Cg14906 (mettl4) mediates m6a methylation of u2 snrna in drosophila

Dear Editor, While the eukaryotic candidate m6A methyltransferases belong to multiple distinct methylase lineages, the most widespread group belongs to the MT-A70 family exemplified by the

yeast messenger RNA (mRNA) adenine methylase complex Ime4/Kar4. At the structural level, all of these enzymes are characterized by a 7-β-strand methyltransferase domain at their C terminus,

fused to a predicted α-helical domain at their N terminus and require _S_-adenosyl-l-methionine (SAM) as a methyl donor. The catalytic motif, [DSH]PP[YFW], present in many members of this

family, has shown to be critical for METTL3/METTL4-mediated mRNA m6A methylation1. The high degree of amino acid sequence conservation among the predicted N6-methyladenosine

methyltransferases motivates further explorations into their potential functional conservation. METTL4 is a member of the MT-A70-like protein family, which is conserved during evolution

(Fig. 1a)2. Previous studies suggested that METTL4 regulates DNA 6mA in vivo and therefore is a candidate DNA 6mA methyltransferase3,4,5. However, the enzymatic activity of METTL4 in vitro

has not been demonstrated. To identify the substrate(s) for METTL4, we purified His-tagged, wild-type (WT) as well as a catalytic mutant (DPPW mutated to NPPW) (Supplementary Fig. S1) of

_Drosophila melanogaster_ mettl4 from _Escherichia coli_ strain BL21 (DE3). In order to unbiasedly identify potential substrates of mettl4, we performed in vitro enzymatic assays using

various substrates, including both DNA and RNA with and without secondary structures. We used deuterated _S_-adenosyl methionine (SAM-d3) in the in vitro enzymatic assays in order to

identify m6A mediated by mettl4. Although we detected a weak enzymatic activity on DNA substrates composed of previously published sequence motifs, mettl4 appears to prefer RNA substrates

potentially with secondary structures (Supplementary Fig. S2). We next performed enhanced crosslinking and immunoprecipitation (IP) followed by high-throughput sequencing (eCLIP-seq), which

was originally developed to map binding sites of RNA-binding proteins on their target RNAs6, to identify the RNA type that is targeted by mettl4 in vivo. Since there are no commercial

antibodies available for fly mettl4, we generated a _Drosophila_ Kc cell line with a FLAG-tagged mettl4 for the eCLIP-seq experiment7. In total, we generated two biological replicates for IP

samples, and their respective input samples, together with one IP-control and input-control sample for the quality control and enrichment analysis8. The two replicates showed a strong

correlation with a Spearman’s correlation coefficient of 0.97, indicating great consistency between the replicates (Supplementary Fig. S3). Thus, we merged the two replicates to increase the

sequencing depth and power for downstream analyses, which showed that mettl4 captured RNA molecules, mostly transfer RNA (tRNA) and small nuclear RNA (snRNA), including U2, U4, and U6atac

(Fig. 1b, c). We next investigated whether the RNAs identified by the eCLIP experiments are indeed substrates of mettl4 by carrying out in vitro enzymatic assays. We synthesized

oligonucleotides containing tRNA and snRNA sequences and various controls, including DNAs with the same sequences (Supplementary Table S1). The in vitro enzymatic activity of mettl4 on each

candidate substrate and control sequences was measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). These in vitro experiments led to the identification of U2 as the

best substrate among all the snRNA subtypes (Fig. 1d). Next, we wished to identify the adenosine residues in U2 that are methylated by mettl4. Previous studies documented that the adenosine

at the 30th position of U2 is frequently methylated in vertebrate U2 snRNA9, with a sequence motif of AA-G as opposed to 29AAAG32 in fly. To identify which adenosine within the motif is

essential for the enzymatic activity in fly, we generated point mutations and deletions of adenosine within and close to this motif and measured the enzymatic activity of mettl4 on these

substrates. We found that when the 29th position adenosine is mutated or deleted, no m6A methylation was detected by LC-MS/MS, whereas other point mutations or deletions (26th and 31st

positions) did not affect substrate methylation or only decreased methylation partially (i.e., the 30th position). These results indicate that adenosine at position 29 is the adenosine in U2

that is methylated by mettl4 in fly (Fig. 1d). In order to better characterize the enzymology of mettl4, we next investigated the kinetics of mettl4 and determined that mettl4 was able to

methylate U2 with a Michaelis–Menten constant (_K_m) of 5.298 μM and a catalytic rate constant (_k_cat) of 46.566 min−1 (Fig. 1e). In addition, the enzyme is inhibited by the substrate at

higher concentrations (Fig. 1e). Next, we investigated whether mettl4 catalyzes U2 m6A in vivo. To accomplish this goal, we generated _mettl4_ knockout (KO) and rescue cell lines (rescued by

either WT or catalytic mutant of mettl4) (Supplementary Figs. S4 and S5). Indeed, the U2 m6A level is decreased dramatically in the _mettl4_ KO cells and restored in the wt, but not in the

catalytic compriomised, _mettl4_ rescued cells (Fig. 1f; Supplementary Figs. S7 and S8a). Furthermore, the same reduction of U2 m6A level was also seen in KO flies (Fig. 1g; Supplementary

Figs. S6 and S8b). The low DNA 6mA levels between WT and KO fly cells for both nuclear and mitochondrial DNA showed no significant differences (Supplementary Fig. S9). These findings suggest

that it is mettl4 that mediates U2 methylation in vivo. Interestingly, the U2 m6A level in WT female flies is significantly higher than that in males, suggesting that mettl4 might play

sex-specific roles (Fig. 1g; Supplementary Fig. S8b), which will be interesting to investigate in the future. Given U2 snRNA is involved in pre-mRNA splicing10, we performed RNA-seq using

both WT and _mettl4_ KO _Drosophila_ Kc cell lines to determine if RNA splicing is affected as a result of mettl4 loss. In total, we identified 2366 transcripts with differential alternative

splicing events, which cover 1771 genes. Gene Ontology Enrichment analysis suggests that _mettl4_ affects a broad set of biological processes, including differentiation, development,

growth, and response to stimulus (Fig. 1h). We next investigated whether there are any significant phenotypic differences between WT and KO cells, given the broad changes in the whole

transcriptome caused by _mettl4_ KO. Indeed, we observed a significant proliferation difference between WT and _mettl4_ KO cells (Fig. 1i; Supplementary Fig. S10). In addition, we analyzed

independently generated _mettl4_ knockdown (KD) cell lines by RNA interference (RNAi), and both KD cell lines displayed enhanced growth/proliferation than control cells (Supplementary Fig.

S11), indicating loss of mettl4 is associated with enhanced cell proliferation. Although both KO and KD cell lines show similar proliferation pattern, genetic rescue experiments are needed

to definitively rule out potential off-target effects. Since U2 is an essential component of the major spliceosomal complex, which plays an important role in pre-RNA splicing, loss of

_mettl4_ might have broad impacts through altered RNA splicing. However, whether the altered RNA splicing events are regulated by mettl4 through methylation of U2 snRNA or other

yet-to-be-identified substrates, or whether mettl4 regulates splicing in an enzymatic activity-independent manner, remain to be determined in the future. In addition, we did not observe any

significant difference during development of _mettl4_ KO flies, although we observed altered proliferation of the Kc cell line lacking _mettl4_. The reason for the discrepancy between cell

line and whole fly studies is unclear at this time. Gene expression at the organismal level is regulated in a spatiotemporal manner and by both genetic and environmental factors11,12. This

complexity may explain why we did not observe overt developmental phenotypes of the _mettl4_ KO fly. At the same time, we also identified METTL4 as a novel methyltransferase for U2 snRNA in

human. Human METTL4 catalyzes Am to m6Am, whereas fly mettl4 catalyzes A to m6A. Although the only difference between m6Am and m6A is the 2′-O-methyl group on the sugar, we demonstrated that

human METTL4 cannot convert A to m6A. Future structural studies will provide insight into how these two highly related enzymes come to possess different substrate requirements for m6A

methylation of U2 snRNA. Furthermore, since the U2 RNA undergoes different modifications (m6A vs m6Am), it is possible that they could have distinct biological functions and significances.

They may affect the structure and function of the U2 RNA or even the spliceosome differently, and require different readers and erasers, as well as a set of Am writers/readers/erasers.

Indeed, while fly cells lacking _mettl4_ show an enhanced proliferation rate, human 293T cells do not. Consistently, pathway analysis shows that cell proliferation genes are affected in

response to _mettl4_ loss only in fly, but not in human cells (293T). Together, these findings raise many intriguing questions, including the origin of the substrate preference, the

structural mechanism that contributes to the recognition of the 2′-O-methyl group on Am, and the biological implications of the mechanistic evolution of METTL4. In summary, we demonstrated

that mettl4 catalyzes U2 m6A in fly both in vitro and in vivo. Furthermore, whole transcriptome profiling revealed that loss of _mettl4_ broadly impacts various biological pathways. Lastly,

we were able to observe a significant difference in cell proliferation between _mettl4_ normal and deficient fly cells. Our work answered a long-standing question regarding the enzymatic

activity of mettl4, and thus paved the way for further investigation of mettl4 functions in different biological settings. REFERENCES * Iyer, L. M., Zhang, D. & Aravind, L. Adenine

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569–576 (2015). Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to all members of the Shi lab for the general support and to _Drosophila_ RNAi Screening

Center (DRSC) for excellent technical support. This work was supported by BCH funds and an epigenetic seed grant from Harvard medical school (601139_2018_Shi_Epigenetics). L.G. is supported

by NIH T32 award (4T32AG000222-25 and 2T32AG000222-26). Y.S. is an American Cancer Society Research Professor. D.R. is a New York Stem Cell Foundation-Robertson Investigator. This work was

also supported by The New York Stem Cell Foundation. AUTHOR INFORMATION Author notes * These authors contributed equally: Lei Gu, Longfei Wang, Hao Chen, Jiaxu Hong AUTHORS AND AFFILIATIONS

* Department of Medicine, Division of Newborn Medicine and Epigenetics Programe, Boston Children’s Hospital, Boston, MA, 02115, USA Lei Gu, Hao Chen, Zhangfei Shen, Abhinav Dhall, Chaozhong

Liu, Zheng Wang, Yifan Xu, Damayanti Chakraborty & Yang Shi * Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA Lei Gu, Hao Chen, Zhangfei Shen, Abhinav Dhall, 

Chaozhong Liu, Zheng Wang, Yifan Xu, Damayanti Chakraborty & Yang Shi * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA

Longfei Wang & Hao Wu * Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, 02115, USA Longfei Wang & Hao Wu * Department of Ophthalmology and Vision

Science, Shanghai Eye, Ear, Nose and Throat Hospital, Fudan University, 200031, Shanghai, China Jiaxu Hong * Department of Ophthalmology, The Affiliated Hospital of Guizhou Medical

University, Guiyang, Guizhou, 550004, China Jiaxu Hong * Division of Rheumatology, Allergy and Immunology, Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital,

Charlestown, MA, 02129, USA Taotao Lao * Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA, 02115, USA Hong-Wen Tang & Norbert Perrimon * CAS Key

Laboratory of Regenerative Biology, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Medical University, Guangzhou,

Guangdong, 510530, China Jiekai Chen * Department of Neurobiology, Harvard Medical School, Boston, MA, 02115, USA Zhihua Liu & Dragana Rogulja * Howard Hughes Medical Institute, 77

Avenue Louis Pasteur, Boston, MA, 02115, USA Norbert Perrimon Authors * Lei Gu View author publications You can also search for this author inPubMed Google Scholar * Longfei Wang View author

publications You can also search for this author inPubMed Google Scholar * Hao Chen View author publications You can also search for this author inPubMed Google Scholar * Jiaxu Hong View

author publications You can also search for this author inPubMed Google Scholar * Zhangfei Shen View author publications You can also search for this author inPubMed Google Scholar * Abhinav

Dhall View author publications You can also search for this author inPubMed Google Scholar * Taotao Lao View author publications You can also search for this author inPubMed Google Scholar

* Chaozhong Liu View author publications You can also search for this author inPubMed Google Scholar * Zheng Wang View author publications You can also search for this author inPubMed Google

Scholar * Yifan Xu View author publications You can also search for this author inPubMed Google Scholar * Hong-Wen Tang View author publications You can also search for this author inPubMed

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can also search for this author inPubMed Google Scholar * Yang Shi View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS L.G. and Y.S. conceived

the project. L.G. designed and coordinated the project, and performed data analysis. L.G. and L.W. performed most of the in vitro experiments. J.H. generated _mettl4_ KO cell line and

performed in vivo analysis. A.D. performed kinetics analysis. T.L. and H.-W.T. performed cell proliferation assay. Z.S., Z.W., C.L., and Y.X. helped the in vitro and in vivo experiments

under the supervision of L.G., L.W., D.C., H.C., Z.L., and H.-W.T. J.C. generated _mettl4_ KO fly. N.P., D.R., H.W., and Y.S. supervised the project in general. L.G. wrote the manuscript

with support from Y.S. and other authors. CORRESPONDING AUTHOR Correspondence to Yang Shi. ETHICS DECLARATIONS CONFLICT OF INTEREST Y.S. is a co-founder and equity holder of Constellation

Pharmaceuticals, Inc., and Athelas Therapeutics, Inc., an equity holder of Imago Biosciences and a consultant for Active Motif, Inc. All other authors declare that they have no conflict of

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THIS ARTICLE CITE THIS ARTICLE Gu, L., Wang, L., Chen, H. _et al._ CG14906 (mettl4) mediates m6A methylation of U2 snRNA in _Drosophila_. _Cell Discov_ 6, 44 (2020).

https://doi.org/10.1038/s41421-020-0178-7 Download citation * Received: 16 March 2020 * Accepted: 09 May 2020 * Published: 30 June 2020 * DOI: https://doi.org/10.1038/s41421-020-0178-7 SHARE

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