A Pol V–Mediated Silencing, Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA

Julien Douet,Sylvette Tourmente,Sylvie Tutois

doi:10.1371/journal.pgen.1000690

Abstract

The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA–directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA–Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA–encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V–loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM–independent and comes specifically at chromosome 4, in addition to the RdDM pathway.

Highlights

The plant-specific RNA polymerases firstly named Pol IVa and Pol IVb and referred as Pol IV and Pol V [1], contribute to short interfering RNAs (siRNAs) production and are essential to RNA-directed DNA methylation (RdDM) [2,3,4,5,6]
Previous results have shown that 5S rRNA–encoding DNA (rDNA) is subject to a variety of overlapping regulation pathways, such as the limiting amount of TFIIIA (TRANSCRIPTION FACTOR IIIA; 5S rDNA specific) [30], the methylation-independent MOM1 pathway [31], the DDM1/MET1- pathway [30,39], as well as the Pol IV/Pol V RNA–directed DNA methylation (RdDM) [4,24,39]
V on 5S rDNA was previously demonstrated on the basis of DNA hypomethylation, decrease of 5S small RNA accumulation and chromatin decompaction [4,24,39], the relative impact of Pol IV and Pol V, their potential selective action on the different 5S arrays and the consequence of their mutation on 5S rDNA silencing of each 5S array were unknown

Summary

Introduction

The plant-specific RNA polymerases firstly named Pol IVa and Pol IVb and referred as Pol IV and Pol V [1], contribute to siRNA production and are essential to RNA-directed DNA methylation (RdDM) [2,3,4,5,6]. In current models of RdDM silencing pathway [7,8,9,10], Pol IV is speculated to produce single-stranded RNA transcripts from heterochromatic repeated regions. These transcripts, converted onto double-stranded RNAs by RDR2 (RNA-DEPENDENT RNA POLYMERASE 2), are processed into siRNA duplexes by DCL3 (DICER-LIKE 3) [11,12,13,14]. Pol V and DRD1 (DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1) were found to be required to mediate production of non-coding transcripts which are necessary for steps downstream of siRNA biogenesis [1]. Recent evidence suggests that siRNAs/AGO4 complexes bind to Pol V transcripts, guiding the de novo DNA methyltransferase DRM2 [16] and histone modifying complexes to the target loci [17,18]

Methods

Results

Conclusion