A genetics screen uncovers the role of RNA-binding protein RBP45D in flowering time under heat and DCL3-independent RNA-directed DNA methylation

In the canonical RdDM, small interfering RNAs (siRNAs) are produced through the slicer activity of DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system in which transcriptional gene silencing (TGS) through RdDM is independent of DLC3. To identify players in the DCL3-independent pathway, a forward genetics screen was performed with the prors1 (LUC) system to identify suppressor mutants in which TGS is released. Among already known components of RdDM, we identified RNA-binding protein RBP45D. RBP45D promotes DNA methylation changes, and a lack of RBP45D results in a late flowering phenotype especially under heat, presumably mediated by elevated FLC levels. A lack of RBP45D did not influence the pre-mRNA splicing of known RdDM genes, suggesting that RBP45D involvement in the RdDM might be direct. RBP45D is localized to the nucleus where it is associated with snRNAs and snoRNAs. RBP45D promotes siRNA production originating from the transgene and we hypothesize that this function is direct and not indirect. Examination of differential gene expression in the Arabidopsis rbp-kd1 and -kd2 mutants in comparison to Col-0