Skip to main content
SpringerLink
Log in

Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing

  • Protocol
  • First Online:
HIV Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2807))

Abstract

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Shukla M, Kizito F, Mbonye U, Nguyen K, Dobrowolski C, Karn J (2022) HIV reservoirs methods and protocols a reliable primary cell model for HIV latency: The QUECEL (Quiescent Effector Cell Latency) method. Springer US New York NY, pp 57–68

    Google Scholar 

  2. Nguyen Quang N, Goudey S, Ségéral E et al (2020) Dynamic nanopore long-read sequencing analysis of HIV-1 splicing events during the early steps of infection. Retrovirology 17:1–24. https://doi.org/10.1186/S12977-020-00533-1/FIGURES/8

    Article  Google Scholar 

  3. Emery A, Zhou S, Pollom E, Swanstrom R (2017) Characterizing HIV-1 splicing by using next-generation sequencing. J Virol 91:2515–2531. https://doi.org/10.1128/JVI.02515-16/ASSET/3185AA31-EF8E-4E28-B852-4ED36B5C54FA/ASSETS/GRAPHIC/ZJV9991824380010.JPEG

    Article  Google Scholar 

  4. Pollard VW, Malim MH (2003) The HIV-1 REV protein. Ann Rev Microbiol 52:491–532. https://doi.org/10.1146/ANNUREV.MICRO.52.1.491

    Article  Google Scholar 

  5. Truman CTS, Järvelin A, Davis I, Castello A (2020) HIV Rev-isited. Open Biol 10:200320. https://doi.org/10.1098/RSOB.200320

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Karn J, Stoltzfus CM (2012) Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harb Perspect Med 2:a006916. https://doi.org/10.1101/CSHPERSPECT.A006916

    Article  PubMed  PubMed Central  Google Scholar 

  7. Crespo R, Rao S, Mahmoudi T (2022) HibeRNAtion: HIV-1 RNA metabolism and viral latency. Front Cell Infect Microbiol 12:855092. https://doi.org/10.3389/FCIMB.2022.855092/BIBTEX

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Riquelme-Barrios S, Pereira-Montecinos C, Valiente-Echeverría F, Soto-Rifo R (2018) Emerging roles of N6-methyladenosine on HIV-1 RNA metabolism and viral replication. Front Microbiol 9:576. https://doi.org/10.3389/FMICB.2018.00576

    Article  PubMed  PubMed Central  Google Scholar 

  9. N’Da Konan S, Ségéral E, Bejjani F et al (2022) YTHDC1 regulates distinct post-integration steps of HIV-1 replication and is important for viral infectivity. Retrovirology 19:4. https://doi.org/10.1186/S12977-022-00589-1

    Article  PubMed  PubMed Central  Google Scholar 

  10. Lu W, Tirumuru N, Gelais CS et al (2018) N6-Methyladenosine-binding proteins suppress HIV-1 infectivity and viral production. J Biol Chem 293:12992–13005. https://doi.org/10.1074/JBC.RA118.004215

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kennedy EM, Bogerd HP, Kornepati AVR et al (2016) Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression. Cell Host Microbe 19:675–685. https://doi.org/10.1016/J.CHOM.2016.04.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lichinchi G, Gao S, Saletore Y et al (2016) Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells. Nat Microbiol 1:16011. https://doi.org/10.1038/NMICROBIOL.2016.11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Tirumuru N, Zhao BS, Lu W et al (2016) N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. elife 5:e15528. https://doi.org/10.7554/ELIFE.15528

    Article  PubMed  PubMed Central  Google Scholar 

  14. Tsai K, Bogerd HP, Kennedy EM et al (2021) Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing. Genes Dev 35:992–1004. https://doi.org/10.1101/GAD.348508.121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang Y, Geng X, Li Q et al (2020) m6A modification in RNA: biogenesis, functions and roles in gliomas. J Exp Clin Cancer Res 39(1):1–16. https://doi.org/10.1186/S13046-020-01706-8

    Article  Google Scholar 

  16. Jiang X, Liu B, Nie Z et al (2021) The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther 6(1):74. https://doi.org/10.1038/s41392-020-00450-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zaccara S, Ries RJ, Jaffrey SR (2019) Reading, writing and erasing mRNA methylation. Nat Rev Mol Cell Biol 20(10):608–624. https://doi.org/10.1038/s41580-019-0168-5

    Article  CAS  PubMed  Google Scholar 

  18. Telwatte S, Morón-López S, Aran D et al (2019) Heterogeneity in HIV and cellular transcription profiles in cell line models of latent and productive infection: implications for HIV latency. Retrovirology 16:32. https://doi.org/10.1186/s12977-019-0494-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bartelt RR, Cruz-Orcutt N, Collins M, Houtman JCD (2009) Comparison of T cell receptor-induced proximal signaling and downstream functions in immortalized and primary T cells. PLoS One 4:e5430. https://doi.org/10.1371/journal.pone.0005430

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lin Z, Fillmore GC, Um TH et al (2003) Comparative microarray analysis of gene expression during activation of human peripheral blood T cells and leukemic Jurkat T cells. Lab Investig 83:765–776. https://doi.org/10.1097/01.LAB.0000073130.58435.E5

    Article  CAS  PubMed  Google Scholar 

  21. Chu CC, Liu B, Plangger R et al (2019) m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB. PLoS One 14:e0224850. https://doi.org/10.1371/JOURNAL.PONE.0224850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dominissini D, Moshitch-Moshkovitz S, Schwartz S et al (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature 485:201–206. https://doi.org/10.1038/NATURE11112

    Article  CAS  PubMed  Google Scholar 

  23. Meyer KD, Saletore Y, Zumbo P et al (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell 149:1635–1646. https://doi.org/10.1016/J.CELL.2012.05.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ke S, Pandya-Jones A, Saito Y et al (2017) m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev 31(10):990–1006. https://doi.org/10.1101/gad.301036

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xiang ZH, Sheng ZX, Sui N (2020) Advances in the profiling of N6-methyladenosine (m6A) modifications. Biotechnol Adv 45:107656. https://doi.org/10.1016/J.BIOTECHADV.2020.107656

    Article  Google Scholar 

  26. Linder B, Grozhik AV, Olarerin-George AO et al (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767–772. https://doi.org/10.1038/NMETH.3453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Liu N, Pan T (2015) Probing RNA modification status at single-nucleotide resolution in total RNA. Methods Enzymol 560:149–159. https://doi.org/10.1016/BS.MIE.2015.03.005

    Article  CAS  PubMed  Google Scholar 

  28. Garcia-Campos MA, Edelheit S, Toth U et al (2019) Deciphering the “m6A code” via antibody-independent quantitative profiling. Cell 178:731–747.e16. https://doi.org/10.1016/J.CELL.2019.06.013

    Article  CAS  PubMed  Google Scholar 

  29. Zhang Z, Chen LQ, Zhao YL et al (2019) Single-base mapping of m6A by an antibody-independent method. Sci Adv 5:250–253. https://doi.org/10.1126/SCIADV.AAX0250/SUPPL_FILE/AAX0250_SM.PDF

    Article  Google Scholar 

  30. Pandey RR, Pillai RS (2019) Counting the cuts: MAZTER-Seq quantifies m6A levels using a methylation-sensitive ribonuclease. Cell 178:515–517. https://doi.org/10.1016/J.CELL.2019.07.006

    Article  CAS  PubMed  Google Scholar 

  31. Price AM, Hayer KE, McIntyre ABR et al (2020) Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing. Nat Commun 11(1):1–17. https://doi.org/10.1038/s41467-020-19787-6

    Article  CAS  Google Scholar 

  32. Parker MT, Knop K, Sherwood AV et al (2020) Nanopore direct RNA sequencing maps the complexity of arabidopsis mRNA processing and m6A modification. elife 9:e49658. https://doi.org/10.7554/ELIFE.49658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Campos JHC, Maricato JT, Braconi CT et al (2021) Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants. Viruses 13:2108. https://doi.org/10.3390/V13112108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Workman RE, Tang AD, Tang PS et al (2019) Nanopore native RNA sequencing of a human poly(A) transcriptome. Nat Methods 16(12):1297–1305. https://doi.org/10.1038/s41592-019-0617-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Leger A, Amaral PP, Pandolfini L et al (2021) RNA modifications detection by comparative nanopore direct RNA sequencing. Nat Commun 12(1):7198. https://doi.org/10.1038/s41467-021-27393-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. White LK, Hesselberth JR (2022) Modification mapping by nanopore sequencing. Front Genet 13:1037134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Depledge DP, Srinivas KP, Sadaoka T et al (2019) Direct RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen. Nat Commun 10:754. https://doi.org/10.1038/s41467-019-08734-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hendra C, Pratanwanich PN, Wan YK et al (2022) Detection of m6A from direct RNA sequencing using a multiple instance learning framework. Nat Methods 19(12):1590–1598. https://doi.org/10.1038/s41592-022-01666-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Liu H, Begik O, Lucas MC et al (2019) Accurate detection of m6A RNA modifications in native RNA sequences. Nat Commun 10(1):1–9. https://doi.org/10.1038/s41467-019-11713-9

    Article  CAS  Google Scholar 

  40. Pratanwanich PN, Yao F, Chen Y et al (2021) Identification of differential RNA modifications from nanopore direct RNA sequencing with xPore. Nat Biotechnol 39(11):1394–1402. https://doi.org/10.1038/s41587-021-00949-w

    Article  CAS  PubMed  Google Scholar 

  41. Huang S, Zhang W, Katanski CD et al (2021) Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling. Genome Biol 22:1–14. https://doi.org/10.1186/S13059-021-02557-Y/FIGURES/2

    Article  Google Scholar 

  42. Hassan D, Acevedo D, Daulatabad SV et al (2022) Penguin: a tool for predicting pseudouridine sites in direct RNA nanopore sequencing data. Methods 203:478–487. https://doi.org/10.1016/J.YMETH.2022.02.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Abebe JS, Price AM, Hayer KE et al (2022) DRUMMER—rapid detection of RNA modifications through comparative nanopore sequencing. Bioinformatics 38:3113–3115. https://doi.org/10.1093/BIOINFORMATICS/BTAC274

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Tan CCS, Maurer-Stroh S, Wan Y et al (2019) A novel method for the capture-based purification of whole viral native RNA genomes. AMB Express 9:45. https://doi.org/10.1186/s13568-019-0772-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jain M, Abu-Shumays R, Olsen HE, Akeson M (2022) Advances in nanopore direct RNA sequencing state-of-the-art nanopore DRS. Nat Methods 19:1160–1164. https://doi.org/10.1038/s41592-022-01633-w

    Article  CAS  PubMed  Google Scholar 

  46. Rousseau-Gueutin M, Belser C, Silva C Da et al (2020) Long-read assembly of the Brassica napus reference genome Darmor-bzh. Gigascience 9:1–16. https://doi.org/10.1093/GIGASCIENCE/GIAA137

    Article  CAS  Google Scholar 

  47. Grünberger F, Ferreira-Cerca S, Grohmann D (2022) Nanopore sequencing of RNA and cDNA molecules in Escherichia coli. RNA 28:400–417. https://doi.org/10.1261/RNA.078937.121

    Article  PubMed  PubMed Central  Google Scholar 

  48. Lucas MC, Pryszcz LP, Medina R et al (2023) Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing. Nat Biotechnol 42:72–86. https://doi.org/10.1038/s41587-023-01743-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Ethan Honeycutt, Fredrick Kizito & Jonathan Karn

  2. Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Thomas Sweet

Authors
  1. Ethan Honeycutt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fredrick Kizito
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jonathan Karn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Sweet
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jonathan Karn .

Editor information

Editors and Affiliations

  1. Albert Einstein College of Medicine, Bronx, NY, USA

    Vinayaka R. Prasad

  2. Albert Einstein College of Medicine, Bronx, NY, USA

    Ganjam V. Kalpana

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Honeycutt, E., Kizito, F., Karn, J., Sweet, T. (2024). Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing. In: Prasad, V.R., Kalpana, G.V. (eds) HIV Protocols . Methods in Molecular Biology, vol 2807. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3862-0_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3862-0_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3861-3

  • Online ISBN: 978-1-0716-3862-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation