A complex of Arabidopsis DRB proteins can impair dsRNA processing

Abstract

Small RNAs play an important role in regulating gene expression through transcriptional and post-transcriptional gene silencing. Biogenesis of small RNAs from longer double-stranded (ds) RNA requires the activity of dicer-like ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant , we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry-based fingerprinting and discovered DRB7.1. This interaction, verified by reciprocal coimmunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY-2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24-nucleotide (nt) small interfering (si) RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21-nt siRNAs produced by DCL4. Overexpression of DRB7.1 in altered accumulation of siRNAs in a manner reminiscent of mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNase three-like (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1-DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants.

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

“…Perfect co-localization most likely reflects the direct association of DRB2 and DRB4 on replicating dsRNA within the VRCs. This result is in line with the experimentally verified ability of DRB2 and DRB4 to bind dsRNA [36, 50] and of DRB4 to bind TYMV dsRNA in vivo [41]. DRB2 was recently shown in A. thaliana to re-localize to cytoplasmic punctate bodies upon infection by TuMV, TSWV and TYMV [8] and DRB4 to re-localize from nuclei to cytoplasmic VRCs upon TYMV infection [41].…”

“…Despite showing low spectral counts in our IPs, two DRBs were identified in our analysis: DRB2 (AT2G28380, total counts: 4, Figure 4A ) and DRB4 (AT3G62800, total counts: 5, Figure 4A ) that were obvious candidates to test ( Figure 4 ). DRB2 was recently shown to localize to the replication complexes of different RNA viruses [8], to be able to bind dsRNA [36] and to play a role in endogenous small RNA biogenesis [32, 37, 38]. In non-infected plants DRB2:tRFP and B2:GFP localized to rather distinct cytoplasmic and nuclear structures, whereas a near-perfect colocalization of DRB2:tRFP and B2:GFP was observed in the VRCs upon TRV-PDS infection ( Figure 4B ), which was particularly evident at high magnification ( Figure 4C) .…”

New DRB complexes for new DRB functions in plants

“…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.…”

“…Indeed, whereas DCL2 and DCL3 have been shown to be purely located in the nucleus, DCL4 was shown to be mostly cytoplasmic. 31,32 By accumulating in the same subcellular compartment where endogenous IRs transcripts are produced, folded in dsRNA and, at least in a certain proportion, sequestered by the nuclear DRB4/DRB7.2 complex, the endoIR-siRNA precursors released in drb4 or drb7.2 mutant plants will be preferentially attacked by DCL3 and DCL2 over DCL4. In parallel, the apparent higher efficiency of DCL3 over DCL2 in processing these released substrates might possibly reflect differences in terms of steady-state level, affinity or activity of these 2 proteins, leading to DCL2 being outcompeted by DCL3.…”

“…Interestingly, DRB7.1, the other member of the DRB7 family, was also found to interact with DRB4 in vivo, and this latter was shown to promote binding of DRB7.1 to various dsRNA substrates in vitro, resulting in impaired production of DCL3dependent 24nt siRNAs in BY2 evacuolated lysate. 32 Unfortunately, DRB7.1 function in vivo could not be formally assessed as neither p4-siRNAs, miRNAs, tasiRNAs nor endoIR-siRNAs accumulation was altered in drb7.1 mutant plants. 30,32 This might be due to the restricted expression pattern of DRB7.1 in plants, which can strongly dilute, and ultimately mask, any potential effect of this protein on small RNAs accumulation in whole tissue analyses.…”