Distinct and concurrent pathways of Pol II- and Pol IV-dependent si RNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

Short interfering RNAs (si RNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation ( Rd DM) and transcriptional gene silencing ( TGS) of target genes. In our system, si RNAs 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 RNAs of 21-24 nt. Primarily 24-nt si RNAs trigger Rd 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 ( Pol II)- and Pol IV-dependent pathways of si RNA biogenesis. At the original silencer locus, Pol- II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant si RNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt si RNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. si RNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing si RNAs revealed the preferential accumulation of si RNAs 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 Pol II and Pol IV use different promoters to transcribe the same template. [ABSTRACT FROM AUTHOR]