Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genomewide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.Transposable elements (TEs) constitute large percentages of both animal and plant genomes. TEs are major targets of multiple endogenous gene-silencing pathways that act to limit their expression and ability to generate new insertions and mutations (for review, see Girard and Hannon, 2008). To study TE silencing, the process has been divided into three distinct mechanisms: the de novo initiation/triggering of silencing, the corrective reestablishment of silencing of TEs that were recently transcriptionally reactivated, and the epigenetic maintenance of TE silencing.In the Arabidopsis (Arabidopsis thaliana) genome, nearly all TEs are found in a transcriptionally silenced state (Lippman et al., 2004). This transcriptional gene silencing is maintained by symmetrical DNA methylation, which is propagated through mitotic cell divisions (for review, see Law and Jacobsen, 2010). In contrast to animals, plants do not erase the DNA methylation patterns of their gametes; therefore, CG and CHG (where H = A, T, or C) symmetrical DNA methylation patterns established in one generation are inherited and act to maintain TE silencing in the next generation through a process termed transgenerational epigenetic inheritance (Mathieu et al., 2007;Becker et al., 2011). In addition to the maintenance of symmetrical methylation, methylation of TEs is continually reinforced through a process of ...

Plant small interfering RNAs (siRNAs) communicate from cell to cell and travel long distances through the vasculature. However, siRNA movement into germ cells has remained controversial, and has gained interest because the terminally differentiated pollen vegetative nurse cell surrounding the sperm cells undergoes a programmed heterochromatin decondensation and transcriptional reactivation of transposable elements (TEs). Transcription of TEs leads to their post-transcriptional degradation into siRNAs, and it has been proposed that the purpose of this TE reactivation is to generate and load TE siRNAs into the sperm cells. Here, we identify the molecular pathway of TE siRNA production in the pollen grain and demonstrate that siRNAs produced from pollen vegetative cell transcripts can silence TE reporters in the sperm cells. Our data demonstrates that TE siRNAs act non-cell-autonomously, inhibiting TE activity in the germ cells and potentially the next generation.

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.

Transposable elements (TEs) generate mutations and chromosomal instability when active. To repress TE activity, eukaryotic cells evolved mechanisms to both degrade TE mRNAs into small interfering RNAs (siRNAs) and modify TE chromatin to epigenetically inhibit transcription. Since the populations of small RNAs that participate in TE post-transcriptional regulation differ from those that establish RNA-directed DNA methylation (RdDM), the mechanism through which transcriptionally active TEs transition from post-transcriptional RNAi regulation to chromatin level control has remained unclear. We have identified the molecular mechanism of a plant pathway that functions to direct DNA methylation to transcriptionally active TEs. We demonstrated that 21-22 nucleotide (nt) siRNA degradation products from the RNAi of TE mRNAs are directly incorporated into the ARGONAUTE 6 (AGO6) protein and direct AGO6 to TE chromatin to guide its function in RdDM. We find that this pathway functions in reproductive precursor cells to primarily target long centromeric high-copy transcriptionally active TEs for RdDM prior to gametogenesis. This study provides a direct mechanism that bridges the gap between the posttranscriptional regulation of TEs and the establishment of TE epigenetic silencing.

A transcript-based annotation of transposable elements allows for reduced bioinformatic complexity and direct hypothesis testing of previously challenging regions of the genome.