| Eukaryotes use small RNAs (sRNAs) to regulate gene expression and silence invasive sequences. Arabidopsis encodes four Dicer-like ribonucleases (DCLs) that process double-stranded RNAs (dsRNAs) into sRNAs. DCL1 processes microRNAs (miRNAs) from imperfectly paired hairpin transcripts miRNAs act to regulate developmental gene expression. DCL2, DCL3, and DCL4 function redundantly to process small interfering RNAs (siRNAs) from perfect dsRNA duplexes produced from transposons and repeats. siRNAs trigger degradation of complementary messenger RNAs, and initiate cytosine methylation and transcriptional silencing on matching genomic DNA sequences. We characterized dcl mutants for effects on cytosine methylation of the endogenous duplicated phosphoribosylanthranilate isomerase (PAI) genes, which include a transcribed inverted repeat duplication that can produce hairpin dsRNA transcripts. We show that a dcl2 dcl3 dcl4 mutant is depleted for PAI sRNAs and fails to maintain full cytosine methylation on the PAI genes. PAI demethylation does not occur through a defect in the known sRNA-dependent initiation pathway. Instead, it occurs through impaired maintenance of methylation at the lysine 9 position of histone H3 tails in PAI-associated nucelosomes, which causes reduced PAI cytosine methylation as a secondary effect. We propose that this sRNA-dependent histone methylation pathway reinforces internal silencing at sequences like the PAI inverted repeat that are transcribed from an outside promoter. We also show that mutations in DCL1 and two associated miRNA processing factors cause reduced PAI cytosine methylation. These results are consistent with a pathway where the DCL1 processing complex trims unpaired sequences from the PAI dsRNA precursor, analogously to early steps in miRNA biogenesis; the trimmed dsRNAs are then cleaved into PAI sRNAs by the combined activity of DCL2, DCL3, and DCL4. Our results suggest that other unusual dsRNA species that are neither miRNA transcripts nor perfect duplexes might require activities from both miRNA and siRNA processing pathways to produce sRNAs. |
