Small RNA-mediated chromatin silencing directed to the 3′ region of the Arabidopsis gene encoding the developmental regulator, FLC

Small RNA-mediated chromatin silencing is well characterized for repeated sequences and transposons, but its role in regulating single-copy endogenous genes is unclear. We have identified two small RNAs (30 and 24 nucleotides) corresponding to the reverse strand 3' to the canonical poly(A) site of FLOWERING LOCUS C(FLC), an Arabidopsis gene encoding a repressor of flowering. Genome searches suggest that these RNAs originate from the FLC locus in a genomic region lacking repeats. The 24-nt small RNA, which is most abundant in developing fruits, is absent in mutants defective in RNA polymerase IVa, RNA-DEPENDENT RNA POLYMERASE 2, and DICER-LIKE 3, components required for RNAi-mediated chromatin silencing. The corresponding genomic region shows histone 3 lysine 9 climethylation, which was reduced in a dc12,3,4 triple mutant. Investigations into the origins of the small RNAs revealed a polymerase IVa-depenclent spliced, antisense transcript covering the 3' FLC region. Mutation of this genomic region by T-DNA insertion led to FLC misexpression and delayed flowering, suggesting that RNAi-mediated chromatin modification is an important component of enclogenous pathways that function to suppress FLC expression.