A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis.

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3' fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5' cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

[1]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[2]  P. Waterhouse,et al.  A Branched Pathway for Transgene-Induced RNA Silencing in Plants , 2002, Current Biology.

[3]  E. Sontheimer,et al.  A Dicer-2-Dependent 80S Complex Cleaves Targeted mRNAs during RNAi in Drosophila , 2004, Cell.

[4]  D. Baulcombe,et al.  Spreading of RNA Targeting and DNA Methylation in RNA Silencing Requires Transcription of the Target Gene and a Putative RNA-Dependent RNA Polymerase Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010480. , 2002, The Plant Cell Online.

[5]  Gang Wu,et al.  Nuclear processing and export of microRNAs in Arabidopsis. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[8]  Christophe Ritzenthaler,et al.  Transitivity‐dependent and ‐independent cell‐to‐cell movement of RNA silencing , 2003, The EMBO journal.

[9]  M. Gorovsky,et al.  Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE). , 1993, Nucleic acids research.

[10]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[11]  C. Pikaard,et al.  Plant Nuclear RNA Polymerase IV Mediates siRNA and DNA Methylation-Dependent Heterochromatin Formation , 2005, Cell.

[12]  Tamas Dalmay,et al.  An RNA-Dependent RNA Polymerase Gene in Arabidopsis Is Required for Posttranscriptional Gene Silencing Mediated by a Transgene but Not by a Virus , 2000, Cell.

[13]  W. Filipowicz,et al.  Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP , 2002, The EMBO journal.

[14]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[15]  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.

[16]  Xuemei Chen,et al.  Methylation as a Crucial Step in Plant microRNA Biogenesis , 2005, Science.

[17]  V. Walbot,et al.  Initiation of silencing of maize MuDR/Mu transposable elements. , 2003, The Plant journal : for cell and molecular biology.

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

[19]  B. Reinhart,et al.  A biochemical framework for RNA silencing in plants. , 2003, Genes & development.

[20]  Eric Westhof,et al.  Single Processing Center Models for Human Dicer and Bacterial RNase III , 2004, Cell.

[21]  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.

[22]  H. Goodman,et al.  Uridine Addition After MicroRNA-Directed Cleavage , 2004, Science.

[23]  Kazuo Shinozaki,et al.  Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in Arabidopsis thaliana , 2004, Plant Molecular Biology.

[24]  Xiaofeng Cao,et al.  ARGONAUTE4 Control of Locus-Specific siRNA Accumulation and DNA and Histone Methylation , 2003, Science.

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

[26]  Ronald H. A. Plasterk,et al.  Transposon silencing in the Caenorhabditis elegans germ line by natural RNAi , 2003, Nature.

[27]  H. L. Sänger,et al.  RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties. , 1993, The Journal of biological chemistry.

[28]  P. Zamore,et al.  A Protein Sensor for siRNA Asymmetry , 2004, Science.

[29]  M. Barton,et al.  MicroRNA binding sites in Arabidopsis class III HD-ZIP mRNAs are required for methylation of the template chromosome. , 2004, Developmental cell.

[30]  A. Hamilton,et al.  Potato Virus X Amplicons in Arabidopsis Mediate Genetic and Epigenetic Gene Silencing , 2000, Plant Cell.

[31]  V. Ambros,et al.  MicroRNAs and Other Tiny Endogenous RNAs in C. elegans , 2003, Current Biology.

[32]  M. Carmell,et al.  Posttranscriptional Gene Silencing in Plants , 2006 .

[33]  R. Amasino,et al.  siRNAs targeting an intronic transposon in the regulation of natural flowering behavior in Arabidopsis. , 2004, Genes & development.

[34]  A. Djikeng,et al.  RNA interference in Trypanosoma brucei: cloning of small interfering RNAs provides evidence for retroposon-derived 24-26-nucleotide RNAs. , 2001, RNA.

[35]  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.

[36]  P. Waterhouse,et al.  Posttranscriptional Gene Silencing Is Not Compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) Mutant, a Homolog of Dicer-1 from Drosophila , 2003, Current Biology.

[37]  D. Marks,et al.  The small RNA profile during Drosophila melanogaster development. , 2003, Developmental cell.

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

[39]  Edwards Allen,et al.  P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. , 2003, Developmental cell.

[40]  G. Macino,et al.  RNAi-dependent and RNAi-independent mechanisms contribute to the silencing of RIPed sequences in Neurospora crassa. , 2004, Nucleic acids research.

[41]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[42]  Karen S. Osmont,et al.  A database analysis method identifies an endogenous trans-acting short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and ARF4 genes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[44]  L. Maquat.  Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics , 2004, Nature Reviews Molecular Cell Biology.

[45]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[46]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[47]  Olivier Voinnet,et al.  Two classes of short interfering RNA in RNA silencing , 2002, The EMBO journal.

[48]  H. Vaucheret,et al.  Geminivirus VIGS of endogenous genes requires SGS2/SDE1 and SGS3 and defines a new branch in the genetic pathway for silencing in plants. , 2004, The Plant journal : for cell and molecular biology.

[49]  R. Pictet,et al.  Messenger RNA deadenylylation precedes decapping in mammalian cells. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

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

[51]  Phillip D Zamore,et al.  Perspective: machines for RNAi. , 2005, Genes & development.

[52]  M. A. Rector,et al.  Endogenous and Silencing-Associated Small RNAs in Plants Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003210. , 2002, The Plant Cell Online.

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

[54]  D. Baulcombe,et al.  RNA Polymerase IV Directs Silencing of Endogenous DNA , 2005, Science.

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

[56]  E. Sontheimer,et al.  Distinct Roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA Silencing Pathways , 2004, Cell.

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

[58]  D. Headon,et al.  A Link Between mRNA Turnover and RNA Interference in Arabidopsis , 2004, Science.

[59]  Philippe Mourrain,et al.  Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance , 2000, Cell.

[60]  A. Aravin,et al.  Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline , 2001, Current Biology.

[61]  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.

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

[63]  Alex Bateman,et al.  The SGS3 protein involved in PTGS finds a family , 2002, BMC Bioinformatics.

[64]  Wei Ge,et al.  Synthetic shRNAs as potent RNAi triggers , 2005, Nature Biotechnology.

[65]  P. Green,et al.  AtXRN4 degrades mRNA in Arabidopsis and its substrates include selected miRNA targets. , 2004, Molecular cell.

[66]  Frédérique Bitton,et al.  Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis , 2004, The Plant Cell Online.

[67]  J. Rossi,et al.  Uncoupling of RNAi from active translation in mammalian cells. , 2005, RNA.