title: Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis.
abstract: In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.
authors.affiliation: Howard Hughes Medical Institute, Indiana University, Bloomington, United States.|Department of Biology, Indiana University, Bloomington, United States.|Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States.
authors.forename: Todd
authors.initials: T
authors.lastname: Blevins
authors.affiliation: Center for Genomics and Bioinformatics, Indiana University, Bloomington, United States.|School of Informatics and Computing, Indiana University, Bloomington, United States.
authors.forename: Ram
authors.initials: R
authors.lastname: Podicheti
authors.affiliation: Department of Biology, Indiana University, Bloomington, United States.|Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States.
authors.forename: Vibhor
authors.initials: V
authors.lastname: Mishra
authors.affiliation: Department of Biology, Indiana University, Bloomington, United States.|Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States.
authors.forename: Michelle
authors.initials: M
authors.lastname: Marasco
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Doug
authors.initials: D
authors.lastname: Rusch
authors.affiliation: School of Informatics and Computing, Indiana University, Bloomington, United States.
authors.forename: Haixu
authors.initials: H
authors.lastname: Tang
authors.affiliation: Howard Hughes Medical Institute, Indiana University, Bloomington, United States.|Department of Biology, Indiana University, Bloomington, United States.|Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, United States.
authors.forename: Craig S
authors.initials: CS
authors.lastname: Pikaard
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D019175:DNA Methylation; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D034741:RNA, Small Interfering / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D043244:Ribonuclease III / Q000172:deficiency / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D034741:RNA, Small Interfering; C499181:RNA polymerase IV, Arabidopsis; C519655:RDR2 protein, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; C519654:DCL3 protein, Arabidopsis; D043244:Ribonuclease III
keywords: RNA interference; RNA silencing; RNA-directed DNA methylation; arabidopsis; chromosomes; epigenetics; genes; noncoding RNA; transcription