PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 105; no. 8; pp. 3145 - 3150
• Main Authors: Mosher, Rebecca A, Schwach, Frank, Studholme, David, Baulcombe, David C
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 26.02.2008; National Acad Sciences
• Subjects: Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - physiology; Arabidopsis thaliana; Base Sequence; Biological Sciences; Blotting, Northern; Chromatin; Chromatin Assembly and Disassembly - physiology; Deoxyribonucleic acid; DNA; DNA Methylation; DNA-directed RNA polymerase; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - physiology; Epigenetics; Flowers & plants; Gene loci; gene silencing; Genetic loci; Genomes; loci; Methylation; Molecular Sequence Data; Mutation; Polls; Protein isoforms; Protein Subunits - genetics; RNA; RNA interference; RNA polymerase; RNA, Small Interfering - biosynthesis; RNA, Small Interfering - genetics; Sequence Analysis, DNA; Small interfering RNA; Transposons
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0709632105

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.