to mammals. As in yeast, MAZTER-seq identified many novel methylation sites in mouse ESCs. However, available data point to a more complex regulation of target-site selection in mammals compared to yeast, via an intimate link to the transcription process itself. For instance, in mammals, transcript- and site-specific m6A methylation is influenced by direct interaction between specific transcription factors and the methylase complex (Bertero et al., 2018Bertero A. Brown S. Madrigal P. Osnato A. Ortmann D. Yiangou L. Kadiwala J. Hubner N.C. de Los Mozos I.R. Sadée C. et al.The SMAD2/3 interactome reveals that TGFβ controls m6A mRNA methylation in pluripotency.Nature. 2018; 555: 256-259Crossref PubMed Scopus (201) Google Scholar), the elongation rate of RNA polymerase (Slobodin et al., 2017Slobodin B. Han R. Calderone V. Vrielink J.A. Loayza-Puch F. Elkon R. Agami R. Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation.Cell. 2017; 169: 326-337.e12Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), and even heat-shock regulation of methylase recruitment (Knuckles et al., 2017Knuckles P. Carl S.H. Musheev M. Niehrs C. Wenger A. Bühler M. RNA fate determination through cotranscriptional adenosine methylation and microprocessor binding.Nat. Struct. Mol. Biol. 2017; 24: 561-569Crossref PubMed Scopus (95) Google Scholar). Thus, a simple sequence code may not explain the deposition of m6A at most sites in the mammalian system. Taken together, Garcia-Campos et al. provide the field with a new tool to accurately quantify and study m6A RNA methylation at a large number of sites. Expanding the MazF toolbox should help overcome the current limitations on the fraction of sites captured. Specifically, protein engineering or isolation of natural variants with expanded sequence specificities beyond the ACA motif should allow a more complete and quantified picture of m6A methylation to emerge. Most importantly, MAZTER-seq provides a complementary approach toward identification and quantification of m6A sites that can, together with orthogonal methods such as m6A-IP-seq, provide a valuable m6A-map across transcriptomes. A quantified view of methylation is only part of the long journey ahead in our complete understanding of m6A biology. We point the readers to a related manuscript using MazF for mapping m6A sites on mammalian RNAs (Zhang et al., bioRxiv. https://doi.org/10.1101/575555), which we couldn't unfortunately discuss here due to space limitations. Deciphering the "m6A Code" via Antibody-Independent Quantitative ProfilingGarcia-Campos et al.CellJune 27, 2019In BriefA new enzymatic approach for precise mapping and measurement of m6A within mRNAs provides insight into how methylation sites are selected and the functional impact of the modifications. Full-Text PDF Open Archive