Skip to main content
SpringerLink
Log in

Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in Arabidopsis thaliana

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CPL:

RNA polymerase II C-terminal domain phosphatase-like protein

DCL:

Dicer-like protein

DRB:

dsRNA–binding protein

dsRBM:

dsRNA-binding motif

dsRBP:

dsRBM-containing protein

dsRNA:

double-stranded RNA

PKR:

dsRNA-dependent protein kinase

eIF-2α:

α subunit of eukaryotic initiation factor 2

EST:

expressed sequence tag

GFP:

green fluorescent protein

GST:

glutathion S-transferase

HEN1:

HUA ENHANCER1

HYL1:

HYPONASTIC LEAVES1

miRNA:

microRNAs

NCBI:

National Center for Biotechnology Information

NLS:

nuclear localization signal

OsDRB :

Oryza sativadsRNA-binding protein

PACT:

protein activator of PKR

PAZ:

Piwi/Argonaute/Zwille

PBS:

phosphate-buffered saline

PTGS:

post-transcriptional gene silencing

RISC:

RNA-induced silencing complex

RNAi:

RNA interference

RTL:

ribonuclease three (III)-like protein

siRNA:

short interfering RNA

ssRNA:

single-stranded RNA

TBS:

Tris-buffered saline

TRBP:

trans-activation region (TAR)-RNA-binding protein

References

  • B. Bartel D.P. Bartel (2003) ArticleTitleMicroRNAs: at the root of plant development? Plant Physiol 132 709–717

    Google Scholar 

  • E Bernstein A.A. Caudy S.M. Hammond G.J. Hannon (2001) ArticleTitleRole for a bidentate ribonuclease in the initiation step of RNA interference Nature 409 363–366 Occurrence Handle10.1038/35053110 Occurrence Handle1:CAS:528:DC%2BD3MXms12ksA%3D%3D Occurrence Handle11201747

    Article  CAS  PubMed  Google Scholar 

  • S. Boutet F. Vazquez J. Liu C. Beclin M. Fagard A. Gratias J.B. Morel P. Crete X. Chen H. Vaucheret (2003) ArticleTitleArabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance Curr Biol 13 843–848

    Google Scholar 

  • G.P. Cosentino S. Venkatesan F.C. Serluca S.R. Green M.B. Mathews N. Sonenberg (1995) ArticleTitleDouble-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo Proc. Natl. Acad. Sci. USA 92 9445–9449

    Google Scholar 

  • S.M. Elbashir J. Harborth W. Lendeckel A. Yalcin K. Weber T. Tuschl (2001) ArticleTitleDuplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 411 494–498 Occurrence Handle10.1038/35078107 Occurrence Handle1:CAS:528:DC%2BD3MXkt1ejt7Y%3D Occurrence Handle11373684

    Article  CAS  PubMed  Google Scholar 

  • I. Fierro-Monti M.B. Mathewsc (2000) ArticleTitleProteins binding to duplexed RNA: one motif, multiple functions Trends Biochem. Sci. 25 241–246

    Google Scholar 

  • A. Fire S. Xu M. Montgomery S. Kostas S. Driver C. Mello (1998) ArticleTitlePotent and specific genetic interference by doubule-strand RNA in Caenorhabitis elegans Nature 391 806–811 Occurrence Handle10.1038/35888 Occurrence Handle1:CAS:528:DyaK1cXhtlCju74%3D Occurrence Handle9486653

    Article  CAS  PubMed  Google Scholar 

  • T.A. Golden S.E. Schauer J.D. Lang S. Pien A.R. Mushegian U. Grossniklaus D.W. Meinke A. Ray (2002) ArticleTitleSHORT INTEGUMENTS1/SUSPENSOR1/CARPEL FACTORY, a Dicer homolog, is a maternal effect gene required for embryo development in Arabidopsis Plant Physiol 130 808–822

    Google Scholar 

  • A. Grishok A.E. Pasquinelli D. Conte N. Li S. Parrish I. Ha D.L. Baillie A. Fire G. Ruvkun C.C. Mello (2001) ArticleTitleGenes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing Cell 106 23–34

    Google Scholar 

  • M.H. Han S. Goud L. Song N. Fedoroff (2004) ArticleTitleThe Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation Proc. Natl. Acad. Sci. USA 101 1093–1098

    Google Scholar 

  • G.J. Hannon (2002) ArticleTitleRNA interference Nature 418 244–251 Occurrence Handle10.1038/418244a Occurrence Handle1:CAS:528:DC%2BD38XltFGmu7Y%3D Occurrence Handle12110901

    Article  CAS  PubMed  Google Scholar 

  • S.E. Jacobsen M.P. Running E.M. Meyerowitz (1999) ArticleTitleDisruption of an RNA helicase/RNaseIII gene in Arabidopsis causes unregulated cell division in floral meristems Development 126 5231–5243

    Google Scholar 

  • R.J. Kaufman (1999) ArticleTitleDouble-stranded RNA-activated protein kinase mediates virus-induced apoptosis: a new role for an old actor Proc. Natl. Acad. Sci. USA 96 11693–11695

    Google Scholar 

  • H. Koiwa A.W. Barb L. Xiong F. Li M.G. McCully B.H. Lee I. Sokolchik J. Zhu Z. Gong M. Reddy A. Sharkhuu Y. Manabe S. Yokoi J.K. Zhu R.A. Bressan P.M. Hasegawa (2002) ArticleTitleC-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development Proc. Natl. Acad. Sci. USA 99 10893–10898

    Google Scholar 

  • Q. Liu T.A. Rand S. Kalidas F. Du H.E. Kim D.P. Smith X. Wang (2003) ArticleTitleR2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway Science 301 1921–1925

    Google Scholar 

  • C. Lu N. Fedoroff (2000) ArticleTitleA mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin Plant Cell 12 2351–2366 Occurrence Handle10.1105/tpc.12.12.2351 Occurrence Handle1:CAS:528:DC%2BD3MXns1Shtw%3D%3D Occurrence Handle11148283

    Article  CAS  PubMed  Google Scholar 

  • L. Manche S.R. Green C. Schmedt M.B. Mathews (1992) ArticleTitleInteractions between double-stranded RNA regulators and the protein kinase DAI Mol. Cell. Biol. 12 5238–5248

    Google Scholar 

  • P.J. Martin M.H. Delmotte P. Formstecher P. Lefebvre (2003) ArticleTitlePLZF is a negative regulator of retinoic acid receptor transcriptional activity Nucl. Recept. 1 6

    Google Scholar 

  • Y. Matsushita O. Miyakawa M. Deguchi M. Nishiguchi H. Nyunoya (2002) ArticleTitleCloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1 that interacts with the tomato mosaic virus movement protein J. Exp. Bot 53 1531–1532

    Google Scholar 

  • E. Meurs K. Chong J. Galabru N.S. Thomas I.M. Kerr B.R. Williams A.G. Hovanessian (1990) ArticleTitleMolecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon Cell 62 379–390

    Google Scholar 

  • W. Park J. Li R. Song J. Messing X. Chen (2002) ArticleTitleCARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana Curr. Biol. 12 1484–1495

    Google Scholar 

  • R.C. Patel G.C. Sen (1998) ArticleTitlePACT, a protein activator of the interferon-induced protein kinase, PKR EMBO J 17 4379–4390

    Google Scholar 

  • R.C. Patel P. Stanton N.M. McMillan B.R. Williams G.C. Sen (1995) ArticleTitleThe interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo Proc. Natl. Acad. Sci. USA 92 8283–8287

    Google Scholar 

  • B.J. Reinhart E.G. Weinstein M.W. Rhoades B. Bartel D.P. Bartel (2002) ArticleTitleMicroRNAs in plants Genes Dev. 16 1616–1626

    Google Scholar 

  • J.M. Ryter S.C. Schultz (1998) ArticleTitleMolecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA EMBO J 17 7505–7513

    Google Scholar 

  • C.E. Samuel (1993) ArticleTitleThe eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans J Biol. Chem. 268 7603–7606

    Google Scholar 

  • L.R. Saunders G.N. Barber (2003) ArticleTitleThe dsRNA binding protein family: critical roles, diverse cellular functions FASEB J. 17 961–983

    Google Scholar 

  • S.E. Schauer S.E. Jacobsen D.W. Meinke A. Ray (2002) ArticleTitleDICER-LIKE1: blind men and elephants in Arabidopsis development Trends Plant Sci. 7 487–491

    Google Scholar 

  • M. Seki M. Narusaka A. Kamiya J. Ishida M. Satou T. Sakurai M. Nakajima A. Enju K. Akiyama Y. Oono M. Muramatsu Y. Hayashizaki J. Kawai P. Carninci M. Itoh Y. Ishii T. Arakawa K. Shibata A. Shinagawa K. Shinozaki (2002) ArticleTitleFunctional annotation of a full-length Arabidopsis cDNA collection Science 296 141–145

    Google Scholar 

  • D. St Johnston N.H. Brown J.G. Gall M. Jantsch (1992) ArticleTitleA conserved double-stranded RNA-binding domain Proc. Nat.l Acad. Sci. USA 89 10979–10983

    Google Scholar 

  • H. Tabara E. Yigit H. Siomi C.C. Mello (2002) ArticleTitleThe dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans Cell 109 861–871

    Google Scholar 

  • J.D. Thompson T.J. Gibson F. Plewniak F. Jeanmougin D.G. Higgins (1997) ArticleTitleThe ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res. 25 4876–4882

    Google Scholar 

  • F. Vazquez V. Gasciolli P. Crete H. Vaucheret (2004) ArticleTitleThe nuclear dsRNA binding protein HYL1 is required for microRNA accumulation and plant development, but not posttranscriptional transgene silencing Curr. Biol. 14 346–351

    Google Scholar 

  • P.M. Waterhouse M.B. Wang T. Lough (2001) ArticleTitleGene silencing as an adaptive defence against viruses Nature 411 834–842 Occurrence Handle10.1038/35081168 Occurrence Handle1:CAS:528:DC%2BD3MXksF2gu7Y%3D Occurrence Handle11459066

    Article  CAS  PubMed  Google Scholar 

  • Z. Xie L.K. Johansen A.M. Gustafson K.D. Kasschau A.D. Lellis D. Zilberman S.E. Jacobsen J.C. Carrington (2004) ArticleTitleGenetic and functional diversification of small RNA pathways in plants PLoS Biol. 2 1–11

    Google Scholar 

  • L. Xiong H. Lee M. Ishitani Y. Tanaka B. Stevenson H. Koiwa R.A. Bressan P.M. Hasegawa J.K. Zhu (2002) ArticleTitleRepression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis Proc. Natl. Acad. Sci. USA 99 10899–10904

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, Saiwaicho 3-5-8, Fuchu, Tokyo, 183-8509, Japan

    Akihiro Hiraguri, Riku Itoh, Naoko Kondo, Yasuko Nomura, Daisuke Aizawa, Yusuke Murai & Toshiyuki Fukuhara

  2. Department of Horticultural Sciences, Texas A&M University, College Station, TX, 77843-2133, USA

    Hisashi Koiwa

  3. Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, 305-0074, Japan

    Motoaki Seki & Kazuo Shinozaki

  4. Plant Mutation Exploration Team, Plant Functional Genomics Research Group, RIKEN Genomic Sciences Center, Yokohama, 230-0045, Japan

    Motoaki Seki & Kazuo Shinozaki

Authors
  1. Akihiro Hiraguri
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Riku Itoh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Naoko Kondo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yasuko Nomura
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Aizawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yusuke Murai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hisashi Koiwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Motoaki Seki
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Kazuo Shinozaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Toshiyuki Fukuhara
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiyuki Fukuhara.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hiraguri, A., Itoh, R., Kondo, N. et al. Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol 57, 173–188 (2005). https://doi.org/10.1007/s11103-004-6853-5

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-004-6853-5

Keywords

Search

Navigation