A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis

RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5' -adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured
with 5' -monophosphates and 3' -misincorporated nucleotides. The 3' -misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a ‘‘one precursor, one siRNA’’ model for the biogenesis of 24-nt siRNAs in Arabidopsis.