Transcriptional gene silencing (TGS) of transgenes by promoter-related RNAs has been known for more than a decade. However, the effectiveness and efficiency of silencing of endogenes by single-stranded and inverted repeat (IR) RNA/silencers remain unclear. Here, we demonstrated that a single-stranded antisense (AS) silencer targeting the promoter region can efficiently silence four Arabidopsis endogenes, with comparable efficiency to an IR silencer. In the case of Too Many Mouths (TMM), single-stranded silencers generated mainly 24 nt small RNAs (smRNAs), whereas IR silencers produced a higher proportion of 21-23 nt smRNAs. Heavy CG, CHG and CHH methylations were detected on the TMM promoter in silenced plant lines. We also demonstrated that the silencing and DNA methylation of the TMM promoter was dependent on the presence of the silencer. Chromatin immunoprecipitation (ChIP) assays showed that DNA methylation was accompanied by formation of repressive chromatin structures. Our results suggest that single-stranded silencer transcripts are converted to double-stranded RNA to enter the RdRM (RNA-directed DNA methylation) pathway for TGS of endogenes.
Keywords: RNA-directed DNA methylation; Single-stranded RNA; Small RNAs; Too Many Mouths; Transcriptional gene silencing.