Abstract
In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.
Highlights
In plants, transposable elements, transgenes, repetitive sequences and endogenous genes can be transcriptionally silenced by RNA-directed DNA methylation (Matzke and Mosher, 2014; Pikaard et al, 2013; Wierzbicki, 2012; Zhang and Zhu, 2011)
Based on sequence alignments and sequence motifs shared by polymerase IV (Pol IV)/RNA-dependent RNA polymerase 2 (RDR2)-dependent RNAs (P4R2 RNAs) and siRNAs, we propose that siRNAs are typically generated by a single internal DICER-like 3 (DCL3) cleavage event whose position is measured from either end of a short P4R2 RNA
This indicates that Pol IV and RDR2 are each required for the biogenesis of the longer RNAs, paralleling their requirement for 24 and 23 nt siRNA biogenesis
Summary
Transposable elements, transgenes, repetitive sequences and endogenous genes can be transcriptionally silenced by RNA-directed DNA methylation (Matzke and Mosher, 2014; Pikaard et al, 2013; Wierzbicki, 2012; Zhang and Zhu, 2011). In this process, 24 nucleotide small interfering RNAs (24 nt siRNAs) bound to an Argonaute family protein, primarily Argonaute 4 (AGO4; [Zilberman et al, 2003]), guide the cytosine methylation and histone modification of corresponding DNA sequences, leading to chromatin states that are refractive to transcription by RNA polymerases I, II, or III (Figure 1).
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