Loss of RNA–Dependent RNA Polymerase 2 (RDR2) Function Causes Widespread and Unexpected Changes in the Expression of Transposons, Genes, and 24-nt Small RNAs

Yi Jia,Dan Nettleton,Michael J Scanlon,Patrick S Schnable,Damon R Lisch,Kazuhiro Ohtsu,Joseph R Ecker

doi:10.1371/journal.pgen.1000737

Yi Jia, Dan Nettleton + Show 5 more

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https://doi.org/10.1371/journal.pgen.1000737

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Abstract

Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA–dependent RNA polymerase 2 (RDR2) is a component of the RNA–directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA–seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2–sensitive and RDR2–resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.

Highlights

Repetitive sequences, including transposable elements (TEs) and tandem repeats, comprise a substantial fraction of many eukaryotic genomes
shoot apical meristems (SAMs) plus leaves at plastochron 0 (P0) and P1 stages were collected via Laser Capture Microdissection (LCM) followed by RNA extraction and amplification according to our previously published procedures [15]
A pooled RNA sample from twelve mutant SAMs and a pooled RNA sample from ten non-mutant SAMs were subjected to Illumina/Solexa sequencing. 2.8 million reads from mop1 mutants and 4.1 million reads from nonmutants could be uniquely mapped to the Maize Genome Sequencing Project’s (MGSP’s) B73 reference genome [16]

Summary

Introduction

Repetitive sequences, including transposable elements (TEs) and tandem repeats, comprise a substantial fraction of many eukaryotic genomes. To protect genome integrity TEs are typically transcriptionally silenced via epigenetic mechanisms [1,2,3]. Various RDRs (e.g. RDR1, RDR2 and RDR6) are functional in different siRNA pathways and most siRNAs are biosynthesized from the heterochromatic loci, a process that generally requires RDR2 and DICER-LIKE3 (DCL3)[4]. In Arabidopsis, these heterochromatic 24 nt siRNAs, predominate the small RNA population. These siRNAs recruit chromatin-targeted RNA silencing components to form transcriptionally silent heterochromatin, which is derived mainly from TEs and tandem repeats [5], by cytosine methylation and various histone modifications, such as histone deacetylation and histone H3 lysine 9 dimethylation. RDR2 is a key component of the chromatin-targeted RNA silencing process, which is called the RNA dependent DNA Methylation (RdDM) pathway

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