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 Arabidopsis, 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 Arabidopsis altered accumulation of siRNAs in a manner reminiscent of drb4 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.

Keywords: DCL; RNA silencing; RNase III; RNase three-like; double-stranded RNA binding protein (DRB); endogenous inverted repeats; in vitro BY-2 lysate.

FIGURE 1.

DRB7.1 interacts with DRB4 and DCL4. (A–E) Bimolecular fluorescence complementation assay (BiFC) performed by transient expression in N. benthamiana leaves and imaged by confocal microscopy. YFP signal emitted from reconstituted half-YFP fusions to the proteins indicated on panels is shown together with bright-field (differential interference contrast) and an overlay of channels. (F) Western blot analysis of DRB7.1:DRB7.1-FHA following immunoprecipitation with anti-Flag antibodies from WT Col-0 or drb4-1 mutant plants. Immunoprecipitated DRB7.1 fusion protein is detected with an HA antibody, and coimmunoprecipitated DCL4 and DRB4 are detected by native antibodies. Total protein staining by Coomassie blue is shown as a loading control for input and supernatant (unbound fraction). Star indicates background band that cross-reacts with DCL4 antibody. Arrow indicates DRB4 band, to distinguish from background cross-reacting bands. (G,H) Colocalization of YFP signals from DRB7.1+DRB4 BiFC with DCL4-mCherry (G) and RDR6-mCherry (H) performed by transient expression in N. benthamiana leaves and imaged by confocal microscopy. Nucleus (N) and cytoplasm (C) are indicated. Arrowheads indicate nuclear foci, arrows indicate punctate cytoplasmic signals. Scale bar = 10 µm.

FIGURE 2.

Combining DRB7.1 with DRB4 in BYL impairs endogenous dsRNA processing. (A–C) DRB7.1, DRB7.1-HA, DRB4, DRB4-Flag, DRB2, and DRB2-Flag were in vitro translated in BYL, mixed together and incubated for 15 min with 34/36-nt (A), or 30 min with 98/100-nt (B), and 510/512-nt (C) radiolabeled dsRNA substrates. RNA was extracted and separated on 15% native polyacrylamide gel. MOCK samples (lysate with no addition of exogenous mRNA) represent the endogenous processing activity of the lysate after addition of radiolabeled dsRNA and is used as a baseline control. Normalized quantification of band intensity is displayed below the gel. Substrates and siRNAs intensity values were normalized to Mock values.

FIGURE 3.

Coimmunopurification of dsRNA with DRB7.1, DRB4, and DRB2. (A–C) DRB7.1-HA, DRB4-Flag, and DRB2-Flag were in vitro translated in BYL and combined as indicated (protein input, bottom), then incubated with radiolabeled dsRNA substrates of various sizes. After 7 min of incubation, RNA input (top) was extracted. For immunoprecipitation (IP), Flag or HA magnetic beads were added to the BYL mixes and incubated for 40 min at 4°C, followed by washes. A fraction of magnetic beads was used for protein extraction (protein IP, bottom) and RNA was extracted from the remaining sample (RNA IP, top). RNA was separated on 15% native polyacrylamide gel. Proteins were resolved on 12% SDS–PAGE gel.

FIGURE 4.

Combining DRB7.1 with DRB4 impairs dsRNA processing by RTL1 and RTL2 on shorter dsRNA substrates. (A–C) DRB7.1, DRB4, Flag-RTL1, and Flag-RTL2 were in vitro translated in BYL, mixed together as indicated and incubated for 15 min (A), 30 min (B), or 20 min (C) with dsRNA substrates of indicated size. RNA was extracted and separated on 15% native polyacrylamide gel. MOCK (- -) samples (lanes 1,12, and 21) represent the endogenous processing activity of the lysate after addition of radiolabeled dsRNA and is used as a control for processing pattern. Flag-RTL1 (- -) or Flag-RTL2 (- -) are used as controls for respective RTL activity in this experiment. (A) DRB7.1 mixed with DRB4 impairs processing of 34/36 RNA duplexes by Flag-RTL1. Flag-RTL2 does not process 34/36 RNA duplexes. (B) DRB7.1 mixed with DRB4 impairs processing of 98/100 RNA duplexes by Flag-RTL1 and Flag-RTL2. (C) Combining DRB7.1 and DRB4 does not impede RTL1 and RTL2 dsRNA processing of 510/512 nt. Normalized quantification of band intensity is displayed below the gel. Substrates and siRNAs intensity values were normalized to Mock values.

FIGURE 5.

Effect of drb7.1-1 and DRB7.1 overexpression on siRNA accumulation. (A) Low-molecular weight RNA blot with RNA extracted from inflorescences of drb7.1-1 and No-0 control, and dcl4-2, drb4-1, and Col-0 control. DCL2/DCL3-dependent IR71, DCL4-dependent miR822, TAS1 and 2, DCL3-dependent SimpleHat and Rep2, and DCL1-dependent miR159 are shown. (B) Low-molecular weight Northern blotting was performed on total RNA extracted from inflorescences of independent, stable transgenic Col-0 WT plants expressing 35S:DRB7.1-GFP or 35S:DRB7.1-FHA. Two independent samples from Col-0 WT control plants are included. DCL2/DCL3-dependent IR71 and IR2039, DCL4-dependent miR822, TAS1 and 2, DCL3-dependent SimpleHat and Rep2, and DCL1-dependent miR172 are shown. (C) Similar analysis as B, but performed with batches of T1 plants and with addition of DCL4-dependent TAS3. (A–C) U6 is included as a loading control. siRNA sizes are indicated. The same Northern blot membrane was stripped and rehybridized with different probes to analyze multiple siRNA species. Normalized quantification of band intensity is displayed below the gel image. Intensity values were normalized to U6 and are displayed as a ratio relative to Col-0 (wild-type) sample. (D) Relative expression of DRB7.1, measured by RT-qPCR, of T1 pools. Average of two biological replicates (batches) ± standard deviation is shown. (E) Bisulfite sequencing PCR (BSP) analysis of IR71 5′ region in all contexts (CG, CHG, and CHH sites) in WT (Col-0) and DRB7.1 OX lines. Error bars represent 95% confidence intervals, calculated using Wilson score interval. (F) Confocal image of 35S:DRB7.1-GFP localization in a live root of Col-0 WT background. Arrow, cytoplasmic punctae; N, nucleus. Scale bar = 10 µm.

FIGURE 6.

Expression pattern and localization of DRB7.1-GFP. (A) Confocal microscope images from a single plane of inflorescence meristems from DRB7.1:DRB7.1-GFP expressing Col-0 WT plants (left and middle) or a drb4-1 mutant plant (right). Images depict GFP signal overlaid with chlorophyll autofluorescence. Scale bar = 50 µm. (B,C) Close-up images of a single plane from inflorescence meristems expressing the DRB7.1:DRB7.1-GFP fusion in Col-0 WT (B) or drb4-1 mutants (C) with GFP signal (middle), chlorophyll autofluorescence (right) and overlay (left) shown. Inset depicts nucleus (N), nuclear foci (arrowhead), and cytoplasmic punctae (arrow). Scale bars = 10 µm.