Distinct and concurrent pathways of Pol II‐ and Pol IV‐dependent siRNA biogenesis at a repetitive trans‐silencer locus in Arabidopsis thaliana

Abstract

SUMMARYShort interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA 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 siRNA 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 siRNAs 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 siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

Battles and hijacks: noncoding transcription in plants

“…These siRNAs trigger DNA methylation only at the homologous sequence (in red) and partial target silencing. Model based on [92]. Distinct and concurrent pathways of Pol II-and Pol IVdependent siRNA biogenesis Transcriptional regulatory regions can be controlled by siRNA-mediated RdDM.…”

“…In Arabidopsis, several endogenous inverted repeat (IR) regions giving rise to hairpinderived siRNAs have been identified [91]. An artificial system was designed to dissect distinct Pol II-and Pol IV-dependent pathways of siRNA biogenesis ( Figure 3A) [92]. Arabidopsis plants were transformed with a construct bearing a GFP gene downstream from a 35S minimal promoter and an enhancer sequence (the target locus).…”

An Rtf2 Domain-Containing Protein Influences Pre-mRNA Splicing and Is Essential for Embryonic Development in Arabidopsis thaliana

“…GSE63238. The raw reads and the processed dataset for the wild-type methylome (Sasaki et al 2014) can be downloaded from NCBI GEO under accession no. GSE47453.…”

“…DNA methylation analysis of the target enhancer by bisulfite sequencing was carried out as described previously (Kanno et al 2008;Daxinger et al 2009;Sasaki et al , 2014. Genomic DNA was isolated using a Plant Genomic DNA Purification kit (GeneMark, Taiwan).…”

“…The silencer locus, S, contains an inverted DNA repeat of distal enhancer sequences (black arrowheads corresponding to thick black bar in T) that is transcribed by RNA polymerase II (Pol II) from a constitutive viral promoter (35S). The resulting hairpin RNA is processed by DICER-LIKE3 (DCL3) to produce 24-nt small interfering RNAs that induce Pol V complex-mediated de novo DNA methylation (blue "m") of the target enhancer region, leading to transcriptional silencing of GFP expression (Kanno et al 2008Sasaki et al 2014). Given a control sample and a mutant sample, the preference of an intron retention event was measured using a x 2 test for goodness of fit by comparing the depths of the intron in the two samples taking the depths of neighboring exons in the two samples as the background.…”