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

Abstract

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)-mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

New DRB complexes for new DRB functions in plants

“…This was characterized by a global increase in the total amount of endoIR-siRNAs produced and a clear change in their accumulation pattern with, chiefly, a strong increase in the accumulation of DCL3-dependent 24nt small RNAs from these endogenous IRs loci, compared with wild-type plants. 31 Interestingly, a similar effect on endoIR-siRNA accumulation was previously reported in drb4 mutant plants, 33 suggesting that, somehow, both DRB4 and DRB7.2 negatively regulate the processing of these endogenous IR transcripts by the siRNAgenerating DCLs. Incidentally, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (co-IP) and RNA immunoprecipitation (RIP) experiments revealed that these 2 DRB proteins selectively interact with each other and that this complex specifically binds endoIR-siRNA precursors.…”

“…Accordingly, overexpression of DRB7.2 triggered a specific decrease of endoIR-siRNA accumulation, without any effect on the other classes of small RNAs. 31 The observation that, in single drb7.2 or drb4 mutant backgrounds, accumulation of DCL3-dependent 24nt endoIR-siR-NAs was the most strongly increased suggested that this complex acts primarily as a repressor of DCL3 processing on IR substrates ( Fig. 1 and ref.…”

“…29 However, the recent identification of a new clade of DRB protein (named DRB7), conserved in all vascular plants, 30 and the characterization of their implication in the formation of specific DRB complexes that selectively regulate the processing of endoIR-siRNA precursors, strongly suggest that endogenous IRs might be bona fide regulators of gene expression. 31,32 Members of the DRB7 family (DRB7.1 and DRB7.2), as opposed to the previously characterized DRB1-5, harbour a single dsRBM. 30 Genome-wide small RNA profiling and northern blot analysis performed on drb7.2 mutant plants revealed that lack of DRB7.2 does not significantly impact the accumulation of p4-siRNAs, miRNAs and tasiRNAs but strongly alters the production of endoIR-siRNAs.…”

“…Incidentally, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (co-IP) and RNA immunoprecipitation (RIP) experiments revealed that these 2 DRB proteins selectively interact with each other and that this complex specifically binds endoIR-siRNA precursors. 31 Binding of the DRB4/DRB7.2 complex most likely protects or sequesters these long dsRNAs and, by doing so, prevents, or at least considerably slows down, their access and cleavage by the DCLs that process them. Accordingly, overexpression of DRB7.2 triggered a specific decrease of endoIR-siRNA accumulation, without any effect on the other classes of small RNAs.…”

Immunocapture of dsRNA-bound proteins provides insight into tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

“…DRB4 has been shown to be both a co-factor of DCL4 in small RNA biogenesis and an inhibitor of DCL3 in endogenous inverted-repeat RNA processing in A. thaliana [39, 40]. More relevantly here, DRB4 is involved in the defense against RNA viruses [41, 42].…”