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Characterization of METTL16 as a cytoplasmic RNA binding protein


Autoři: Daniel J. Nance aff001;  Emily R. Satterwhite aff001;  Brinda Bhaskar aff001;  Sway Misra aff001;  Kristen R. Carraway aff001;  Kyle D. Mansfield aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, United States of America aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0227647

Souhrn

mRNA modification by N6-methyladenosine (m6A) is involved in many post-transcriptional regulation processes including mRNA stability, splicing and promotion of translation. Accordingly, the recently identified mRNA methylation complex containing METTL3, METTL14, and WTAP has been the subject of intense study. However, METTL16 (METT10D) has also been identified as an RNA m6A methyltransferase that can methylate both coding and noncoding RNAs, but its biological role remains unclear. While global studies have identified many potential RNA targets of METTL16, only a handful, including the long noncoding RNA MALAT1, the snRNA U6, as well as the mRNA MAT2A have been verified and/or studied to any great extent. In this study we identified/verified METTL16 targets by immunoprecipitation of both endogenous as well as exogenous FLAG-tagged protein. Interestingly, exogenously overexpressed METTL16 differed from the endogenous protein in its relative affinity for RNA targets which prompted us to investigate METTL16's localization within the cell. Surprisingly, biochemical fractionation revealed that a majority of METTL16 protein resides in the cytoplasm of a number of cells. Furthermore, siRNA knockdown of METTL16 resulted in expression changes of a few mRNA targets suggesting that METTL16 may play a role in regulating gene expression. Thus, while METTL16 has been reported to be a nuclear protein, our findings suggest that METTL16 is also a cytoplasmic methyltransferase that may alter its RNA binding preferences depending on its cellular localization. Future studies will seek to confirm differences between cytoplasmic and nuclear RNA targets in addition to exploring the physiological role of METTL16 through long-term knockdown.

Klíčová slova:

Cytoplasm – Immunohistochemistry techniques – Immunoprecipitation – Messenger RNA – Methylation – Non-coding RNA sequences – Small interfering RNAs – Small nuclear RNA


Zdroje

1. Zhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Biol. 2017;18(1):31–42. doi: 10.1038/nrm.2016.132 27808276; PubMed Central PMCID: PMC5167638.

2. Roundtree IA, He C. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Trends Genet. 2016;32(6):320–1. doi: 10.1016/j.tig.2016.03.006 27050931.

3. Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015;518(7540):560–4. doi: 10.1038/nature14234 25719671; PubMed Central PMCID: PMC4355918.

4. Alarcon CR, Goodarzi H, Lee H, Liu X, Tavazoie S, Tavazoie SF. HNRNPA2B1 Is a Mediator of m(6)A-Dependent Nuclear RNA Processing Events. Cell. 2015;162(6):1299–308. doi: 10.1016/j.cell.2015.08.011 26321680; PubMed Central PMCID: PMC4673968.

5. Du H, Zhao Y, He J, Zhang Y, Xi H, Liu M, et al. YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex. Nature communications. 2016;7:12626. doi: 10.1038/ncomms12626 27558897; PubMed Central PMCID: PMC5007331.

6. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117–20. doi: 10.1038/nature12730 24284625; PubMed Central PMCID: PMC3877715.

7. Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 2017. doi: 10.1038/cr.2017.15 28106072.

8. Li A, Chen YS, Ping XL, Yang X, Xiao W, Yang Y, et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 2017. doi: 10.1038/cr.2017.10 28106076.

9. Fry NJ, Law BA, Ilkayeva OR, Holley CL, Mansfield KD. N6-methyladenosine is required for the hypoxic stabilization of specific mRNAs. RNA. 2017;23(9):1444–55. Epub June 13, 2017. doi: 10.1261/rna.061044.117 28611253.

10. Liu K, Ding Y, Ye W, Liu Y, Yang J, Liu J, et al. Structural and Functional Characterization of the Proteins Responsible for N(6)-Methyladenosine Modification and Recognition. Curr Protein Pept Sci. 2016;17(4):306–18. 26323656.

11. Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24(2):177–89. doi: 10.1038/cr.2014.3 24407421; PubMed Central PMCID: PMC3915904.

12. Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. Cell reports. 2014;8(1):284–96. doi: 10.1016/j.celrep.2014.05.048 24981863; PubMed Central PMCID: PMC4142486.

13. Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012;149(7):1635–46. doi: 10.1016/j.cell.2012.05.003 22608085; PubMed Central PMCID: PMC3383396.

14. Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature. 2016;537. doi: 10.1038/nature19342 27602518

15. Patil DP, Chen CK, Pickering BF, Chow A, Jackson C, Guttman M, et al. m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature. 2016;537(7620):369–73. doi: 10.1038/nature19342 27602518.

16. Yue Y, Liu J, He C. RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation. Genes Dev. 2015;29(13):1343–55. doi: 10.1101/gad.262766.115 26159994; PubMed Central PMCID: PMC4511210.

17. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, et al. N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell. 2015;161(6):1388–99. doi: 10.1016/j.cell.2015.05.014 26046440; PubMed Central PMCID: PMC4825696.

18. Jaffrey SR, Kharas MG. Emerging links between m6A and misregulated mRNA methylation in cancer. Genome Medicine. 2017;9(1):2. doi: 10.1186/s13073-016-0395-8 28081722

19. Lin S, Choe J, Du P, Triboulet R, Gregory RI. The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell. 2016;62(3):335–45. doi: 10.1016/j.molcel.2016.03.021 27117702; PubMed Central PMCID: PMC4860043.

20. Zhang C, Samanta D, Lu H, Bullen JW, Zhang H, Chen I. Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m(6)A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A. 2016;113. doi: 10.1073/pnas.1618558114

21. Zhang C, Zhi WI, Lu H, Samanta D, Chen I, Gabrielson E, et al. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells. Oncotarget. 2016;7(40):64527–42. doi: 10.18632/oncotarget.11743 27590511.

22. Aguilo F, Zhang F, Sancho A, Fidalgo M, Di Cecilia S, Vashisht A, et al. Coordination of m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Pluripotency and Reprogramming. Cell Stem Cell. 2015;17(6):689–704. doi: 10.1016/j.stem.2015.09.005 26526723; PubMed Central PMCID: PMC4671830.

23. Wu Y, Zhang S, Yuan Q. N6-Methyladenosine Methyltransferases and Demethylases: New Regulators of Stem Cell Pluripotency and Differentiation. Stem Cells Dev. 2016. doi: 10.1089/scd.2016.0062 27216987.

24. Xiao Z, Guifang J. RNA epigenetic modification: N6-methyladenosine. Yi Chuan. 2016;38(4):275–88. doi: 10.16288/j.yczz.16-049 27103452.

25. Zhao BS, He C. Fate by RNA methylation: m6A steers stem cell pluripotency. Genome Biol. 2015;16:43. doi: 10.1186/s13059-015-0609-1 25723450; PubMed Central PMCID: PMC4336730.

26. Cui Q, Shi H, Ye P, Li L, Qu Q, Sun G, et al. m6A RNA Methylation Regulates the Self-Renewal and Tumorigenesis of Glioblastoma Stem Cells. Cell reports. 2017;18(11):2622–34. doi: 10.1016/j.celrep.2017.02.059 28297667.

27. Fry NJ, Law BA, Ilkayeva OR, Carraway KR, Holley CL, Mansfield KD. N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression. Oncotarget. 2018;9:31231–43. doi: 10.18632/oncotarget.25782 30131850

28. Warda AS, Kretschmer J, Hackert P, Lenz C, Urlaub H, Hobartner C, et al. Human METTL16 is a N(6)-methyladenosine (m(6)A) methyltransferase that targets pre-mRNAs and various non-coding RNAs. EMBO Rep. 2017;18(11):2004–14. doi: 10.15252/embr.201744940 29051200; PubMed Central PMCID: PMC5666602.

29. Pendleton KE, Chen B, Liu K, Hunter OV, Xie Y, Tu BP, et al. The U6 snRNA m(6)A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention. Cell. 2017;169(5):824–35 e14. doi: 10.1016/j.cell.2017.05.003 28525753; PubMed Central PMCID: PMC5502809.

30. Brown JA, Kinzig CG, DeGregorio SJ, Steitz JA. Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA. Proc Natl Acad Sci U S A. 2016;113(49):14013–8. doi: 10.1073/pnas.1614759113 27872311; PubMed Central PMCID: PMC5150381.

31. Mendel M, Chen KM, Homolka D, Gos P, Pandey RR, McCarthy AA, et al. Methylation of Structured RNA by the m(6)A Writer METTL16 Is Essential for Mouse Embryonic Development. Mol Cell. 2018;71(6):986–1000 e11. doi: 10.1016/j.molcel.2018.08.004 30197299; PubMed Central PMCID: PMC6162343.

32. Shima H, Matsumoto M, Ishigami Y, Ebina M, Muto A, Sato Y, et al. S-Adenosylmethionine Synthesis Is Regulated by Selective N(6)-Adenosine Methylation and mRNA Degradation Involving METTL16 and YTHDC1. Cell reports. 2017;21(12):3354–63. doi: 10.1016/j.celrep.2017.11.092 29262316.

33. Doxtader KA, Wang P, Scarborough AM, Seo D, Conrad NK, Nam Y. Structural Basis for Regulation of METTL16, an S-Adenosylmethionine Homeostasis Factor. Mol Cell. 2018;71(6):1001–11 e4. doi: 10.1016/j.molcel.2018.07.025 30197297; PubMed Central PMCID: PMC6367934.

34. Ruszkowska A, Ruszkowski M, Dauter Z, Brown JA. Structural insights into the RNA methyltransferase domain of METTL16. Sci Rep. 2018;8(1):5311. doi: 10.1038/s41598-018-23608-8 29593291; PubMed Central PMCID: PMC5871880.

35. Barbieri I, Tzelepis K, Pandolfini L, Shi J, Millan-Zambrano G, Robson SC, et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature. 2017;552(7683):126–31. doi: 10.1038/nature24678 29186125; PubMed Central PMCID: PMC6217924.

36. Wang T, Birsoy K, Hughes NW, Krupczak KM, Post Y, Wei JJ, et al. Identification and characterization of essential genes in the human genome. Science. 2015;350(6264):1096–101. doi: 10.1126/science.aac7041 26472758; PubMed Central PMCID: PMC4662922.

37. Bertomeu T, Coulombe-Huntington J, Chatr-Aryamontri A, Bourdages KG, Coyaud E, Raught B, et al. A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome. Mol Cell Biol. 2018;38(1). doi: 10.1128/MCB.00302-17 29038160; PubMed Central PMCID: PMC5730719.

38. Blomen VA, Majek P, Jae LT, Bigenzahn JW, Nieuwenhuis J, Staring J, et al. Gene essentiality and synthetic lethality in haploid human cells. Science. 2015;350(6264):1092–6. doi: 10.1126/science.aac7557 26472760.

39. Hart T, Chandrashekhar M, Aregger M, Steinhart Z, Brown KR, MacLeod G, et al. High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities. Cell. 2015;163(6):1515–26. doi: 10.1016/j.cell.2015.11.015 26627737.

40. Silva JM, Marran K, Parker JS, Silva J, Golding M, Schlabach MR, et al. Profiling essential genes in human mammary cells by multiplex RNAi screening. Science. 2008;319(5863):617–20. doi: 10.1126/science.1149185 18239125; PubMed Central PMCID: PMC2981861.

41. Dorsett M, Schedl T. A role for dynein in the inhibition of germ cell proliferative fate. Mol Cell Biol. 2009;29(22):6128–39. doi: 10.1128/MCB.00815-09 19752194; PubMed Central PMCID: PMC2772574.

42. Pauley RJ, Soule HD, Tait L, Miller FR, Wolman SR, Dawson PJ, et al. The MCF10 family of spontaneously immortalized human breast epithelial cell lines: models of neoplastic progression. Eur J Cancer Prev. 1993;2 Suppl 3:67–76. 7507749.

43. Dawson PJ, Wolman SR, Tait L, Heppner GH, Miller FR. MCF10AT: a model for the evolution of cancer from proliferative breast disease. The American journal of pathology. 1996;148(1):313–9. 8546221; PubMed Central PMCID: PMC1861604.

44. Ma H, Wang X, Cai J, Dai Q, Natchiar SK, Lv R, et al. N(6-)Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation. Nat Chem Biol. 2019;15(1):88–94. doi: 10.1038/s41589-018-0184-3 30531910; PubMed Central PMCID: PMC6463480.

45. van Tran N, Ernst FGM, Hawley BR, Zorbas C, Ulryck N, Hackert P, et al. The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112. Nucleic Acids Res. 2019. doi: 10.1093/nar/gkz619 31328227.

46. Fury MG, Zieve GW. U6 snRNA maturation and stability. Exp Cell Res. 1996;228(1):160–3. doi: 10.1006/excr.1996.0311 8892983.

47. Zieve GW, Sauterer RA, Feeney RJ. Newly synthesized small nuclear RNAs appear transiently in the cytoplasm. J Mol Biol. 1988;199(2):259–67. doi: 10.1016/0022-2836(88)90312-9 3351925.

48. Madhani HD, Bordonne R, Guthrie C. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 1990;4(12B):2264–77. doi: 10.1101/gad.4.12b.2264 2149118.


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