Full-length autonomous transposable elements are preferentially targeted by expression-dependent forms of RNA-directed DNA methylation

Abstract

BackgroundChromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a “non-canonical” form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM.ResultsWe performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs.ConclusionsExpression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1032-y) contains supplementary material, which is available to authorized users.

Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

“…When the solo Tto LTR was transformed into the iTto line ( Figure 2E), increased CHG and CHH methylation occurs compared with either the solo Tto LTR Dpro transgene transformed into iTto (identity-based silencing only) ( Figure 2E) or the solo Tto LTR transgene transformed into wild-type Col (expression-dependent silencing only) ( Figure 2C). This is in line with the observation that transcriptionally active endogenous TEs are the strongest targets of RdDM (Panda et al, 2016), since both identity-based and expression-dependent mechanisms simultaneously target them.…”

“…Additionally, Dicer-independent small RNAs have been shown to function in RdDM (Ye et al, 2016;Yang et al, 2016). These three noncanonical RdDM pathways have only limited roles in wild-type plants that lack TE activity but play a considerable role in silencing transcriptionally reactivated TEs (Panda et al, 2016). Consequently, each of these noncanonical RdDM pathways has been hypothesized to function in the original establishment of TE silencing.…”

“…In addition to TEs, the non-protein-coding single-copy TAS loci are targets of this pathway in wild-type TE-silenced plants Panda et al, 2016). Pol II mRNAs can also be directly degraded by DCL3 into 24-nucleotide siRNAs, which target RdDM in a pathway termed DCL3-RdDM (Panda et al, 2016). Additionally, Dicer-independent small RNAs have been shown to function in RdDM (Ye et al, 2016;Yang et al, 2016).…”

“…These 21-to 22-nucleotide siRNAs guide RdDM through their incorporation into AGO6 (McCue et al, 2015). In addition to TEs, the non-protein-coding single-copy TAS loci are targets of this pathway in wild-type TE-silenced plants Panda et al, 2016). Pol II mRNAs can also be directly degraded by DCL3 into 24-nucleotide siRNAs, which target RdDM in a pathway termed DCL3-RdDM (Panda et al, 2016).…”

“…For example, RDR6-RdDM functions on transcriptionally active TEs through the production of 21-to 22-nucleotide siRNAs directly generated from the degradation of TE mRNAs (Nuthikattu et al, 2013;Panda et al, 2016). These 21-to 22-nucleotide siRNAs guide RdDM through their incorporation into AGO6 (McCue et al, 2015).…”