Advertisement

Plant Molecular Biology

, Volume 74, Issue 6, pp 591–603 | Cite as

Pol II-directed short RNAs suppress the nuclear export of mRNA

  • Tatiana V. Komarova
  • Anton M. Schwartz
  • Olga Y. Frolova
  • Anna S. Zvereva
  • Yuri Y. Gleba
  • Vitaly Citovsky
  • Yuri L. Dorokhov
Article

Abstract

The synthesis and subsequent nuclear export of non-coding RNA (ncRNA) directed by RNA polymerase (Pol) II is very sensitive to abiotic and biotic external stimuli including pathogen challenges. To assess whether stress-induced ncRNAs may suppress the nuclear export of mRNA, we exploited the ability of Agrobacterium tumefaciens to co-deliver Pol I, II and III promoter-based vectors for the transcription of short (s) ncRNAs, GFP mRNA or genomic RNA of plant viruses (Tobacco mosaic virus, TMV; or Potato virus X, PVX) into the nucleus of Nicotiana benthamiana cells. We showed that, in contrast to Pol I- and Pol III-derived sncRNAs, all tested Pol II-derived sncRNAs (U6 RNA, tRNA or artificial RNAs) resulted in decreased expression of GFP and host mRNA. The level of this inhibitory effect depended on the non-coding transcript length and promoter strength. Short coding RNA (scRNA) can also compete with mRNA for nuclear export. We showed that scRNA, an artificial 117-nt short sequence encoding Elastin-Like peptide element tandems with FLAG sequence (ELF) and the 318-nt N. benthamiana antimicrobial peptide thionin (defensin) gene efficiently decreased GFP expression. The stress-induced export of Pol II-derived sncRNA and scRNA into the cytoplasm via the mRNA export pathway may block nucleocytoplasmic traffic including the export of mRNA responsible for antivirus protection. Consistent with this model, we observed that Pol II-derived sncRNAs as well as scRNA, thionin and ELF strongly enhanced the cytoplasmic reproduction of TMV and PVX RNA.

Keywords

Non-coding RNAs Nuclear export mRNA Plant virus RNA polymerase II Short coding RNA 

Notes

Acknowledgments

We thank Drs. C. S. Pikaard (at Washington University St. Louis), T. Okuno (Kyoto University), A. E. Simon (University of Maryland, College Park) and H. Beier (University of Würzburg) for generously providing the Pol I promoter containing plasmid pBor2, pBICmiR171prec, GNC encoding plasmid, and plasmid pNtY1 encoding Nicotiana rustica pre-tRNATyr, respectively. We also thank members of the MSU Department of Virology for helpful discussions and technical assistance. This work was partly supported by the Russian Foundation for Basic Research (grants 08-04-00106 and 08-04-12073) and Icon Genetics GmbH. The work in the VC laboratory is supported by grants from NIH, NSF, NRI USDA CSREE, BARD, and BSF.

Supplementary material

11103_2010_9700_MOESM1_ESM.doc (32 kb)
Supplementary Table S1 (DOC 32 kb)
11103_2010_9700_MOESM2_ESM.tif (280 kb)
Fig. S1 An MS2-GFP RNA-tagging technique for identification of the Pol I-, Pol II- and Pol III promoter-directed sncRNAs in the plant cell cytoplasm. (A) Organization of the TMV-(MS2-H)4 vector: A. thaliana Act2 promoter-based full-length infectious clone of TMV U1, containing RNA-dependent RNA-polymerase (RdRp), movement protein (MP), and MS2 hairpin (H) repeats (MS2-H)4. (B,C) Confocal images (B) and their overlays over the respective false-transmission images (C) showing GFP in epidermal cells of N. benthamiana leaves co-agroinjected with Pol II35S- GNC and TMV-(MS2-H)4. (TIF 281 kb)
11103_2010_9700_MOESM3_ESM.tif (95 kb)
Fig. S2 Intron insertion into the GFP gene relieves its suppression by Pol II35S-(MS2-H)4-derived sncRNA. (TIF 95 kb)
11103_2010_9700_MOESM4_ESM.tif (53 kb)
Fig. S3 Relative quantity of (GAAA)16 sncRNA and Lnc-(MS2-H)4 as determined by real-time PCR in leaf areas co-agroinjected with respective plasmids. (TIF 54 kb)
11103_2010_9700_MOESM5_ESM.tif (385 kb)
Fig. S4 The fluorimetric analysis of GFP expression in N. benthamiana leaves co-agroinjected with PVX:GFP and the Pol II35S-(GAAA)16 binary vector. Control, co-agroinjection with empty pBin19. The dilution factor for Agrobacterium cultures harboring the PVX vector is indicated. (TIF 385 kb)

References

  1. Bartlett J, Blagojevic J, Carter D, Eskiw C, Fromaget M, Job C, Shamsher M, Trindade IF, Xu M, Cook PR (2006) Specialized transcription factories. Biochem Soc Symp 73:67–75PubMedGoogle Scholar
  2. Bazzini AA, Hopp HE, Beachy RN, Asurmendi S (2007) Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development. Proc Natl Acad Sci USA 104:12157–12162CrossRefPubMedGoogle Scholar
  3. Ben Amor B, Wirth S, Merchan F, Laporte P, d’Aubenton-Carafa Y, Hirsch J, Maizel A, Mallory A, Lucas A, Deragon JM, Vaucheret H, Thermes C, Crespi M (2009) Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses. Genome Res 19:57–69CrossRefPubMedGoogle Scholar
  4. Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI (2007) The multifunctional nucleolus. Nat Rev Mol Cell Biol 8:574–585CrossRefPubMedGoogle Scholar
  5. Bond U (2006) Stressed out! Effects of environmental stress on mRNA metabolism. FEMS Yeast Res 6:160–170CrossRefPubMedGoogle Scholar
  6. Brown JW, Marshall DF, Echeverria M (2008) Intronic noncoding RNAs and splicing. Trends Plant Sci 13:335–342CrossRefPubMedGoogle Scholar
  7. Carmody SR, Tran EJ, Apponi LH, Corbett AH, Wente SR. (2010) The MAP kinase Slt2 regulates nuclear retention of non-heat shock mRNAs during heat shock-induced stress. Mol Cell Biol Sep 7 (Epub ahead of print)Google Scholar
  8. Chinnusamy V, Gong Z, Zhu JK (2008) Nuclear RNA export and its importance in abiotic stress responses of plants. Curr Top Microbiol Immunol 326:235–355CrossRefPubMedGoogle Scholar
  9. Chinnusamy V, Zhu JK, Sunkar R (2010) Gene regulation during cold stress acclimation in plants. Methods Mol Biol 639:39–55CrossRefPubMedGoogle Scholar
  10. Csorba T, Bovi A, Dalmay T, Burgyán J (2007) The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA-and microRNA-mediated pathways. J Virol 81:11768–11780CrossRefPubMedGoogle Scholar
  11. Dorokhov YL, Skulachev MV, Ivanov PA, Zvereva SD, Tjulkina LG, Merits A, Gleba YY, Hohn T, Atabekov JG (2002) Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry. Proc Natl Acad Sci USA 99:5301–5306CrossRefPubMedGoogle Scholar
  12. Dorokhov YL, Frolova OY, Skurat EV, Ivanov PA, Gasanova TV, Sheveleva AS, Ravin NV, Mäkinen K, Klimyuk VI, Skryabin KG, Gleba YY, Atabekov JG (2006) A novel function for a ubiquitous plant enzyme pectin methylesterase: the enhancer of RNA silencing. FEBS Lett 580:3872–3878CrossRefPubMedGoogle Scholar
  13. Faro-Trindade I, Cook PR (2006) Transcription factories: structures conserved during differentiation and evolution. Biochem Soc Trans 34:1133–1137CrossRefPubMedGoogle Scholar
  14. Gallouzi IE, Brennan CM, Stenberg MG, Swanson MS, Eversole A, Maizels N, Steitz JA (2000) HuR binding to cytoplasmic mRNA is perturbed by heat shock. Proc Natl Acad Sci USA 97:3073–3078CrossRefPubMedGoogle Scholar
  15. Gibbs AJ, Fargette D, García-Arenal F, Gibbs MJ (2010) Time—the emerging dimension of plant virus studies. J Gen Virol 91:13–22CrossRefPubMedGoogle Scholar
  16. Grünwald D, Singer RH (2010) In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport. Nature 467:604–607CrossRefPubMedGoogle Scholar
  17. Hiscox JA (2007) RNA viruses: hijacking the dynamic nucleolus. Nat Rev Microbiol 5:119–127CrossRefPubMedGoogle Scholar
  18. Izawa S, Takemura R, Miki T, Inoue Y (2005) Characterization of the export of bulk poly(A) + mRNA in Saccharomyces cerevisiae during the wine-making process. Appl Environ Microbiol 71:2179–2182CrossRefPubMedGoogle Scholar
  19. Jarmolowski A, Boelens WC, Izaurralde E, Mattaj IW (1994) Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol 124:627–635CrossRefPubMedGoogle Scholar
  20. Kimura T, Hashimoto I, Nishikawa M, Yamada H (2009) Nucleocytoplasmic transport of luciferase gene mRNA requires CRM1/Exportin1 and RanGTPase. Med Mol Morphol 42:70–81CrossRefPubMedGoogle Scholar
  21. Komarova TV, Skulachev MV, Zvereva AS, Schwartz AM, Dorokhov YL, Atabekov JG (2006) New viral vector for efficient production of target proteins in plants. Biochemistry (Moscow) 71:846–850CrossRefGoogle Scholar
  22. Krebber H, Taura T, Lee MS, Silver PA (1999) Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export. Genes Dev 13:1994–2004CrossRefPubMedGoogle Scholar
  23. Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126:93–106CrossRefPubMedGoogle Scholar
  24. Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836–843CrossRefPubMedGoogle Scholar
  25. Lózsa R, Csorba T, Lakatos L, Burgyán J (2008) Inhibition of 3′ modification of small RNAs in virus-infected plants requires spatial and temporal co-expression of small RNAs and viral silencing-suppressor proteins. Nucleic Acids Res 36:4099–4107CrossRefPubMedGoogle Scholar
  26. MacEwan SR, Chilkoti A (2010) Elastin-like polypeptides: biomedical applications of tunable biopolymers. Biopolymers 94:60–77CrossRefPubMedGoogle Scholar
  27. Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol 23:718–723CrossRefPubMedGoogle Scholar
  28. Moore MJ, Proudfoot NJ (2009) Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136:688–700CrossRefPubMedGoogle Scholar
  29. Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y (2010) Dynamics of single mRNP nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol 12:543–552CrossRefPubMedGoogle Scholar
  30. Navarro L, Jay F, Nomura K, He SY, Voinnet O (2008) Suppression of the microRNA pathway by bacterial effector proteins. Science 321:964–967CrossRefPubMedGoogle Scholar
  31. Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA 102:3691–3696CrossRefPubMedGoogle Scholar
  32. Pelegrini PB, Franco OL (2005) Plant gamma-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins. Int J Biochem Cell Biol 37:2239–2253CrossRefPubMedGoogle Scholar
  33. Pontes O, Pikaard CS (2008) siRNA and miRNA processing: new functions for Cajal bodies. Curr Opin Genet Dev 18:197–203CrossRefPubMedGoogle Scholar
  34. Saavedra C, Tung KS, Amberg DC, Hopper AK, Cole CN (1996) Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. Genes Dev 10:1608–1620CrossRefPubMedGoogle Scholar
  35. Saavedra CA, Hammell CM, Heath CV, Cole CN (1997) Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p. Genes Dev 11:2845–2856CrossRefPubMedGoogle Scholar
  36. Schwartz AM, Komarova TV, Skulachev MV, Zvereva AS, Dorokhov YL, Atabekov JG (2006) Stability of plant mRNAs depends on the length of the 3′-untranslated region. Biochemistry (Moscow) 71:1377–1384CrossRefGoogle Scholar
  37. Sels J, Mathys J, De Coninck BM, Cammue BP, De Bolle MF (2008) Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiol Biochem 46:941–950CrossRefPubMedGoogle Scholar
  38. Stewart M (2009) Nuclear export of small RNAs. Science 326:1195–1196CrossRefPubMedGoogle Scholar
  39. Stewart M (2010) Nuclear export of mRNA. Trends Biochem Sci Aug 16 (Epub ahead of print)Google Scholar
  40. Vaucheret H (2006) Post-transcriptional small RNA pathways in plants: mechanisms and regulations. Genes Dev 20:759–771CrossRefPubMedGoogle Scholar
  41. Voinnet O (2008) Post-transcriptional RNA silencing in plant-microbe interactions: a touch of robustness and versatility. Curr Opin Plant Biol 11:464–470CrossRefPubMedGoogle Scholar
  42. Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104CrossRefPubMedGoogle Scholar
  43. Yelina NE, Smith LM, Jones AM, Patel K, Kelly KA, Baulcombe DC (2010) Putative Arabidopsis THO/TREX mRNA export complex is involved in transgene and endogenous siRNA biosynthesis. Proc Natl Acad Sci USA 107:13948–13953CrossRefPubMedGoogle Scholar
  44. Zhang F, Simon AE (2003) A novel procedure for the localization of viral RNAs in protoplasts and whole plants. Plant J 35:665–673CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Tatiana V. Komarova
    • 1
  • Anton M. Schwartz
    • 2
  • Olga Y. Frolova
    • 1
    • 2
  • Anna S. Zvereva
    • 1
    • 5
  • Yuri Y. Gleba
    • 3
  • Vitaly Citovsky
    • 4
  • Yuri L. Dorokhov
    • 1
    • 2
  1. 1.A. N. Belozersky Institute of Physico-Chemical BiologyMoscow State UniversityMoscowRussia
  2. 2.N. I. Vavilov Institute of General Genetics, Russian Academy of ScienceMoscowRussia
  3. 3.Nomad Bioscience GmbH, Biozentrum HalleHalleGermany
  4. 4.Department of Biochemistry and Cell BiologyState University of New YorkStony BrookUSA
  5. 5.Botanical InstituteUniversity of BaselBaselSwitzerland

Personalised recommendations