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

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.

[1]  R. Bressan,et al.  Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  B. Williams,et al.  The interferon-inducible double-stranded RNA-activated protein kinase self-associates in vitro and in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Mathews,et al.  Interactions between double-stranded RNA regulators and the protein kinase DAI , 1992, Molecular and cellular biology.

[4]  Xiaodong Wang,et al.  R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway , 2003, Science.

[5]  G. Hannon.  RNA interference : RNA , 2002 .

[6]  H. Nyunoya,et al.  Cloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1 that interacts with the tomato mosaic virus movement protein. , 2002, Journal of experimental botany.

[7]  H. Vaucheret,et al.  Arabidopsis HEN1 A Genetic Link between Endogenous miRNA Controlling Development and siRNA Controlling Transgene Silencing and Virus Resistance , 2003, Current Biology.

[8]  L. Excoffier,et al.  Modern Humans Did Not Admix with Neanderthals during Their Range Expansion into Europe , 2004, PLoS biology.

[9]  G. Barber,et al.  The dsRNA binding protein family: critical roles, diverse cellular functions , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[10]  H. Vaucheret,et al.  The Nuclear dsRNA Binding Protein HYL1 Is Required for MicroRNA Accumulation and Plant Development, but Not Posttranscriptional Transgene Silencing , 2004, Current Biology.

[11]  P. Lefebvre,et al.  PLZF is a negative regulator of retinoic acid receptor transcriptional activity , 2003, Nuclear receptor.

[12]  David P. Bartel,et al.  MicroRNAs: At the Root of Plant Development?1 , 2003, Plant Physiology.

[13]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[14]  C. Mello,et al.  The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans , 2002, Cell.

[15]  K. Akiyama,et al.  Functional Annotation of a Full-Length Arabidopsis cDNA Collection , 2002, Science.

[16]  M. Mathews,et al.  Proteins binding to duplexed RNA: one motif, multiple functions. , 2000, Trends in biochemical sciences.

[17]  S C Schultz,et al.  Molecular basis of double‐stranded RNA‐protein interactions: structure of a dsRNA‐binding domain complexed with dsRNA , 1998, The EMBO journal.

[18]  B. Reinhart,et al.  MicroRNAs in plants. , 2002, Genes & development.

[19]  D. St Johnston,et al.  A conserved double-stranded RNA-binding domain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Holmgren,et al.  Science on the Rise in Developing Countries , 2004, PLoS biology.

[21]  A. Mushegian,et al.  Short Integuments1/suspensor1/carpel Factory, a Dicer Homolog, Is a Maternal Effect Gene Required for Embryo Development in Arabidopsis1 , 2002, Plant Physiology.

[22]  C. Samuel,et al.  The eIF-2 alpha protein kinases, regulators of translation in eukaryotes from yeasts to humans. , 1993, The Journal of biological chemistry.

[23]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[24]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[25]  Jianhua Zhu,et al.  C-terminal domain phosphatase-like family members (AtCPLs) differentially regulate Arabidopsis thaliana abiotic stress signaling, growth, and development , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[26]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[27]  Animesh Ray,et al.  DICER-LIKE1: blind men and elephants in Arabidopsis development. , 2002, Trends in plant science.

[28]  J. Messing,et al.  CARPEL FACTORY, a Dicer Homolog, and HEN1, a Novel Protein, Act in microRNA Metabolism in Arabidopsis thaliana , 2002, Current Biology.

[29]  N. Sonenberg,et al.  Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[30]  G. Sen,et al.  PACT, a protein activator of the interferon‐induced protein kinase, PKR , 1998, The EMBO journal.

[31]  A. Pasquinelli,et al.  Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing , 2001, Cell.

[32]  Gregory J. Hannon,et al.  Insight Review Articles , 2022 .

[33]  I. Kerr,et al.  Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon , 1990, Cell.

[34]  R. Kaufman,et al.  Double-stranded RNA-activated protein kinase mediates virus-induced apoptosis: a new role for an old actor. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Ming-Bo Wang,et al.  Gene silencing as an adaptive defence against viruses , 2001, Nature.

[36]  Adam M. Gustafson,et al.  Genetic and Functional Diversification of Small RNA Pathways in Plants , 2004, PLoS biology.

[37]  N. Fedoroff,et al.  A Mutation in the Arabidopsis HYL1 Gene Encoding a dsRNA Binding Protein Affects Responses to Abscisic Acid, Auxin, and Cytokinin , 2000, Plant Cell.

[38]  S. Jacobsen,et al.  Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. , 1999, Development.

[39]  N. Fedoroff,et al.  The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.