Advertisement

Plant Molecular Biology

, Volume 93, Issue 3, pp 283–298 | Cite as

The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events

  • Brian B. Sørensen
  • Hans F. Ehrnsberger
  • Silvia Esposito
  • Alexander Pfab
  • Astrid Bruckmann
  • Judith Hauptmann
  • Gunter Meister
  • Rainer Merkl
  • Thomas Schubert
  • Gernot Längst
  • Michael Melzer
  • Marion Grasser
  • Klaus D. Grasser
Article

Abstract

Key message

We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development.

Abstract

TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.

Keywords

mRNA processing/export Chromatin Protein complexes RNA polymerase II Development 

Notes

Acknowledgements

We thank Jelle Van Leene and Geert De Jaeger for advice regarding cell culture transformation and for providing Arabidopsis PSB-D cells, Liang-zi Zhou for help with the protoplast transformation experiment, Eduard Hochmuth for recording mass spectra, Patrick Motte for plasmids pBI35S::atRSZp22-mRFP1 and pBI35S::atRSZp33-mRFP1, Claus Schwechheimer for pid-14 seeds, the Nottingham Arabidopsis Stock Centre (NASC) for providing Arabidopsis T-DNA insertion lines. This work was supported by the German Research Foundation (DFG) through grant SFB960 to M. G and K. D. G.

Author contributions

BBS, MG, KDG conceived and designed the experiments. BBS, HFE, SE, AP, TS, MM, MG performed the experiments. BBS, HFE, SE, AP, AB, GM, RM, TS, GL, MM, MG, KDG analysed the data. JH, GM, RM, TS, GL, MM, MG, KDG contributed reagents/materials/analysis tools. MG, KDG wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2016_561_MOESM1_ESM.pdf (850 kb)
Supplementary material 1 (PDF 850 KB)

References

  1. Albertazzi L, Arosio D, Marchetti L, Ricci F, Beltram F (2009) Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair. Photochem Photobiol 85:287–297CrossRefPubMedGoogle Scholar
  2. Antosch M, Schubert V, Holzinger P, Houben A, Grasser KD (2015) Mitotic lifecycle of chromosomal 3xHMG-box proteins and the role of their N-terminal domain in the association with rDNA loci and proteolysis. New Phytol 208:1067–1077CrossRefPubMedGoogle Scholar
  3. Bennett SRM, Alvarez J, Bossinger G, Smyth DR (1995) Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J 8:505–520CrossRefGoogle Scholar
  4. Chanarat S, Seizl M, Sträßer K (2011) The Prp19 complex is a novel transcription elongation factor required for TREX occupancy at transcribed genes. Genes Dev 25:1147–1158CrossRefPubMedPubMedCentralGoogle Scholar
  5. Chávez S, Beilharz T, Rondón AG, Erdjument-Bromage H, Tempst P, Svejstrup JQ, Lithgow T, Aguilera A (2000) A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J 19:5824–5834CrossRefPubMedPubMedCentralGoogle Scholar
  6. Chen YI, Moore RE, Ge HY, Young MK, Lee TD, Stevens WW (2007) Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors. Nucleic Acids Res 35:3928–3944CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cheng Y, Qin G, Dai X, Zhao Y (2007) NPY1, a BTB-NPH3-like protein, plays a critical role in auxin-regulated organogenesis in Arabidopsis. Proc Natl Acad Sci USA 104:18825–18829CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cheng H, Dufu K, Lee CS, Hsu JL, Dias A, Reed R (2006) Human mRNA export machinery recruited to the 5′ end of mRNA. Cell 127:1389–1400CrossRefPubMedGoogle Scholar
  9. Chi B, Wang Q, Wu G, Tan M, Wang L, Shi M, Chang X, Cheng H (2013) Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA. Nucleic Acids Res 41:1294–1306CrossRefPubMedGoogle Scholar
  10. Dias AP, Dufu K, Lei H, Reed R (2010) A role for TREX components in the release of spliced mRNA from nuclear speckle domains. Nat Commun 1:97CrossRefPubMedPubMedCentralGoogle Scholar
  11. Dufu K, Livingstone MJ, Seebacher J, Gygi SP, Wilson SA, Reed R (2010) ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex. Genes Dev 24:2043–2053CrossRefPubMedPubMedCentralGoogle Scholar
  12. Dürr J, Lolas IB, Sørensen BB, Schubert V, Houben A, Melzer M, Deutzmann R, Grasser M, Grasser KD (2014) The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis. Nucleic Acids Res 42:4332–4347CrossRefPubMedPubMedCentralGoogle Scholar
  13. Ellisdon AM, Dimitrova L, Hurt E, Stewart M (2012) Structural basis for the assembly and nucleic acid binding of the TREX-2 transcription-export complex. Nat Struct Mol Biol 19:328–336CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fischer T, Sträßer K, Rácz A, Rodriguez-Navarro S, Oppizzi M, Ihrig P, Lechner J, Hurt E (2002) The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J 21:5843–5852CrossRefPubMedPubMedCentralGoogle Scholar
  15. Fleckner J, Zhang M, Valcarcel J, Green MR (1997) U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev 11:1864–1872CrossRefPubMedGoogle Scholar
  16. Francisco-Mangilet AG, Karlsson P, Kim MH, Eo HJ, Oh SA, Kim JH, Kulcheski FR, Park SK, Manavella PA (2015) THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis. Plant J 82:1018–1029CrossRefPubMedGoogle Scholar
  17. Furumizu C, Tsukaya H, Komeda Y (2010) Characterization of EMU, the Arabidopsis homolog of the yeast THO complex member HPR1. RNA 16:1809–1817CrossRefPubMedPubMedCentralGoogle Scholar
  18. García-Rubio M, Chávez S, Huertas P, Tous C, Jimeno S, Luna R, Aguilera A (2008) Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity. Mol Genet Genomics 279:123–132CrossRefPubMedGoogle Scholar
  19. Gatfield D, Le Hir H, Schmitt C, Braun IC, Köcher T, Wilm M, Izaurralde E (2001) The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila. Curr Biol 11:1716–1721CrossRefPubMedGoogle Scholar
  20. Germain H, Qu N, Cheng YT, Lee E, Huang Y, Dong OX, Gannon P, Huang S, Ding P, Li Y, Sack F, Zhang Y, Li X (2010) MOS11: a new component in the mRNA export pathway. PLoS Genet 6:e1001250CrossRefPubMedPubMedCentralGoogle Scholar
  21. Gong Z, Dong CH, Lee H, Zhu J, Xiong L, Gong D, Stevenson B, Zhu JK (2005) A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. Plant Cell 17:256–267CrossRefPubMedPubMedCentralGoogle Scholar
  22. Grasser KD, Grimm R, Ritt C (1996) Maize chromosomal HMGc: two closely related structure-specific DNA-binding proteins specify a second type of plant HMG-box protein. J Biol Chem 271:32900–32906CrossRefPubMedGoogle Scholar
  23. Hamperl S, Brown CR, Perez-Fernandez J, Huber K, Wittner M, Babl V, Stöckl U, Boeger H, Tschochner H, Milkereit P, Griesenbeck J (2014) Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol Biol 1094:329–341CrossRefPubMedGoogle Scholar
  24. Herold N, Will CL, Wolf E, Kastner B, Urlaub H, Lührmann R (2009) Conservation of the protein composition and electron microscopy structure of Drosophila melanogaster and human spliceosomal complexes. Mol Cell Biol 29:281–301CrossRefPubMedGoogle Scholar
  25. Hsin JP, Manley JL (2012) The RNA polymerase II CTD coordinates transcription and RNA processing. Genes Dev 26:2119–2137CrossRefPubMedPubMedCentralGoogle Scholar
  26. Jani D, Lutz S, Hurt E, Laskey RA, Stewart M, Wickramasinghe VO (2012) Functional and structural characterization of the mammalian TREX-2 complex that links transcription with nuclear messenger RNA export. Nucleic Acids Res 40:4562–4573CrossRefPubMedPubMedCentralGoogle Scholar
  27. Jauvion V, Elmayan T, Vaucheret H (2010) The conserved RNA trafficking proteins HPR1 and TEX1 are involved in the production of endogenous and exogenous small interfering RNA in Arabidopsis. Plant Cell 22:2697–2709CrossRefPubMedPubMedCentralGoogle Scholar
  28. Jimeno S, Luna R, García-Rubio M, Aguilera A (2006) Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis in yeast THO mutants. Mol Cell Biol 26:4387–4398CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kammel C, Thomaier M, Sørensen BB, Schubert T, Längst G, Grasser M, Grasser KD (2013) Arabidopsis DEAD-box RNA helicase UAP56 interacts with both RNA and DNA as well as with mRNA export factors. PLoS One 8:e60644CrossRefPubMedPubMedCentralGoogle Scholar
  30. Karpova TS, Baumann CT, He L, Wu X, Grammer A, Sipsky P, Hager GL, NcNally JG (2003) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J Microsc 209:56–70CrossRefPubMedGoogle Scholar
  31. Katahira J (2012) mRNA export and the TREX complex. Biochim Biophys Acta 1819:507–513CrossRefPubMedGoogle Scholar
  32. Köhler A, Hurt E (2007) Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 8:761–773CrossRefPubMedGoogle Scholar
  33. Launholt D, Merkle T, Houben A, Schulz A, Grasser KD (2006) Arabidopsis chromatin-associated HMGA and HMGB use different nuclear targeting signals and display highly dynamic localization within the nucleus. Plant Cell 18:2904–2918CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lildballe DL, Pedersen DS, Kalamajka R, Emmersen J, Houben A, Grasser KD (2008) The expression level of the chromatin-associated HMGB1 protein influences growth, stress tolerance and transcriptome in Arabidopsis. J Mol Biol 384:9–21CrossRefPubMedGoogle Scholar
  35. Lolas IB, Himanen K, Grønlund JT, Lynggaard C, Houben A, Melzer M, Van Lijsebettens M, Grasser KD (2010) The transcript elongation factor FACT affects Arabidopsis vegetative and reproductive development and genetically interacts with HUB1/2. Plant J 61:686–697CrossRefPubMedGoogle Scholar
  36. Lu Q, Tang X, Tian G, Wang F, Liu K, Nguyen V, Kohalmi SE, Keller WA, Tsang EW, Harada JJ, Rothstein SJ, Cui Y (2010) Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J 61:259–270CrossRefPubMedGoogle Scholar
  37. Luna R, Jimeno S, Marín M, Huertas P, García-Rubio M, Aguilera A (2005) Interdependence between transcription and mRNP processing and export, and its impact on genetic stability. Mol Cell 10:711–722CrossRefGoogle Scholar
  38. Luna R, Rondón AG, Aguilera A (2012) New clues to understand the role of THO and other functionally related factors in mRNP biogenesis. Biochim Biophys Acta 1819:514–520CrossRefPubMedGoogle Scholar
  39. Masuda S, Das R, Cheng H, Hurt E, Dorman N, Reed R (2005) Recruitment of the human TREX complex to mRNA during splicing. Genes Dev 19:1512–1517CrossRefPubMedPubMedCentralGoogle Scholar
  40. Monaghan J, Xu F, Gao M, Palma K, Long C, Chen S, Zhang Y, Li X (2009) Two Prp19-like U-box proteins in the MOS4-associated complex play redundant roles in plant innate immunity. PLoS Pathog 5:e1000526CrossRefPubMedPubMedCentralGoogle Scholar
  41. Moore MJ, Proudfoot NJ (2009) Pre-mRNA processing reaches back to transcription and ahead to translation. Cell 136:688–700CrossRefPubMedGoogle Scholar
  42. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  43. Nelissen H, DeGroeve S, Fleury D, Neyt P, Bruno L, Bitonti MB, Vandenbussche F, Van Der Straeten D, Yamaguchi T, Tsukaya H, Witters E, de Jaeger G, Houben A, Lijsebettens M (2010) Plant Elongator regulates auxin-related genes during RNA polymerase II-mediated transcription elongation. Proc Natl Acad Sci USA 107:1678–1683.CrossRefPubMedPubMedCentralGoogle Scholar
  44. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084CrossRefPubMedGoogle Scholar
  45. Palusa SG, Ali GS, Reddy AS (2007) Alternative splicing of pre-mRNAs of Arabidopsis serine/arginine-rich proteins: regulation by hormones and stresses. Plant J 49:1091–1107CrossRefPubMedGoogle Scholar
  46. Pan H, Liu S, Tang D (2012) HPR1, a component of the THO/TREX complex, plays an important role in disease resistance and senescence in Arabidopsis. Plant J 69:831–843CrossRefPubMedGoogle Scholar
  47. Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Pérez AC, Chico JM, Bossche RV, Sewell J, Gil E, García-Casado G, Witters E, Inzé D, Long JA, de Jaeger G, Solano R, Goossens A (2010) NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature 464:788–791CrossRefPubMedPubMedCentralGoogle Scholar
  48. Peña A, Gewartowski K, Mroczek S, Cuéllar J, Szykowska A, Prokop A, Czarnocki-Cieciura M, Piwowarski J, Tous C, Aguilera A, Carrascosa JL, Valpuesta JM (2012) Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor. EMBO J 31:1605–1616CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rappsilber J, Ryder U, Lamond AI, Mann M (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12:1231–1245CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rehwinkel J, Herold A, Gari K, Köcher T, Rode M, Ciccarelli FL, Wilm M, Izaurralde E (2004) Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster. Nat Struct Mol Biol 11:558–566CrossRefPubMedGoogle Scholar
  51. Schnittger A, Hülskamp M (2002) Trichome morphogenesis: a cell-cycle perspective. Phil Trans R Soc Land Biol Sci 357:826Google Scholar
  52. Shen H (2009) UAP56—a key player with surprisingly diverse roles in pre-mRNA splicing and nuclear export. BMB Rep 42:185–188CrossRefPubMedGoogle Scholar
  53. Shen H, Zheng X, Shen J, Zhang L, Zhao R, Green MR (2008) Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome. Genes Dev 22:1796–1803CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sträßer K, Masuda S, Mason P, Pfannstiel J, Oppizzi M, Rodriguez-Navarro S, Rondón AG, Aguilera A, Struhl K, Reed R, Hurt E (2002) TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417:304–308CrossRefPubMedGoogle Scholar
  55. Sugiura T, Sakurai K, Nagano Y (2007) Intracellular characterization of DDX39, a novel growth-associated RNA helicase. Exp Cell Res 313:782–790CrossRefPubMedGoogle Scholar
  56. Tillemans V, Dispa L, Remacle C, Motte P (2005) Functional distribution and dynamics of Arabidopsis SR splicing factors in living plant cells. Plant J 41:567–582CrossRefPubMedGoogle Scholar
  57. van Leene J, Eeckhout D, Cannoot B, De Winne N, Persiau G, van de Slijke E, Vercruysse L, Dedecker M, Vandepoele K, Martens L, Witters E, Gevaert K, de Jaeger G (2015) An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. Nat Protoc 10:169–187CrossRefPubMedGoogle Scholar
  58. Walsh MJ, Hautbergue GM, Wilson SA (2010) Structure and function of mRNA export adaptors. Biochem Soc Trans 38:232–236CrossRefPubMedGoogle Scholar
  59. Wickramasinghe VO, Laskey RA (2015) Control of mammalian gene expression by selective mRNA export. Nat Rev Mol Cell Biol 16:431–442CrossRefPubMedGoogle Scholar
  60. Wu FH, Shen SC, Lee LY, Lee SH, Chan MT, Lin CS (2009) Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isolation method. Plant Meth 5:16CrossRefGoogle Scholar
  61. Xu C, Zhou X, Wen CK (2015) HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of the REVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis. PLoS Genet 11:e1004956CrossRefPubMedPubMedCentralGoogle Scholar
  62. Yamazaki T, Fujiwara N, Yukinaga H, Ebisuya M, Shiki T, Kurihara T, Kioka N, Kambe T, Nagao N, Nishida E, Masuda S (2010) The closely related RNA helicases, UAP56 and URH49, preferentially form distinct mRNA export machineries and coordinately regulate mitotic progression. Mol Biol Cell 21:2953–2965CrossRefPubMedPubMedCentralGoogle Scholar
  63. Yelina NE, Smith LM, Jones AME, 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 107:13948–13953CrossRefPubMedPubMedCentralGoogle Scholar
  64. Zhai Z, Jung HI, Vatamaniuk OK (2009) Isolation of protoplasts from tissues of 14-day-old seedlings of Arabidopsis thaliana. J Vis Exp 17:1149Google Scholar
  65. Zhou Z, Licklider LJ, Gygi SP, Reed R (2002) Comprehensive proteomic analysis of the human spliceosome. Nature 419:182–185CrossRefPubMedGoogle Scholar
  66. Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Längst G, Baaske P (2012) Microscale thermophoresis as a sensitive method to quantify protein:nucleic acid interactions in solution. Methods Mol Biol 815:241–252CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Brian B. Sørensen
    • 1
  • Hans F. Ehrnsberger
    • 1
  • Silvia Esposito
    • 1
  • Alexander Pfab
    • 1
  • Astrid Bruckmann
    • 2
  • Judith Hauptmann
    • 2
  • Gunter Meister
    • 2
  • Rainer Merkl
    • 3
  • Thomas Schubert
    • 4
  • Gernot Längst
    • 4
  • Michael Melzer
    • 5
  • Marion Grasser
    • 1
  • Klaus D. Grasser
    • 1
  1. 1.Department of Cell Biology and Plant Biochemistry, Biochemistry CentreUniversity of RegensburgRegensburgGermany
  2. 2.Department for Biochemistry I, Biochemistry CentreUniversity of RegensburgRegensburgGermany
  3. 3.Department for Biochemistry II, Biochemistry CentreUniversity of RegensburgRegensburgGermany
  4. 4.Department for Biochemistry III, Biochemistry CentreUniversity of RegensburgRegensburgGermany
  5. 5.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) GaterslebenStadt SeelandGermany

Personalised recommendations