A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes.

Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA-auxin response factor (tasiR-ARF), that shares a 21- and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3/ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs.

[1]  J. Nemhauser,et al.  ETTIN patterns the Arabidopsis floral meristem and reproductive organs. , 1997, Development.

[2]  Yang Li,et al.  Computational identification of novel family members of microRNA genes in Arabidopsis thaliana and Oryza sativa. , 2005, Acta biochimica et biophysica Sinica.

[3]  S. Jayasena,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[4]  Scott A. Givan,et al.  ASRP: the Arabidopsis Small RNA Project Database , 2004, Nucleic Acids Res..

[5]  Thomas Tuschl,et al.  Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. , 2004, RNA.

[6]  V. Ambros,et al.  Role of MicroRNAs in Plant and Animal Development , 2003, Science.

[7]  A. Mccarthy.  Development , 1996, Current Opinion in Neurobiology.

[8]  D. Bartel,et al.  Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. , 2004, Molecular cell.

[9]  O. Voinnet,et al.  In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. , 2004, Genes & development.

[10]  J. Krol,et al.  Structural Features of MicroRNA (miRNA) Precursors and Their Relevance to miRNA Biogenesis and Small Interfering RNA/Short Hairpin RNA Design* , 2004, Journal of Biological Chemistry.

[11]  Junjie Li,et al.  HUA1, a Regulator of Stamen and Carpel Identities in Arabidopsis, Codes for a Nuclear RNA Binding Protein , 2001, The Plant Cell Online.

[12]  Gi-Ho Sung,et al.  Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana , 2004, Nature Genetics.

[13]  S. Hake.  MicroRNAs: A Role in Plant Development , 2003, Current Biology.

[14]  H. Vaucheret,et al.  MicroRNAs: something important between the genes. , 2004, Current opinion in plant biology.

[15]  Terry Gaasterland,et al.  Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets , 2004, Genome Biology.

[16]  Franck Vazquez,et al.  Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. , 2004, Molecular cell.

[17]  Javier F. Palatnik,et al.  Control of leaf morphogenesis by microRNAs , 2003, Nature.

[18]  Adam M. Gustafson,et al.  microRNA-Directed Phasing during Trans-Acting siRNA Biogenesis in Plants , 2005, Cell.

[19]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[20]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[21]  Charlie Chang,et al.  Functional Genomic Analysis of the AUXIN RESPONSE FACTOR Gene Family Members in Arabidopsis thaliana: Unique and Overlapping Functions of ARF7 and ARF19w⃞ , 2005, The Plant Cell Online.

[22]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[23]  T. Tuschl,et al.  Identification of Tissue-Specific MicroRNAs from Mouse , 2002, Current Biology.

[24]  J. Botto,et al.  The plant cell , 2007, Plant Molecular Biology Reporter.

[25]  Joseph M. Dale,et al.  Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome , 2003, Science.

[26]  Hajime Sakai,et al.  Regulation of Flowering Time and Floral Organ Identity by a MicroRNA and Its APETALA2-Like Target Genes Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.016238. , 2003, The Plant Cell Online.

[27]  Liang-Hu Qu,et al.  Identification of 20 microRNAs from Oryza sativa. , 2004, Nucleic acids research.

[28]  V. Colot,et al.  Arabidopsis epigenetics: when RNA meets chromatin. , 2005, Current opinion in plant biology.

[29]  B. Reinhart,et al.  Prediction of Plant MicroRNA Targets , 2002, Cell.

[30]  C. Kidner,et al.  The developmental role of microRNA in plants. , 2005, Current opinion in plant biology.

[31]  A. Adai,et al.  Computational prediction of miRNAs in Arabidopsis thaliana. , 2005, Genome research.

[32]  L. Lim,et al.  An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans , 2001, Science.

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

[34]  Lin He,et al.  MicroRNAs: small RNAs with a big role in gene regulation , 2004, Nature reviews genetics.

[35]  John L. Bowman,et al.  Gene regulation: Ancient microRNA target sequences in plants , 2004, Nature.

[36]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[37]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[38]  P. Rouzé,et al.  Detection of 91 potential conserved plant microRNAs in Arabidopsis thaliana and Oryza sativa identifies important target genes. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  W. McDonald,et al.  Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe. , 2000, BioTechniques.

[40]  C. Burge,et al.  The microRNAs of Caenorhabditis elegans. , 2003, Genes & development.

[41]  Gang Wu,et al.  SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. , 2004, Genes & development.

[42]  Xuemei Chen,et al.  A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development , 2004, Science.

[43]  J. Nemhauser,et al.  Auxin and ETTIN in Arabidopsis gynoecium morphogenesis. , 2000, Development.

[44]  R. Sunkar,et al.  Novel and Stress-Regulated MicroRNAs and Other Small RNAs from Arabidopsis , 2004, The Plant Cell Online.

[45]  B. Cullen,et al.  Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. , 2004, RNA.

[46]  R. Martienssen,et al.  The role of RNA interference in heterochromatic silencing , 2004, Nature.