DNA Methylation Influences the Expression of <i>DICER-LIKE4</i> Isoforms, Which Encode Proteins of Alternative Localization and Function

Abstract

Plant RNA silencing operates via RNA-directed DNA-methylation (RdDM) to repress transcription or by targeting mRNAs via posttranscriptional gene silencing (PTGS). These pathways rely on distinct Dicer-like (DCL) proteins that process double-stranded RNA (dsRNA) into small-interfering RNAs (siRNAs). Here, we explored the expression and subcellular localization of Arabidopsis thaliana DCL4. DCL4 expression predominates as a transcription start site isoform encoding a cytoplasmic protein, which also represents the ancestral form in plants. A longer DCL4 transcript isoform encoding a nuclear localization signal, DCL4^NLS, is present in Arabidopsis, but DNA methylation normally suppresses its expression. Hypomethylation caused by mutation, developmental reprogramming, and biotic stress correlates with enhanced DCL4^NLS expression, while hypermethylation of a DCL4 transgene causes a reduction in DCL4^NLS expression. DCL4^NLS functions in a noncanonical siRNA pathway, producing a unique set of 21-nucleotide-long "disiRNAs," for DCL4^NLS isoform-dependent siRNAs, through the nuclear RdDM dsRNA synthesis pathway. disiRNAs originate mostly from transposable elements (TEs) and TE-overlapping/proximal genes, load into the PTGS effector ARGONAUTE1 (AGO1), and display a subtle effect on transcript accumulation together with overlapping 24-nucleotide siRNAs. We propose that, via PTGS, disiRNAs could help to tighten the expression of epigenetically activated TEs and genes using the methylation-state-responsive DCL4^NLS.