Global Detection of RNA Methylation by Click Degradation
By hijacking methyltransferase activity, a novel click-based method utilizes degradation to map RNA methylation across the transcriptome.
- Allen C. ZhuAllen C. ZhuDepartment of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United StatesDepartment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, United StatesHoward Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United StatesMedical Scientist Training Program/Committee on Cancer Biology, The University of Chicago, Chicago, Illinois 60637, United StatesMore by Allen C. Zhu and
- Chuan He Chuan HeEmail: [email protected]Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United StatesDepartment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, United StatesHoward Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United StatesMore by Chuan He
Gene expression regulation is central to sustain life. Post-transcriptional modifications of RNA have recently emerged as a major regulatory layer of gene expression and, thus, a large extent of physiological and disease processes. To understand how an RNA modification contributes to cellular function and gene expression, it is critical to be able to detect the sites of modification in cells. In this issue of ACS Central Science, Mikutis and colleagues present a new approach called methylation CLICK-degradation sequencing (meCLICK-seq), which relies on small-molecule click degraders to identify sites of methylated RNA via transcript depletion.(1) Their work provides a new way to identify RNA species that may be methylated in previously inaccessible regions of the transcriptome.
Over 170 post-transcriptional modifications of RNA have been discovered. While their existence has been known for decades, more recently some of these modifications have been found to play broad regulatory roles in gene expression. For instance, N6-methyladenosine (m6A) regulates many aspects of mRNA metabolism, including transcript stability, nuclear processing, translation, and splicing(2) (Figure 1A). While several other modifications such as pseudouridine, m7G, and m1A have also been studied, the authors focus on m6A, as it is the most abundant internal mRNA modification and the most studied. A majority of mRNA m6A’s are co-transcriptionally and site-selectively installed by a methyltransferase complex of METTL3-METTL14 that uses the S-adenosyl methionine (SAM) cofactor. METTL3 is the catalytic subunit of this main m6A mRNA writer complex.(3) Another methyltransferase, METTL16, was later discovered as an m6A writer that binds U6 snRNA or structured RNA, but its substrate scope is still unclear.(4)
Figure 1
Figure 1. (A) N6-Methyladenosine (m6A) results from the addition of a methyl group onto the nitrogen-6 position of the adenosine nucleoside. Depending on the regulatory protein that binds to m6A, various effects can be exerted on RNA metabolism. (B) Most m6A-sequencing experiments rely on an m6A antibody–bead complex that binds to m6A methylated RNA. The immunoprecipitated RNA can then be prepared as a library for high-throughput sequencing.
To study how RNA modifications contribute to cellular function and gene expression, transcriptome-wide detection of these sites is critical, and the advent of high-throughput sequencing methods now permits such deeper study of their functions. To date, most researchers have relied on antibody immunoprecipitation to enrich methylated RNAs (Figure 1B). This approach, however, has several drawbacks, such as large sample quantity requirements, nonspecific antibody binding, low resolution, and lack of quantitative information. As a result, simpler, antibody-free methods are needed. Global m6A detection has relied on antibody-based methods due to the inert reactivity of m6A. In this study, however, the authors alter this chemical property by introducing a surrogate of SAM into cells, so that a propargyl group is installed onto the RNA substrate in place of a methyl group (Figure 2A). A previous work installed an allyl group to m6A sites using a SAM homologue for m6A detection.(5) The authors here developed a more general and sensitive approach. The propargyl-modified RNA (Pr6A) undergoes a Cu(I)-catalyzed cycloaddition reaction with azide to form an artificial click-based RNA modification that catalyzes degradation of the RNA (Figure 2B). This click-degrader approach links RNA methylation to degradation and provides the basis for a unique readout of meCLICK-seq: methylated RNAs should exhibit reduced transcript abundance upon RNA-sequencing. Degradation serves as a useful readout because it is easy to identify reduced transcript levels with standard RNA-sequencing technology.
Figure 2
Figure 2. (A) After methionine starvation, the meCLICK-seq protocol begins with introduction of a SAM cofactor surrogate (SeAdoYn) that is used by RNA methyltransferases to form a propargyl (alkyne-tagged) modification on an RNA substrate. (B) After the RNA is tagged with a propargyl group, the addition of an azide click degrader leads to a Cu(I)-catalyzed cycloaddition reaction that tags the RNA molecule for general base and copper-mediated degradation, which can be easily identified with standard RNA-sequencing. Reproduced with permission from ref (1). Copyright 2020 American Chemical Society.
The authors first verify in vitro that extensive degradation occurs specifically on RNA functionalized with the click-degrader in the presence of copper and then show that Pr6A can be introduced with subsequent degradation in MOLM13 leukemia and HEK293T kidney cancer cells. Thereafter, the authors apply meCLICK-seq in MOLM13 cells with induced knockdown of either m6A writer METTL3 or METTL16 to map their mRNA substrates. Since meCLICK-seq would label any methylation site that depends on SAM, knocking down a specific methyltransferase helps to identify its substrates. They not only validate that meCLICK-seq yields degradation of known m6A-containing transcripts but also consistently find that a majority of their transcripts overlap with peaks reported from previous m6A cross-linking and immunoprecipitation (miCLIP) sequencing datasets. From their meCLICK-seq method, the authors also report new findings on m6A methylation. For instance, they find a greater number of lncRNA substrates of METTL3 and METTL16 than previous miCLIP data showed. Furthermore, the addition of a bulky propargyl moiety likely hinders RNA processing and recycling pathways. As a result, the authors find m6A methylation on intronic and intergenic regions that were click-degraded with far more peaks primarily due to METTL16. From their intronic sequencing data, the authors discover that intronic polyadenylation sites are linked to methylation by METTL16.
The use of click chemistry for degradation of sites of RNA methylation is clever and simple. After methionine starvation and treatment with the click-degrader, the only steps required to detect methylated sites are RNA extraction followed by sequencing. A drawback of meCLICK-seq, however, may be limited base resolution, or lack of specificity for a particular RNA modification due to its reliance on a SAM surrogate. This issue can be circumvented by knocking down or expressing a particular RNA modification writer, as the authors did in their study. meCLICK-seq also provides a quantifiable output of methylation, since it harnesses an artificial modification for measurable degradation output. Thus, it may offer a way to study modification stoichiometry in a relatively easy and unbiased manner. Perhaps the more interesting finding is the ability to use meCLICK-seq to study different methylations in general. The authors showed that they can also map internal m7G on RNA and map m6A on introns and noncoding RNAs, which could be installed by different methyltransferases. Recent studies have shown that chromatin-associated RNAs, which contain pre-mRNA and noncoding regulatory RNAs, are m6A methylated and play important roles in chromatin regulation.(6) Studying these less abundant RNA species with m6A potentially installed by different methyltransferases may be streamlined by meCLICK-seq.
Within the field of m6A-sequencing, meCLICK-seq joins a series of other recently developed, antibody-free methods. For example, m6A-REF-seq and MAZTER-seq use a bacterial single-stranded endoribonuclease in which m6A methylated sites remain uncleaved.(7,8) DART-seq tethers a cytidine deaminase to a YTH m6A-binding domain to induce mutations adjacent to m6A sites.(9) Similar to meCLICK-seq, m6A-label-seq introduces a SAM analog to form N6-allyladenosine, which results in mutations in place of an m6A site upon sequencing.(5) Meanwhile, m6A-SEAL co-opts an m6A demethylase (FTO) to oxidatively modify m6A for streptavidin-based pulldown.(10) These techniques have benefits, such as lower required input, and limitations, such as required sequence motifs for mutation, complicated post-treatment procedures, or inability to detect all m6A sites. meCLICK-seq fits a unique niche in that it can identify peaks and genes containing RNA modifications installed by a particular methyltransferase or demethylase relatively easily. Overall, these methods complement one another. Together with existing approaches, meCLICK-seq will help advance our understanding and distribution of the roles of RNA modifications in the epitranscriptome.
The authors declare no competing financial interest.
Author Information
- Chuan He - Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, United States; Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United States;
http://orcid.org/0000-0003-4319-7424;
Email: [email protected]
- Allen C. Zhu - Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, United States; Howard Hughes Medical Institute, The University of Chicago, Chicago, Illinois 60637, United States; Medical Scientist Training Program/Committee on Cancer Biology, The University of Chicago, Chicago, Illinois 60637, United States;http://orcid.org/0000-0002-1490-7063
The authors declare no competing financial interest.
References
This article references 10 other publications.
- 1Mikutis, S.; Gu, M.; Sendinc, E.; Hazemi, M. E.; Kiely-Collins, H.; Aspris, D.; Vassiliou, G. S.; Shi, Y.; Tzelepis, K.; Bernardes, G. J. L. meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation. ACS Cent. Sci. 2020, DOI: 10.1021/acscentsci.0c01094
- 2Frye, M.; Harada, B. T.; Behm, M.; He, C. RNA modifications modulate gene expression during development. Science 2018, 361 (6409), 1346– 1349, DOI: 10.1126/science.aau1646[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOrurrI&md5=c901de52de03491c6c3cec5d60b44d59RNA modifications modulate gene expression during developmentFrye, Michaela; Harada, Bryan T.; Behm, Mikaela; He, ChuanScience (Washington, DC, United States) (2018), 361 (6409), 1346-1349CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. RNA modifications have recently emerged as crit. posttranscriptional regulators of gene expression programs. They affect diverse eukaryotic biol. processes, and the correct deposition of many of these modifications is required for normal development. MRNA modifications regulate various aspects of mRNA metab. For example, N6-methyladenosine (m6A) affects the translation and stability of the modified transcripts, thus providing a mechanism to coordinate the regulation of groups of transcripts during cell state maintenance and transition. Similarly, some modifications in tRNAs are essential for RNA structure and function. Others are deposited in response to external cues and adapt global protein synthesis and gene-specific translational accordingly and thereby facilitate proper development.
- 3Liu, J.; Yue, Y.; Han, D.; Wang, X.; Fu, Y.; Zhang, L.; Jia, G.; Yu, M.; Lu, Z.; Deng, X.; Dai, Q.; Chen, W.; He, C. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 2014, 10 (2), 93– 5, DOI: 10.1038/nchembio.1432[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvV2hsbfF&md5=f07bc7352b4254a12dfa8f6e27088432A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylationLiu, Jianzhao; Yue, Yanan; Han, Dali; Wang, Xiao; Fu, Ye; Zhang, Liang; Jia, Guifang; Yu, Miao; Lu, Zhike; Deng, Xin; Dai, Qing; Chen, Weizhong; He, ChuanNature Chemical Biology (2014), 10 (2), 93-95CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)N6-methyladenosine (m6A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m6A RNA methylation. Together with METTL3, the only previously known m6A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m6A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.
- 4Pendleton, K. E.; Chen, B.; Liu, K.; Hunter, O. V.; Xie, Y.; Tu, B. P.; Conrad, N. K. The U6 snRNA m(6)A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention. Cell 2017, 169 (5), 824– 835, DOI: 10.1016/j.cell.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlOqtr4%253D&md5=e18b9d1155f4db389d87875a48d50dd6The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retentionPendleton, Kathryn E.; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V.; Xie, Yang; Tu, Benjamin P.; Conrad, Nicholas K.Cell (Cambridge, MA, United States) (2017), 169 (5), 824-835.e14CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Maintenance of proper levels of the Me donor S-adenosylmethionine (SAM) is crit. for a wide variety of biol. processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an addnl. function in vertebrates to regulate SAM homeostasis.
- 5Shu, X.; Cao, J.; Cheng, M.; Xiang, S.; Gao, M.; Li, T.; Ying, X.; Wang, F.; Yue, Y.; Lu, Z.; Dai, Q.; Cui, X.; Ma, L.; Wang, Y.; He, C.; Feng, X.; Liu, J. A metabolic labeling method detects m(6)A transcriptome-wide at single base resolution. Nat. Chem. Biol. 2020, 16 (8), 887– 895, DOI: 10.1038/s41589-020-0526-9[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjur0%253D&md5=1793171884fef97b342913c2b6b847d3A metabolic labeling method detects m6A transcriptome-wide at single base resolutionShu, Xiao; Cao, Jie; Cheng, Mohan; Xiang, Siying; Gao, Minsong; Li, Ting; Ying, Xiner; Wang, Fengqin; Yue, Yanan; Lu, Zhike; Dai, Qing; Cui, Xiaolong; Ma, Lijia; Wang, Yizhen; He, Chuan; Feng, Xinhua; Liu, JianzhaoNature Chemical Biology (2020), 16 (8), 887-895CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resoln. remains an issue, impeding the authors' understanding of m6A roles at the nucleotide level. Here, the authors report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resoln., called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the Me group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. The authors pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. The authors identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.
- 6Liu, J.; Dou, X.; Chen, C.; Chen, C.; Liu, C.; Xu, M. M.; Zhao, S.; Shen, B.; Gao, Y.; Han, D.; He, C. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 2020, 367 (6477), 580– 586, DOI: 10.1126/science.aay6018[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVGiurw%253D&md5=e2a4f15c7c401604e94665c93960cd01N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcriptionLiu, Jun; Dou, Xiaoyang; Chen, Chuanyuan; Chen, Chuan; Liu, Chang; Xu, Meng Michelle; Zhao, Siqi; Shen, Bin; Gao, Yawei; Han, Dali; He, ChuanScience (Washington, DC, United States) (2020), 367 (6477), 580-586CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)N6-methyladenosine (m6A) regulates stability and translation of mRNA in various biol. processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-assocd. regulatory RNAs (carRNAs), including promoter-assocd. RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, esp. elements of the long interspersed element-1 family, through the nuclear exosome targeting-mediated nuclear degrdn. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.
- 7Garcia-Campos, M. A.; Edelheit, S.; Toth, U.; Safra, M.; Shachar, R.; Viukov, S.; Winkler, R.; Nir, R.; Lasman, L.; Brandis, A.; Hanna, J. H.; Rossmanith, W.; Schwartz, S. Deciphering the ″m(6)A Code″ via Antibody-Independent Quantitative Profiling. Cell 2019, 178 (3), 731– 747, DOI: 10.1016/j.cell.2019.06.013[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1OjsrfE&md5=5a62d1ebc9f1a6186859906c99ed770dDeciphering the "m6A Code" via Antibody-Independent Quantitative ProfilingGarcia-Campos, Miguel Angel; Edelheit, Sarit; Toth, Ursula; Safra, Modi; Shachar, Ran; Viukov, Sergey; Winkler, Roni; Nir, Ronit; Lasman, Lior; Brandis, Alexander; Hanna, Jacob H.; Rossmanith, Walter; Schwartz, SchragaCell (Cambridge, MA, United States) (2019), 178 (3), 731-747.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in crit. roles in development, physiol., and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, the authors develop MAZTER-seq for systematic quant. profiling of m6A at single-nucleotide resoln. at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quant. tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. M6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quant. study of m6A regulation in subcellular fractions, diverse cell types, and disease states.
- 8Zhang, Z.; Chen, L. Q.; Zhao, Y. L.; Yang, C. G.; Roundtree, I. A.; Zhang, Z.; Ren, J.; Xie, W.; He, C.; Luo, G. Z. Single-base mapping of m(6)A by an antibody-independent method. Sci. Adv. 2019, 5 (7), eaax0250 DOI: 10.1126/sciadv.aax0250
- 9Meyer, K. D. DART-seq: an antibody-free method for global m(6)A detection. Nat. Methods 2019, 16 (12), 1275– 1280, DOI: 10.1038/s41592-019-0570-0[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVarsb3I&md5=2578ff8833ee276fc2bb75929fa9fcb3DART-seq: an antibody-free method for global m6A detectionMeyer, Kate D.Nature Methods (2019), 16 (12), 1275-1280CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the mRNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A mapping methods, which use antibodies to immunoppt. methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using std. RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Addnl., we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.
- 10Wang, Y.; Xiao, Y.; Dong, S.; Yu, Q.; Jia, G. Antibody-free enzyme-assisted chemical approach for detection of N(6)-methyladenosine. Nat. Chem. Biol. 2020, 16 (8), 896– 903, DOI: 10.1038/s41589-020-0525-x[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjur8%253D&md5=9d542adbb5a0272f274f6f6c5af60208Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosineWang, Ye; Xiao, Yu; Dong, Shunqing; Yu, Qiong; Jia, GuifangNature Chemical Biology (2020), 16 (8), 896-903CODEN: NCBABT; ISSN:1552-4450. (Nature Research)The inert chem. property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunopptn. and cannot distinguish m6A and N6,2'-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, the authors present an antibody-free, FTO-assisted chem. labeling method termed m6A-SEAL for specific m6A detection. The authors applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO's oxidn. property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications.
Cited By
Abstract
Figure 1
Figure 1. (A) N6-Methyladenosine (m6A) results from the addition of a methyl group onto the nitrogen-6 position of the adenosine nucleoside. Depending on the regulatory protein that binds to m6A, various effects can be exerted on RNA metabolism. (B) Most m6A-sequencing experiments rely on an m6A antibody–bead complex that binds to m6A methylated RNA. The immunoprecipitated RNA can then be prepared as a library for high-throughput sequencing.
Figure 2
Figure 2. (A) After methionine starvation, the meCLICK-seq protocol begins with introduction of a SAM cofactor surrogate (SeAdoYn) that is used by RNA methyltransferases to form a propargyl (alkyne-tagged) modification on an RNA substrate. (B) After the RNA is tagged with a propargyl group, the addition of an azide click degrader leads to a Cu(I)-catalyzed cycloaddition reaction that tags the RNA molecule for general base and copper-mediated degradation, which can be easily identified with standard RNA-sequencing. Reproduced with permission from ref (1). Copyright 2020 American Chemical Society.
References
ARTICLE SECTIONSThis article references 10 other publications.
- 1Mikutis, S.; Gu, M.; Sendinc, E.; Hazemi, M. E.; Kiely-Collins, H.; Aspris, D.; Vassiliou, G. S.; Shi, Y.; Tzelepis, K.; Bernardes, G. J. L. meCLICK-Seq, a Substrate-Hijacking and RNA Degradation Strategy for the Study of RNA Methylation. ACS Cent. Sci. 2020, DOI: 10.1021/acscentsci.0c01094
- 2Frye, M.; Harada, B. T.; Behm, M.; He, C. RNA modifications modulate gene expression during development. Science 2018, 361 (6409), 1346– 1349, DOI: 10.1126/science.aau1646[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhslOrurrI&md5=c901de52de03491c6c3cec5d60b44d59RNA modifications modulate gene expression during developmentFrye, Michaela; Harada, Bryan T.; Behm, Mikaela; He, ChuanScience (Washington, DC, United States) (2018), 361 (6409), 1346-1349CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. RNA modifications have recently emerged as crit. posttranscriptional regulators of gene expression programs. They affect diverse eukaryotic biol. processes, and the correct deposition of many of these modifications is required for normal development. MRNA modifications regulate various aspects of mRNA metab. For example, N6-methyladenosine (m6A) affects the translation and stability of the modified transcripts, thus providing a mechanism to coordinate the regulation of groups of transcripts during cell state maintenance and transition. Similarly, some modifications in tRNAs are essential for RNA structure and function. Others are deposited in response to external cues and adapt global protein synthesis and gene-specific translational accordingly and thereby facilitate proper development.
- 3Liu, J.; Yue, Y.; Han, D.; Wang, X.; Fu, Y.; Zhang, L.; Jia, G.; Yu, M.; Lu, Z.; Deng, X.; Dai, Q.; Chen, W.; He, C. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 2014, 10 (2), 93– 5, DOI: 10.1038/nchembio.1432[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvV2hsbfF&md5=f07bc7352b4254a12dfa8f6e27088432A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylationLiu, Jianzhao; Yue, Yanan; Han, Dali; Wang, Xiao; Fu, Ye; Zhang, Liang; Jia, Guifang; Yu, Miao; Lu, Zhike; Deng, Xin; Dai, Qing; Chen, Weizhong; He, ChuanNature Chemical Biology (2014), 10 (2), 93-95CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)N6-methyladenosine (m6A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m6A RNA methylation. Together with METTL3, the only previously known m6A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m6A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.
- 4Pendleton, K. E.; Chen, B.; Liu, K.; Hunter, O. V.; Xie, Y.; Tu, B. P.; Conrad, N. K. The U6 snRNA m(6)A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention. Cell 2017, 169 (5), 824– 835, DOI: 10.1016/j.cell.2017.05.003[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXotlOqtr4%253D&md5=e18b9d1155f4db389d87875a48d50dd6The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retentionPendleton, Kathryn E.; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V.; Xie, Yang; Tu, Benjamin P.; Conrad, Nicholas K.Cell (Cambridge, MA, United States) (2017), 169 (5), 824-835.e14CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Maintenance of proper levels of the Me donor S-adenosylmethionine (SAM) is crit. for a wide variety of biol. processes. We demonstrate that the N6-adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an addnl. function in vertebrates to regulate SAM homeostasis.
- 5Shu, X.; Cao, J.; Cheng, M.; Xiang, S.; Gao, M.; Li, T.; Ying, X.; Wang, F.; Yue, Y.; Lu, Z.; Dai, Q.; Cui, X.; Ma, L.; Wang, Y.; He, C.; Feng, X.; Liu, J. A metabolic labeling method detects m(6)A transcriptome-wide at single base resolution. Nat. Chem. Biol. 2020, 16 (8), 887– 895, DOI: 10.1038/s41589-020-0526-9[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjur0%253D&md5=1793171884fef97b342913c2b6b847d3A metabolic labeling method detects m6A transcriptome-wide at single base resolutionShu, Xiao; Cao, Jie; Cheng, Mohan; Xiang, Siying; Gao, Minsong; Li, Ting; Ying, Xiner; Wang, Fengqin; Yue, Yanan; Lu, Zhike; Dai, Qing; Cui, Xiaolong; Ma, Lijia; Wang, Yizhen; He, Chuan; Feng, Xinhua; Liu, JianzhaoNature Chemical Biology (2020), 16 (8), 887-895CODEN: NCBABT; ISSN:1552-4450. (Nature Research)Transcriptome-wide mapping of N6-methyladenosine (m6A) at base resoln. remains an issue, impeding the authors' understanding of m6A roles at the nucleotide level. Here, the authors report a metabolic labeling method to detect mRNA m6A transcriptome-wide at base resoln., called 'm6A-label-seq'. Human and mouse cells could be fed with a methionine analog, Se-allyl-L-selenohomocysteine, which substitutes the Me group on the enzyme cofactor SAM with the allyl. Cellular RNAs could therefore be metabolically modified with N6-allyladenosine (a6A) at supposed m6A-generating adenosine sites. The authors pinpointed the mRNA a6A locations based on iodination-induced misincorporation at the opposite site in complementary DNA during reverse transcription. The authors identified a few thousand mRNA m6A sites in human HeLa, HEK293T and mouse H2.35 cells, carried out a parallel comparison of m6A-label-seq with available m6A sequencing methods, and validated selected sites by an orthogonal method. This method offers advantages in detecting clustered m6A sites and holds promise to locate nuclear nascent RNA m6A modifications.
- 6Liu, J.; Dou, X.; Chen, C.; Chen, C.; Liu, C.; Xu, M. M.; Zhao, S.; Shen, B.; Gao, Y.; Han, D.; He, C. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science 2020, 367 (6477), 580– 586, DOI: 10.1126/science.aay6018[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVGiurw%253D&md5=e2a4f15c7c401604e94665c93960cd01N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcriptionLiu, Jun; Dou, Xiaoyang; Chen, Chuanyuan; Chen, Chuan; Liu, Chang; Xu, Meng Michelle; Zhao, Siqi; Shen, Bin; Gao, Yawei; Han, Dali; He, ChuanScience (Washington, DC, United States) (2020), 367 (6477), 580-586CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)N6-methyladenosine (m6A) regulates stability and translation of mRNA in various biol. processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-assocd. regulatory RNAs (carRNAs), including promoter-assocd. RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, esp. elements of the long interspersed element-1 family, through the nuclear exosome targeting-mediated nuclear degrdn. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.
- 7Garcia-Campos, M. A.; Edelheit, S.; Toth, U.; Safra, M.; Shachar, R.; Viukov, S.; Winkler, R.; Nir, R.; Lasman, L.; Brandis, A.; Hanna, J. H.; Rossmanith, W.; Schwartz, S. Deciphering the ″m(6)A Code″ via Antibody-Independent Quantitative Profiling. Cell 2019, 178 (3), 731– 747, DOI: 10.1016/j.cell.2019.06.013[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1OjsrfE&md5=5a62d1ebc9f1a6186859906c99ed770dDeciphering the "m6A Code" via Antibody-Independent Quantitative ProfilingGarcia-Campos, Miguel Angel; Edelheit, Sarit; Toth, Ursula; Safra, Modi; Shachar, Ran; Viukov, Sergey; Winkler, Roni; Nir, Ronit; Lasman, Lior; Brandis, Alexander; Hanna, Jacob H.; Rossmanith, Walter; Schwartz, SchragaCell (Cambridge, MA, United States) (2019), 178 (3), 731-747.e16CODEN: CELLB5; ISSN:0092-8674. (Cell Press)N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in crit. roles in development, physiol., and disease. A major limitation has been the inability to quantify m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, the authors develop MAZTER-seq for systematic quant. profiling of m6A at single-nucleotide resoln. at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quant. tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. M6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quant. study of m6A regulation in subcellular fractions, diverse cell types, and disease states.
- 8Zhang, Z.; Chen, L. Q.; Zhao, Y. L.; Yang, C. G.; Roundtree, I. A.; Zhang, Z.; Ren, J.; Xie, W.; He, C.; Luo, G. Z. Single-base mapping of m(6)A by an antibody-independent method. Sci. Adv. 2019, 5 (7), eaax0250 DOI: 10.1126/sciadv.aax0250
- 9Meyer, K. D. DART-seq: an antibody-free method for global m(6)A detection. Nat. Methods 2019, 16 (12), 1275– 1280, DOI: 10.1038/s41592-019-0570-0[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhvVarsb3I&md5=2578ff8833ee276fc2bb75929fa9fcb3DART-seq: an antibody-free method for global m6A detectionMeyer, Kate D.Nature Methods (2019), 16 (12), 1275-1280CODEN: NMAEA3; ISSN:1548-7091. (Nature Research)N6-methyladenosine (m6A) is a widespread RNA modification that influences nearly every aspect of the mRNA lifecycle. Our understanding of m6A has been facilitated by the development of global m6A mapping methods, which use antibodies to immunoppt. methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m6A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m6A residues, which are detected using std. RNA-seq. DART-seq identifies thousands of m6A sites in cells from as little as 10 ng of total RNA and can detect m6A accumulation in cells over time. Addnl., we use long-read DART-seq to gain insights into m6A distribution along the length of individual transcripts.
- 10Wang, Y.; Xiao, Y.; Dong, S.; Yu, Q.; Jia, G. Antibody-free enzyme-assisted chemical approach for detection of N(6)-methyladenosine. Nat. Chem. Biol. 2020, 16 (8), 896– 903, DOI: 10.1038/s41589-020-0525-x[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXotVCjur8%253D&md5=9d542adbb5a0272f274f6f6c5af60208Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosineWang, Ye; Xiao, Yu; Dong, Shunqing; Yu, Qiong; Jia, GuifangNature Chemical Biology (2020), 16 (8), 896-903CODEN: NCBABT; ISSN:1552-4450. (Nature Research)The inert chem. property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunopptn. and cannot distinguish m6A and N6,2'-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, the authors present an antibody-free, FTO-assisted chem. labeling method termed m6A-SEAL for specific m6A detection. The authors applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO's oxidn. property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications.
