m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB


Autoři: Chia-Chieh Chu aff001;  Bei Liu aff001;  Raphael Plangger aff002;  Christoph Kreutz aff002;  Hashim M. Al-Hashimi aff001
Působiště autorů: Department of Biochemistry, Duke University School of Medicine, Durham, NC, United States of America aff001;  Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Universität Innsbruck, Innsbruck, Austria aff002;  Department of Chemistry, Duke University, Durham, NC, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224850

Souhrn

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m6A sites have not been observed in other studies mapping m6A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m6A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro. Our results indicate that if present in stem IIB, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.

Klíčová slova:

HIV-1 – Melting – Methylation – NMR spectroscopy – RNA structure – Sodium phosphate – Viral replication – Fluorescence polarization


Zdroje

1. 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.

2. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, et al. Topology of the human and mouse m(6)A RNA methylomes revealed by m(6)A-seq. Nature. 2012;485(7397):201–U84. doi: 10.1038/nature11112 WOS:000303799800033. 22575960

3. Desrosiers R, Friderici K, Rottman F. Identification of Methylated Nucleosides in Messenger-Rna from Novikoff Hepatoma-Cells. Proceedings of the National Academy of Sciences of the United States of America. 1974;71(10):3971–5. doi: 10.1073/pnas.71.10.3971 WOS:A1974U614000040. 4372599

4. Li X, Xiong X, Yi C. Epitranscriptome sequencing technologies: decoding RNA modifications. Nat Methods. 2016;14(1):23–31. doi: 10.1038/nmeth.4110 28032622.

5. Wang X, Zhao BS, Roundtree IA, Lu ZK, Han DL, Ma HH, et al. N-6-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell. 2015;161(6):1388–99. doi: 10.1016/j.cell.2015.05.014 WOS:000355935000017. 26046440

6. Wang X, Lu ZK, Gomez A, Hon GC, Yue YN, Han DL, et al. N-6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117–+. doi: 10.1038/nature12730 WOS:000329163300035. 24284625

7. Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 2014;24(12):1403–19. doi: 10.1038/cr.2014.151 WOS:000345894800006. 25412662

8. Xiao W, Adhikari S, Dahal U, Chen YS, Hao YJ, Sun BF, et al. Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Molecular cell. 2016;61(4):507–19. doi: 10.1016/j.molcel.2016.01.012 WOS:000372326300005. 26876937

9. Zhao BXS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications. Nat Rev Mol Cell Bio. 2017;18(1):31–42. doi: 10.1038/nrm.2016.132 WOS:000393267500008. 27808276

10. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA Modifications in Gene Expression Regulation. Cell. 2017;169(7):1187–200. doi: 10.1016/j.cell.2017.05.045 WOS:000403332400009. 28622506

11. Meyer KD, Jaffrey SR. Rethinking m(6)A Readers, Writers, and Erasers. Annu Rev Cell Dev Bi. 2017;33:319–42. doi: 10.1146/annurev-cellbio-100616-060758 WOS:000413584700015. 28759256

12. Deng XL, Su R, Feng XS, Wei MJ, Chen JJ. Role of N-6-methyladenosine modification in cancer. Curr Opin Genet Dev. 2018;48:1–7. doi: 10.1016/j.gde.2017.10.005 WOS:000428232100002. 29040886

13. Chen T, Hao Y-J, Zhang Y, Li M-M, Wang M, Han W, et al. m6A RNA Methylation Is Regulated by MicroRNAs and Promotes Reprogramming to Pluripotency. Cell stem cell. 2015;16(3):289–301. doi: 10.1016/j.stem.2015.01.016 25683224

14. Weng Y-L, Wang X, An R, Cassin J, Vissers C, Liu Y, et al. Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System. Neuron. 2018;97(2):313–25.e6. doi: 10.1016/j.neuron.2017.12.036 29346752

15. Gokhale NS, McIntyre ABR, McFadden MJ, Roder AE, Kennedy EM, Gandara JA, et al. N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection. Cell Host Microbe. 2016;20(5):654–65. Epub 2016/10/25. doi: 10.1016/j.chom.2016.09.015 27773535; PubMed Central PMCID: PMC5123813.

16. Gokhale NS, McIntyre ABR, McFadden MJ, Roder AE, Kennedy EM, Gandara JA, et al. N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection. Cell Host Microbe. 2016;20(5):654–65. doi: 10.1016/j.chom.2016.09.015 WOS:000389472000014. 27773535

17. Lichinchi G, Zhao BS, Wu YA, Lu ZK, Qin Y, He C, et al. Dynamics of Human and Viral RNA Methylation during Zika Virus Infection. Cell Host Microbe. 2016;20(5):666–73. doi: 10.1016/j.chom.2016.10.002 WOS:000389472000015. 27773536

18. Tirumuru N, Zhao BS, Lu WX, Lu ZK, He C, Wu L. N-6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. eLife. 2016;5. ARTN e15528 10.7554/eLife.15528. WOS:000380858000001.

19. Hesser CR, Karijolich J, Dominissini D, He C, Glaunsinger BA. N-6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection. Plos Pathog. 2018;14(4). ARTN e1006995 10.1371/journal.ppat.1006995. WOS:000431135400036.

20. Lichinchi G, Gao S, Saletore Y, Gonzalez GM, Bansal V, Wang Y, et al. Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells. Nature Microbiology. 2016;1. doi: 10.1038/nmicrobiol.2016.11 27572442

21. Kennedy EM, Bogerd HP, Kornepati AVR, Kang D, Ghoshal D, Marshall JB, et al. Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression. Cell Host Microbe. 2016;19(5):675–85. doi: 10.1016/j.chom.2016.04.002 WOS:000375595500017. 27117054

22. Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989;338(6212):254–7. doi: 10.1038/338254a0 2784194.

23. Malim MH, Tiley LS, McCarn DF, Rusche JR, Hauber J, Cullen BR. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell. 1990;60(4):675–83. doi: 10.1016/0092-8674(90)90670-a 2406030.

24. Malim MH, Cullen BR. HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell. 1991;65(2):241–8. doi: 10.1016/0092-8674(91)90158-u 2015625.

25. Bartel DP, Zapp ML, Green MR, Szostak JW. HIV-1 Rev regulation involves recognition of non-Watson-Crick base pairs in viral RNA. Cell. 1991;67(3):529–36. doi: 10.1016/0092-8674(91)90527-6 1934059.

26. Heaphy S, Finch JT, Gait MJ, Karn J, Singh M. Human immunodeficiency virus type 1 regulator of virion expression, rev, forms nucleoprotein filaments after binding to a purine-rich "bubble" located within the rev-responsive region of viral mRNAs. Proceedings of the National Academy of Sciences of the United States of America. 1991;88(16):7366–70. doi: 10.1073/pnas.88.16.7366 1871141; PubMed Central PMCID: PMC52296.

27. Huang XJ, Hope TJ, Bond BL, McDonald D, Grahl K, Parslow TG. Minimal Rev-response element for type 1 human immunodeficiency virus. Journal of virology. 1991;65(4):2131–4. 2002556; PubMed Central PMCID: PMC240087.

28. Chu CC, Plangger R, Kreutz C, Al-Hashimi HM. Dynamic ensemble of HIV-1 RRE stem IIB reveals non-native conformations that disrupt the Rev-binding site. Nucleic acids research. 2019;47(13):7105–17. doi: 10.1093/nar/gkz498 31199872; PubMed Central PMCID: PMC6649712.

29. Ganser LR, Chu C-C, Bogerd HP, Kelly ML, Cullen BR, Al-Hashimi HM. Probing RNA conformational equilibria within the functional cellular context. bioRxiv. 2019:634576. doi: 10.1101/634576

30. Spitale RC, Flynn RA, Zhang QC, Crisalli P, Lee B, Jung JW, et al. Structural imprints in vivo decode RNA regulatory mechanisms. Nature. 2015;519(7544):486–90. doi: 10.1038/nature14263 25799993; PubMed Central PMCID: PMC4376618.

31. Kierzek E, Kierzek R. The thermodynamic stability of RNA duplexes and hairpins containing N6-alkyladenosines and 2-methylthio-N6-alkyladenosines. Nucleic acids research. 2003;31(15):4472–80. doi: 10.1093/nar/gkg633 12888507; PubMed Central PMCID: PMC169893.

32. Roost C, Lynch SR, Batista PJ, Qu K, Chang HY, Kool ET. Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification. Journal of the American Chemical Society. 2015;137(5):2107–15. doi: 10.1021/ja513080v 25611135; PubMed Central PMCID: PMC4405242.

33. Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T. N6-methyladenosine-dependent RNA structural switches regulate RNA–protein interactions. Nature. 2015;518:560. doi: 10.1038/nature14234 25719671

34. Roost C, Lynch SR, Batista PJ, Qu K, Chang HY, Kool ET. Structure and Thermodynamics of N6-Methyladenosine in RNA: A Spring-Loaded Base Modification. Journal of the American Chemical Society. 2015;137(5):2107–15. doi: 10.1021/ja513080v 25611135

35. Kierzek E, Kierzek R. The thermodynamic stability of RNA duplexes and hairpins containing N-6-alkyladenosines and 2-methylthio-N-6-alkyladenosines. Nucleic acids research. 2003;31(15):4472–80. doi: 10.1093/nar/gkg633 WOS:000184532900031. 12888507

36. Huang L, Ashraf S, Wang J, Lilley DM. Control of box C/D snoRNP assembly by N(6)-methylation of adenine. EMBO Rep. 2017;18(9):1631–45. doi: 10.15252/embr.201743967 28623187; PubMed Central PMCID: PMC5579392.

37. Liu B, Merriman DK, Choi SH, Schumacher MA, Plangger R, Kreutz C, et al. A potentially abundant junctional RNA motif stabilized by m(6)A and Mg(2). Nat Commun. 2018;9(1):2761. doi: 10.1038/s41467-018-05243-z 30018356; PubMed Central PMCID: PMC6050335.

38. Neuner S, Santner T, Kreutz C, Micura R. The "Speedy" Synthesis of Atom-Specific (15)N Imino/Amido-Labeled RNA. Chemistry. 2015;21(33):11634–43. doi: 10.1002/chem.201501275 26237536; PubMed Central PMCID: PMC4946632.

39. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR. 1995;6(3):277–93. doi: 10.1007/bf00197809 8520220.

40. Luedtke NW, Tor Y. Fluorescence-based methods for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors. Biopolymers. 2003;70(1):103–19. doi: 10.1002/bip.10428 12925996.

41. Draper DE, Grilley D, Soto AM. Ions and RNA folding. Annu Rev Bioph Biom. 2005;34:221–43. doi: 10.1146/annurev.biophys.34.040204.144511 WOS:000230099600010. 15869389

42. Ippolito JA, Steitz TA. The structure of the HIV-1 RRE high affinity rev binding site at 1.6 A resolution. Journal of molecular biology. 2000;295(4):711–7. doi: 10.1006/jmbi.1999.3405 10656783.

43. Hung LW, Holbrook EL, Holbrook SR. The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(10):5107–12. doi: 10.1073/pnas.090588197 10792052; PubMed Central PMCID: PMC25789.

44. Battiste JL, Tan R, Frankel AD, Williamson JR. Binding of an HIV Rev peptide to Rev responsive element RNA induces formation of purine-purine base pairs. Biochemistry. 1994;33(10):2741–7. doi: 10.1021/bi00176a001 8130185.

45. Peterson RD, Bartel DP, Szostak JW, Horvath SJ, Feigon J. 1H NMR studies of the high-affinity Rev binding site of the Rev responsive element of HIV-1 mRNA: base pairing in the core binding element. Biochemistry. 1994;33(18):5357–66. doi: 10.1021/bi00184a001 8180157.

46. Fang X, Wang J, O'Carroll IP, Mitchell M, Zuo X, Wang Y, et al. An unusual topological structure of the HIV-1 Rev response element. Cell. 2013;155(3):594–605. doi: 10.1016/j.cell.2013.10.008 24243017; PubMed Central PMCID: PMC3918456.

47. Bai Y, Tambe A, Zhou K, Doudna JA. RNA-guided assembly of Rev-RRE nuclear export complexes. eLife. 2014;3:e03656. doi: 10.7554/eLife.03656 25163983; PubMed Central PMCID: PMC4142337.

48. Siegfried NA, Metzger SL, Bevilacqua PC. Folding cooperativity in RNA and DNA is dependent on position in the helix. Biochemistry. 2007;46(1):172–81. doi: 10.1021/bi061375l WOS:000243157300018. 17198387

49. Getz MM, Andrews AJ, Fierke CA, Al-Hashimi HM. Structural plasticity and Mg2+ binding properties of RNase P P4 from combined analysis of NMR residual dipolar couplings and motionally decoupled spin relaxation. Rna. 2007;13(2):251–66. Epub 2006/12/30. doi: 10.1261/rna.264207 17194721; PubMed Central PMCID: PMC1781369.

50. Palmer AG 3rd, Massi F. Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem Rev. 2006;106(5):1700–19. doi: 10.1021/cr0404287 16683750.

51. Korzhnev DM, Orekhov VY, Kay LE. Off-resonance R(1rho) NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain. Journal of the American Chemical Society. 2005;127(2):713–21. doi: 10.1021/ja0446855 15643897.

52. Rangadurai A, Szymaski ES, Kimsey IJ, Shi H, Al-Hashimi HM. Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1rho relaxation dispersion. Prog Nucl Magn Reson Spectrosc. 2019;112–113:55–102. Epub 2019/09/05. doi: 10.1016/j.pnmrs.2019.05.002 31481159; PubMed Central PMCID: PMC6727989.

53. Daugherty MD, D'Orso I, Frankel AD. A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA. Molecular cell. 2008;31(6):824–34. doi: 10.1016/j.molcel.2008.07.016 18922466; PubMed Central PMCID: PMC2651398.

54. Battiste JL, Mao H, Rao NS, Tan R, Muhandiram DR, Kay LE, et al. Alpha helix-RNA major groove recognition in an HIV-1 rev peptide-RRE RNA complex. Science. 1996;273(5281):1547–51. doi: 10.1126/science.273.5281.1547 8703216.

55. Jayaraman B, Crosby DC, Homer C, Ribeiro I, Mavor D, Frankel AD. RNA-directed remodeling of the HIV-1 protein Rev orchestrates assembly of the Rev-Rev response element complex. eLife. 2014;3:e04120. doi: 10.7554/eLife.04120 25486594; PubMed Central PMCID: PMC4360532.

56. Kjems J, Calnan BJ, Frankel AD, Sharp PA. Specific binding of a basic peptide from HIV-1 Rev. The EMBO journal. 1992;11(3):1119–29. 1547776; PubMed Central PMCID: PMC556554.


Článek vyšel v časopise

PLOS One


2019 Číslo 12