A complex of Arabidopsis DRB proteins can impair dsRNA processing

Area of Science:

• Plant molecular biology
• RNA interference pathways
• Epigenetics

Background:

• Small RNAs regulate gene expression via transcriptional and post-transcriptional gene silencing.
• Dicer-like ribonucleases (DCLs) process double-stranded RNA (dsRNA) into small RNAs, with dsRNA binding proteins (DRBs) assisting this in plants.

Purpose of the Study:

• To investigate the function of DRB proteins in small RNA biogenesis in the model plant *Arabidopsis*.
• To identify interactors of DRB4 and elucidate their role in RNA silencing pathways.

Main Methods:

• Immunoprecipitation followed by mass spectrometry to identify DRB4 interactors.
• Reciprocal co-immunoprecipitation and bimolecular fluorescence complementation to verify interactions.
• In vitro assays using tobacco BY-2 cell lysate to assess dsRNA cleavage and small interfering RNA (siRNA) production.
• Analysis of siRNA accumulation and DNA methylation in *Arabidopsis* overexpressing DRB7.1.

Main Results:

• DRB7.1 was identified as a novel interactor of DRB4.
• The DRB7.1/DRB4 complex inhibits DCL3-mediated production of 24-nt siRNAs and antagonizes DRB4's enhancement of DCL4-mediated 21-nt siRNA production.
• Overexpression of DRB7.1 in *Arabidopsis* mimics *drb4* mutant phenotypes regarding siRNA accumulation.
• DRB7.1/DRB4 complex impairs the dsRNA cleavage activity of RNase three-like (RTL) proteins RTL1 and RTL2.
• Enhanced siRNA accumulation from an endogenous inverted repeat correlated with increased DNA methylation.

Conclusions:

• The DRB7.1/DRB4 complex acts as a negative regulator of siRNA biogenesis in plants.
• DRB7.1 antagonizes the function of DRB4 in siRNA production, impacting both RNA interference and epigenetic regulation.
• This study reveals a novel mechanism where DRB complexes can inhibit RNase III activity, thereby modulating siRNA pathways and epigenetic marks.