Evolutionary history of double-stranded RNA binding proteins in plants: identification of new cofactors involved in easiRNA biogenesis

Abstract

In this work, we retrace the evolutionary history of plant double-stranded RNA binding proteins (DRBs), a group of non-catalytic factors containing one or more double-stranded RNA binding motif (dsRBM) that play important roles in small RNA biogenesis and functions. Using a phylogenetic approach, we show that multiple dsRBM DRBs are systematically composed of two different types of dsRBMs evolving under different constraints and likely fulfilling complementary functions. In vascular plants, four distinct clades of multiple dsRBM DRBs are always present with the exception of Brassicaceae species, that do not possess member of the newly identified clade we named DRB6. We also identified a second new and highly conserved DRB family (we named DRB7) whose members possess a single dsRBM that shows concerted evolution with the most C-terminal dsRBM domain of the Dicer-like 4 (DCL4) proteins. Using a BiFC approach, we observed that Arabidopsis thaliana DRB7.2 (AtDRB7.2) can directly interact with AtDRB4 but not with AtDCL4 and we provide evidence that both AtDRB7.2 and AtDRB4 participate in the epigenetically activated siRNAs pathway.

A specific dsRNA-binding protein complex selectively sequesters endogenous inverted-repeat siRNA precursors and inhibits their processing

“…Next, we sorted sRNAs into their major functional categories and normalized their abundance to the amount of the evolutionarily conserved DCL1-dependent miRNAs (68), whose accumulation is not affected by the drb7.2 mutation (Figure 2A; (51)). We found that lack of DRB7.2 does not significantly impact the accumulation of ta-siRNAs or p4-siRNAs (Figure 1B).…”

“…The 35S-GFP line in Columbia ecotype (Col-0) was kindly provided by M. Jean Molinier (IBMP). The mutant drb7.2 (GABI 525B11) was obtained from the Arabidopsis Biological Ressource Center (ABRC) and was described previously (51). Arabidopsis thaliana reference ecotype used was Columbia.…”

“…Counts were made with intersetBed, from bedtools suite (v.2.25.0) (67). Data were normalized with there own conserved miRNA counts (miR156, 157, 159, 160, 162, 164, 165, 166, 167, 168, 169, 170, 171, 172, 319, 390, 391, 393, 394, 395, 396, 397, 398, 399, 403, 408, 472) (68) under the assumption that they were globally unaffected (Figure 2 and (51)). miRNA annotation was downloaded from miRBase v.21 (69).…”

“…Besides those five DRBs containing two dsRBMs, we have recently identified a new DRB family (named DRB7) conserved in all vascular plants and harboring a single dsRBM that shows concerted evolution with the most C-terminal dsRBM of DCL4 (51). We showed that one of the two Arabidopsis DRB7 proteins (DRB7.2) interacts with DRB4 but does not seem to be required for the production of any DCL4-dependent sRNAs, including the newly identified class of epigenetically activated (ea)siRNAs that accumulate mostly in the vegetative nucleus of pollen grains or in Decreased DNA Methylation 1 (ddm1) mutants (52–56).…”

“…By contrast, loss of DRB7.2 triggered an increase in the accumulation of DCL3-dependent 24 nt siRNAs from easiRNA-generating loci. However, given that this observation was only made in a ddm1 mutant background, and considering the pleiotropic nature of this mutation, the reason of this change is still unclear, as is the function of DRB7.2 (51). …”