DNA methylation is an important epigenetic mark in many eukaryotes1-5. In plants, 24-nt small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4) can direct de novo DNA methylation by the methyltransferase DRM22,4-6. Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nt siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that appears to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II, AGO4 and DRM2 in the nucleus. Our results suggest that RDM1 is a component of the RdDM effector complex and may play a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also suggest that although RDM1 and Pol V may function together at some RdDM target sites in the peri-nucleolar siRNA processing center, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

MicroRNAs (miRNAs) are important for plant development and stress responses. However, factors regulating miRNA metabolism are not completely understood. SICKLE (SIC), a proline-rich protein critical for development and abiotic stress tolerance of Arabidopsis, was identified in this study. Loss-of-function sic-1 mutant plants exhibited a serrated, sickle-like leaf margin, reduced height, delayed flowering, and abnormal inflorescence phyllotaxy, which are common characteristics of mutants involved in miRNA biogenesis. The sic-1 mutant plants accumulated lower levels of a subset of miRNAs and transacting siRNAs but higher levels of corresponding primary miRNAs than the WT. The SIC protein colocalizes with the miRNA biogenesis component HYL1 in distinct subnuclear bodies. sic-1 mutant plants also accumulated higher levels of introns from hundreds of loci. In addition, sic-1 mutant plants are hypersensitive to chilling and salt stresses. These results suggest that SIC is a unique factor required for the biogenesis of some miRNAs and degradation of some spliced introns and important for plant development and abiotic stress responses.cold stress | hydroxyproline-rich glycoprotein | intron decay | mRNA stability M icroRNAs (miRNAs) are a class of endogenous small RNAs that function in gene regulation by guiding mRNA cleavage and translational repression and are critical for plant development and stress responses (1-9). The core components involved in miRNA biogenesis have been identified in plants. RNA polymerase II transcribes MIR genes; a 5′ 7-methyl guanosine cap and a 3′ poly(A) tail are added to produce primary miRNA (primiRNA) transcripts, which form imperfect stem-loop secondary structures by Watson-Crick base pairing between self-complementary foldback regions. In the Arabidopsis nucleus, the stemloop structure of the pri-miRNA is processed by the RNase III enzyme DICER-LIKE1 (DCL1) to produce a pre-miRNA, which is further processed to generate a 21-nt-long miRNA/miRNA* duplex. For accurate dicing, DCL1 requires the help of HYPO-NASTIC LEAVES1 (HYL1, a dsRNA-binding protein) and SERRATE (SE, a C2H2zinc-finger protein) (10). The HUA EN-HANCER 1 (HEN1) methyltransferase catalyzes 2'-O-methylation of the ribose sugar in the 3′ termini of miRNA/miRNA* duplexes (11). HASTY (HST), a homolog of mammalian EXPORTIN 5, helps export methylated miRNA/miRNA* duplexes from the nucleus to the cytosol (12). The mature miRNA is incorporated into ARGONAUTE1 (AGO1), forming an RNA-induced silencing complex, which scans for miRNA-complementary mRNAs and directs the cleavage or translational repression of the target mRNAs (1). miR173 and miRNA390 direct the biogenesis of transacting siRNAs (ta-siRNAs). Noncoding transcripts from TRANS-ACT-ING siRNA genes (TAS) are cleaved by the miRNA-containing AGO1/AGO7 complex (13, 14). The cleaved transcripts are converted into dsRNA by RDR6, and these dsRNAs are processed by DCL4 to yield ∼21-nt ta-siRNAs. Like miRNAs, tasiRNAs negatively regulate gene expression posttranscriptionally (15-18).N...