Skip to main content
SpringerLink
Log in

Integrated Genome-Scale Analysis and Northern Blot Detection of Retrotransposon siRNAs Across Plant Species

  • Protocol
  • First Online:
RNA Tagging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2166))

Abstract

Cells have sophisticated RNA-directed mechanisms to regulate genes, destroy viruses, or silence transposable elements (TEs). In terrestrial plants, a specialized non-coding RNA machinery involving RNA polymerase IV (Pol IV) and small interfering RNAs (siRNAs) targets DNA methylation and silencing to TEs. Here, we present a bioinformatics protocol for annotating and quantifying siRNAs that derive from long terminal repeat (LTR) retrotransposons. The approach was validated using small RNA northern blot analyses, comparing the species Arabidopsis thaliana and Brachypodium distachyon. To assist hybridization probe design, we configured a genome browser to show small RNA-seq mappings in distinct colors and shades according to their nucleotide lengths and abundances, respectively. Samples from wild-type and pol IV mutant plants, cross-species negative controls, and a conserved microRNA control validated the detected siRNA signals, confirming their origin from specific TEs and their Pol IV-dependent biogenesis. Moreover, an optimized labeling method yielded probes that could detect low-abundance siRNAs from B. distachyon TEs. The integration of de novo TE annotation, small RNA-seq profiling, and northern blotting, as outlined here, will facilitate the comparative genomic analysis of RNA silencing in crop plants and non-model species.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. https://doi.org/10.1038/35888

    Article  CAS  PubMed  Google Scholar 

  2. Hamilton AJ, Baulcombe DC (1999) A species of small antisense RNA in posttranscriptional gene silencing in plants. Science (80-) 286:950–952. https://doi.org/10.1126/science.286.5441.950

    Article  CAS  Google Scholar 

  3. Mette MF, Aufsatz W, van der Winden J et al (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201. https://doi.org/10.1093/emboj/19.19.5194

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J 21:4671–4679. https://doi.org/10.1093/emboj/cdf464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Xie Z, Johansen LK, Gustafson AM et al (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104. https://doi.org/10.1371/journal.pbio.0020104

    Article  PubMed  PubMed Central  Google Scholar 

  6. Onodera Y, Haag JR, Ream T et al (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120:613–622. https://doi.org/10.1016/j.cell.2005.02.007

    Article  CAS  PubMed  Google Scholar 

  7. Vazquez F, Vaucheret H, Rajagopalan R et al (2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol Cell 16:69–79. https://doi.org/10.1016/j.molcel.2004.09.028

    Article  CAS  PubMed  Google Scholar 

  8. Zilberman D, Cao X, Johansen LK et al (2004) Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr Biol 14:1214–1220. https://doi.org/10.1016/j.cub.2004.06.055

    Article  CAS  PubMed  Google Scholar 

  9. Reinhart BJ, Weinstein EG, Rhoades MW et al (2002) MicroRNAs in plants. Genes Dev 16:1616–1626. https://doi.org/10.1101/gad.1004402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056. https://doi.org/10.1126/science.1076311

    Article  CAS  PubMed  Google Scholar 

  11. Fagard M, Boutet S, Morel JB et al (2000) AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. Proc Natl Acad Sci U S A 97:11650–11654. https://doi.org/10.1073/pnas.200217597

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Park W, Li J, Song R et al (2002) CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12:1484–1495. https://doi.org/10.1016/s0960-9822(02)01017-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Palatnik JF, Allen E, Wu X et al (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263. https://doi.org/10.1038/nature01958

    Article  CAS  PubMed  Google Scholar 

  14. Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741. https://doi.org/10.1105/tpc.016238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Peragine A, Yoshikawa M, Wu G et al (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev 18:2368–2379. https://doi.org/10.1101/gad.1231804

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vaucheret H, Vazquez F, Crete P, Bartel DP (2004) The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes Dev 18:1187–1197. https://doi.org/10.1101/gad.1201404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102:11928–11933. https://doi.org/10.1073/pnas.0505461102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303:2022–2025. https://doi.org/10.1126/science.1088060

    Article  CAS  PubMed  Google Scholar 

  19. Allen E, Xie Z, Gustafson AM, Carrington JC (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121:207–221. https://doi.org/10.1016/j.cell.2005.04.004

    Article  CAS  PubMed  Google Scholar 

  20. Fahlgren N, Montgomery TA, Howell MD et al (2006) Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr Biol 16:939–944. https://doi.org/10.1016/j.cub.2006.03.065

    Article  CAS  PubMed  Google Scholar 

  21. Bennetzen JL, Park M (2018) Distinguishing friends, foes, and freeloaders in giant genomes. Curr Opin Genet Dev 49:49–55. https://doi.org/10.1016/j.gde.2018.02.013

    Article  CAS  PubMed  Google Scholar 

  22. Matzke MA, Mosher RA (2014) RNA-directed DNA methylation: an epigenetic pathway of increasing complexity. Nat Rev Genet 15:394–408. https://doi.org/10.1038/nrg3683

    Article  CAS  PubMed  Google Scholar 

  23. Ferrafiat L, Pflieger D, Singh J et al (2019) The NRPD1 N-terminus contains a Pol IV-specific motif that is critical for genome surveillance in Arabidopsis. Nucleic Acids Res. https://doi.org/10.1093/nar/gkz618

  24. Law JA, Du J, Hale CJ et al (2013) Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1. Nature 498:385–389. https://doi.org/10.1038/nature12178

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wendte JM, Haag JR, Pontes OM et al (2019) The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation. Nucleic Acids Res. https://doi.org/10.1093/nar/gkz615

  26. Ream TS, Haag JR, Wierzbicki AT et al (2009) Subunit compositions of the RNA-silencing enzymes Pol IV and Pol V reveal their origins as specialized forms of RNA polymerase II. Mol Cell 33:192–203. https://doi.org/10.1016/j.molcel.2008.12.015

    Article  CAS  PubMed  Google Scholar 

  27. Haag JR, Ream TS, Marasco M et al (2012) In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol Cell 48:811–818. https://doi.org/10.1016/J.MOLCEL.2012.09.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Singh J, Mishra V, Wang F et al (2019) Reaction mechanisms of Pol IV, RDR2, and DCL3 drive RNA channeling in the siRNA-directed DNA methylation pathway. Mol Cell 75:576–589.e5. https://doi.org/10.1016/j.molcel.2019.07.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhai J, Bischof S, Wang H et al (2015) A one precursor one siRNA model for Pol IV-dependent siRNA biogenesis. Cell 163:445–455. https://doi.org/10.1016/j.cell.2015.09.032

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Blevins T, Podicheti R, Mishra V et al (2015) Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. elife 4. https://doi.org/10.7554/eLife.09591

  31. Wierzbicki AT, Ream TS, Haag JR, Pikaard CS (2009) RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat Genet 41:630–634. https://doi.org/10.1038/ng.365

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Blevins T, Pontvianne F, Cocklin R et al (2014) A two-step process for epigenetic inheritance in Arabidopsis. Mol Cell 54:30–42. https://doi.org/10.1016/j.molcel.2014.02.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ito H, Gaubert H, Bucher E et al (2011) An siRNA pathway prevents transgenerational retrotransposition in plants subjected to stress. Nature 472:115–119. https://doi.org/10.1038/nature09861

    Article  CAS  PubMed  Google Scholar 

  34. Mirouze M, Reinders J, Bucher E et al (2009) Selective epigenetic control of retrotransposition in Arabidopsis. Nature 461:427–430. https://doi.org/10.1038/nature08328

    Article  CAS  PubMed  Google Scholar 

  35. Lanciano S, Mirouze M (2018) Transposable elements: all mobile, all different, some stress responsive, some adaptive? Curr Opin Genet Dev 49:106–114. https://doi.org/10.1016/j.gde.2018.04.002

    Article  CAS  PubMed  Google Scholar 

  36. Grandbastien M-A (2015) LTR retrotransposons, handy hitchhikers of plant regulation and stress response. Biochim Biophys Acta 1849:403–416. https://doi.org/10.1016/j.bbagrm.2014.07.017

    Article  CAS  PubMed  Google Scholar 

  37. Cavrak VV, Lettner N, Jamge S et al (2014) How a retrotransposon exploits the plant’s heat stress response for its activation. PLoS Genet 10:e1004115. https://doi.org/10.1371/journal.pgen.1004115

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Pietzenuk B, Markus C, Gaubert H et al (2016) Recurrent evolution of heat-responsiveness in Brassicaceae COPIA elements. Genome Biol 17:209. https://doi.org/10.1186/s13059-016-1072-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Platt RN 2nd, Blanco-Berdugo L, Ray DA (2016) Accurate transposable element annotation is vital when analyzing new genome assemblies. Genome Biol Evol 8:403–410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Underwood CJ, Henderson IR, Martienssen RA (2017) Genetic and epigenetic variation of transposable elements in Arabidopsis. Curr Opin Plant Biol 36:135–141. https://doi.org/10.1016/J.PBI.2017.03.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. El Baidouri M, Do KK, Abernathy B et al (2015) A new approach for annotation of transposable elements using small RNA mapping. Nucleic Acids Res e84:43. https://doi.org/10.1093/nar/gkv257

    Article  CAS  Google Scholar 

  42. Ahmed I, Sarazin A, Bowler C et al (2011) Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis. Nucleic Acids Res 39:6919–6931. https://doi.org/10.1093/nar/gkr324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Forestan C, Farinati S, Aiese Cigliano R et al (2017) Maize RNA PolIV affects the expression of genes with nearby TE insertions and has a genome-wide repressive impact on transcription. BMC Plant Biol 17:161. https://doi.org/10.1186/s12870-017-1108-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Slotkin RK, Martienssen R (2007) Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet 8:272–285. https://doi.org/10.1038/nrg2072

    Article  CAS  PubMed  Google Scholar 

  45. Axtell MJ (2013) Classification and comparison of small RNAs from plants. Annu Rev Plant Biol 64:137–159. https://doi.org/10.1146/annurev-arplant-050312-120043

    Article  CAS  PubMed  Google Scholar 

  46. Meyers BC, Souret FF, Lu C, Green PJ (2006) Sweating the small stuff: microRNA discovery in plants. Curr Opin Biotechnol 17:139–146. https://doi.org/10.1016/j.copbio.2006.01.008

    Article  CAS  PubMed  Google Scholar 

  47. Lutzmayer S, Enugutti B, Nodine MD (2017) Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data. Sci Rep 7:5913. https://doi.org/10.1038/s41598-017-06174-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Goodstein DM, Shu S, Howson R et al (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:D1178–D1186. https://doi.org/10.1093/nar/gkr944

    Article  CAS  PubMed  Google Scholar 

  49. International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768. https://doi.org/10.1038/nature08747

    Article  CAS  Google Scholar 

  50. Jeong D-H, Schmidt SA, Rymarquis LA et al (2013) Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon. Genome Biol 14:R145. https://doi.org/10.1186/gb-2013-14-12-r145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gremme G, Steinbiss S, Kurtz S (2013) GenomeTools: a comprehensive software library for efficient processing of structured genome annotations. IEEE/ACM Trans Comput Biol Bioinform 10:645–656. https://doi.org/10.1109/TCBB.2013.68

    Article  PubMed  Google Scholar 

  52. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10. https://doi.org/10.14806/ej.17.1.200

    Article  Google Scholar 

  53. Johnson NR, Yeoh JM, Coruh C, Axtell MJ (2016) Improved placement of multi-mapping small RNAs. G3 (Bethesda) 6:2103–2111. https://doi.org/10.1534/G3.116.030452

    Article  CAS  Google Scholar 

  54. Axtell MJ (2013) ShortStack: comprehensive annotation and quantification of small RNA genes. RNA 19:740–751. https://doi.org/10.1261/rna.035279.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25. https://doi.org/10.1186/gb-2009-10-3-r25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Li H, Handsaker B, Wysoker A et al (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. https://doi.org/10.1093/bioinformatics/btp352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Buels R, Yao E, Diesh CM et al (2016) JBrowse: a dynamic web platform for genome visualization and analysis. Genome Biol 17:66. https://doi.org/10.1186/s13059-016-0924-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Thorvaldsdottir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192. https://doi.org/10.1093/bib/bbs017

    Article  CAS  PubMed  Google Scholar 

  59. Robinson JT, Thorvaldsdóttir H, Winckler W et al (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26. https://doi.org/10.1038/nbt.1754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Brkljacic J, Grotewold E, Scholl R et al (2011) Brachypodium as a model for the grasses: today and the future. Plant Physiol 157:3–13. https://doi.org/10.1104/pp.111.179531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Hirochika H, Okamoto H, Kakutani T (2000) Silencing of retrotransposons in Arabidopsis and reactivation by the ddm1 mutation. Plant Cell 12:357–369. https://doi.org/10.1105/tpc.12.3.357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Tsukahara S, Kobayashi A, Kawabe A et al (2009) Bursts of retrotransposition reproduced in Arabidopsis. Nature 461:423–426. https://doi.org/10.1038/nature08351

    Article  CAS  PubMed  Google Scholar 

  63. Thieme M, Lanciano S, Balzergue S et al (2017) Inhibition of RNA polymerase II allows controlled mobilisation of retrotransposons for plant breeding. Genome Biol 18:134. https://doi.org/10.1186/s13059-017-1265-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Akbergenov R, Si-Ammour A, Blevins T et al (2006) Molecular characterization of geminivirus-derived small RNAs in different plant species. Nucleic Acids Res 34:462–471. https://doi.org/10.1093/nar/gkj447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Blevins T (2017) Northern blotting techniques for small RNAs. Methods Mol Biol 1456:141–162. https://doi.org/10.1007/978-1-4899-7708-3_12

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

M.B., A.G., and D.P. developed the bioinformatics pipeline for TE annotation, genome browser display, and northern blot probe design. M.B., B.R., and C.H. performed the molecular genetic and benchtop experiments. R.S., D. L.-C., A.C., and J.V. provided B. distachyon mutant germplasm identified using high-throughput sequencing and bioinformatics; work at the US DOE Joint Genome Institute is supported by the Office of Science of the US Department of Energy under Contract no. DE-AC02-05CH1123 and by user agreement FP00004794 between JGI and the USDA Agricultural Research Service. M.B., B.R., and T.B. assembled the figures and wrote the manuscript. The authors thank Hugues Renault for advice on culturing B. distachyon and for providing wild-type seed (Bd21-3). This study relied upon the dedicated support of the IBMP gardeners, bioinformatics platform, and sequencing facility staff. The Blevins Group is supported by the LabEx consortium ANR-10-LABX-0036_NETRNA (“Investissements d’Avenir”) and by the French Agence Nationale de la Recherche (ANR) Grant ANR-17-CE20-0004-01.

Author information

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes, CNRS UPR2357, Université de Strasbourg, Strasbourg, France

    Marcel Böhrer, Bart Rymen, Christophe Himber, Aude Gerbaud, David Pflieger & Todd Blevins

  2. USDA ARS Western Regional Research Center, Albany, CA, USA

    Debbie Laudencia-Chingcuanco

  3. DOE Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Amy Cartwright & John Vogel

  4. INRAE, UR BIA, Nantes, France

    Richard Sibout

  5. Institut Jean-Pierre Bourgin, UMR 1318, INRAE, AgroParisTech, CNRS, Université Paris-Saclay, Versailles, France

    Richard Sibout

Authors
  1. Marcel Böhrer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bart Rymen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Christophe Himber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aude Gerbaud
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David Pflieger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Debbie Laudencia-Chingcuanco
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Amy Cartwright
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. John Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Richard Sibout
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Todd Blevins
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Todd Blevins .

Editor information

Editors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes (IBMP-CNRS), Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Böhrer, M. et al. (2020). Integrated Genome-Scale Analysis and Northern Blot Detection of Retrotransposon siRNAs Across Plant Species. In: Heinlein, M. (eds) RNA Tagging. Methods in Molecular Biology, vol 2166. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0712-1_23

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0712-1_23

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0711-4

  • Online ISBN: 978-1-0716-0712-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation