Distinct and concurrent pathways of P ol II ‐ and Pol IV ‐dependent si RNA biogenesis at a repetitive trans ‐silencer locus in A rabidopsis thaliana

Summary Short interfering RNA s (si RNA s) homologous to transcriptional regulatory regions can induce RNA ‐directed DNA methylation ( R d DM ) and transcriptional gene silencing ( TGS ) of target genes. In our system, si RNA s are produced by transcribing an inverted DNA repeat ( IR ) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into si RNA s of 21–24 nt. Primarily 24‐nt si RNA s trigger R d DM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed si RNA accumulation from two different structural forms of a trans ‐silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase  II ( P ol  II )‐ and P ol  IV ‐dependent pathways of si RNA biogenesis. At the original silencer locus, P ol‐ II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant si RNA s of 21–24 nt. A silencer variant lacking the 35S promoter revealed a normally masked P ol  IV ‐dependent pathway that produces low levels of 24‐nt si RNA s from the tandem repeats. Both pathways operate concurrently at the original silencer locus. si RNA s accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing si RNA s revealed the preferential accumulation of si RNA s at GC ‐rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and si RNA accumulation, our results highlight the complexity of si RNA biogenesis at repetitive loci and show that P ol  II and P ol  IV use different promoters to transcribe the same template.