The siRNA suppressor RTL1 is redox-regulated through glutathionylation of a conserved cysteine in the double-stranded-RNA-binding domain

Abstract

RNase III enzymes cleave double stranded (ds)RNA. This is an essential step for regulating the processing of mRNA, rRNA, snoRNA and other small RNAs, including siRNA and miRNA. Arabidopsis thaliana encodes nine RNase III: four DICER-LIKE (DCL) and five RNASE THREE LIKE (RTL). To better understand the molecular functions of RNase III in plants we developed a biochemical assay using RTL1 as a model. We show that RTL1 does not degrade dsRNA randomly, but recognizes specific duplex sequences to direct accurate cleavage. Furthermore, we demonstrate that RNase III and dsRNA binding domains (dsRBD) are both required for dsRNA cleavage. Interestingly, the four DCL and the three RTL that carry dsRBD share a conserved cysteine (C230 in Arabidopsis RTL1) in their dsRBD. C230 is essential for RTL1 and DCL1 activities and is subjected to post-transcriptional modification. Indeed, under oxidizing conditions, glutathionylation of C230 inhibits RTL1 cleavage activity in a reversible manner involving glutaredoxins. We conclude that the redox state of the dsRBD ensures a fine-tune regulation of dsRNA processing by plant RNase III.

Figure 1.

RTL1 site-specifically cleaves a near-perfect duplex structure in vitro and in planta. (A) Left, the hairpin structure predicted from 3′UTR At3g18145 mRNA sequence is shown. The positions of p1 and p2 primers used in primer extension experiments are indicated. The dashed rectangle indicates the sequence corresponding to the RNA substrate-1 shown in (E). The sequence corresponding to a 24-nt small RNA is shown by a bar. Arrows show cleavage sites a–d mapped by primer extension. The RNA duplex consensus region rcr1 are boxed (B) Primer extension analysis using in vitro transcribed 3′UTR -At3g18145 and p1 or p2 primers. Arrows (a–d) show mapped cleavage sites in both strands. (C) Primer extension analysis on total RNA extracted from Col0 and overexpressing RTL1-Flag #1 plants with p1 primer. Arrows show the rcr1 cleavage positions. (D) Cleavage assay of ³²P-CTP RNA substrate-1 using His-RTL1 or His-RTL1m3 proteins. Arrows indicate non cleaved RNA substrate-1 (f0) and cleavage fragment products (f1–f3). A low exposure insert is shown to better visualize the f1 and f2 fragments. DNA size markers are indicated on the left. (E) The predicted ³²P-CTP RNA substrate-1 structure and f1–f3 products are represented.

Figure 2.

RNA sequence and structure requirements for RTL1 cleavage. Primer extension analysis using in vitro transcribed 3′UTR RNA (WT and mutated V1-V4) incubated with His-RTL1 protein (+) or buffer alone (−). In the version V1 the A::U and the G::U base pairs within and next to the RNA duplex motif were mutated to G::U and G::C respectively; in V2, three of the A::U base within the RNA duplex motif were mutated to C::G; in V3 the U:: A base pair located in the loop structure was mutated to C::G and in the -V4, the UCG nucleotides in loop structure were mutated to GCU. The Wt and mutated sequences are green and red boxed respectively. Arrow shows cleavage site mapped with p1 primer. Note the two novel cleavage sites (black arrows) adjacent to the a and b sites (black and white arrows) in V1 and the novel cleavage sites adjacent to the a site in V2 and V4. The ΔG (kcal/mol) determined by RNA Mfold for each RNA substrate is indicated.

Figure 3.

The native dsRBD and conserved cysteine C230 are required for RTL1 cleavage of RNA in vitro. (A) Coomassie blue staining and schematic representation of Wt His-RTL1 (R1D1), His-RTL2 (R2D2), swapped His-R1D2 and His-R2D1 or His-RTL1 with mutated cysteines C230S and C260S or truncated HisRTL1ΔdsRBD proteins. Red and yellow boxes correspond to RNase III domains while blue and light blue boxes correspond to dsRBD from RTL1 (R1 and D1) and RTL2 (R2 and D2a and b) proteins respectively. Asterisks show position of mutated Cys 230 and Cys260. (B) ³²P-CTP RNA substrate-1 was incubated with 100 ng of His-RTL1(R1D1), His-R1D2, His-R2D1, His-RTL2 (R2D2), His-RTL1C230S, His-RTL1C260S or His-RTL1ΔdsRBD (lane 10) recombinant proteins or with buffer alone. Arrowhead indicates full-length RNA substrate (f0) and major cleavage fragment products (f1 and f2). DNA size markers are indicated on the left. (C) Amino acid sequence alignment of dsRBD from RTL1 (F4JK37), RTL2 (Q9LTQ0) and DCL1 (NP_171612), DCL2 (NP_566199), DCL3 (NP_189978) and DCL4 (NP_197532). Vertical arrows heads show highly conserved Cys230 and non-conserved Cys250 and Cys260 in the RTL1 sequence.

Figure 4.

The native dsRBD and its conserved cysteine C230 are required for RTL1 cleavage of RNA in planta. (A) Top, schematic representation of the GU-UG RNA and of the siRNAs produced from the processing of the dsRNA stem by DCL2, DCL3 and DCL4. The GU-UG siRNA construct contains promoter (p35) and terminator (t35S) sequences derived from 35S Cauliflower mosaic virus (CaMV) Bottom, schematic representation of native DCL1, RTL1 and RTL2 and of the truncated proteins DCL1-RD and DCL1-RDD mimicking RTL1 and RTL2, respectively. (B) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct (GU-UG) and either a control 35S-driven construct (GFP), a WT-tagged 35S:RTL1-Myc construct (RTL1) or a mutant 35S:RTL1ΔdsRBD-Myc construct lacking the dsRBD (RTL1ΔdsRBD). Low molecular weight RNAs were extracted and hybridized with a GU probe. Ethidium bromide-stained RNAs are shown as loading control. Proteins were extracted and hybridized with a Myc antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 2–6). L, indicates protein ladder. (C). Left, N. benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S). Right, western blot analysis show comparable expression of 35S:RTL1 C230), 35S:RTL1 and 35S:RTL1-Myc constructs in N. benthamiana leaves. Proteins were extracted and hybridized with α RTL1 antibody. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3). (D) Nicotiana benthamiana leaves co-infiltrated with a 35S:GU-UG construct and either a control 35S-driven construct (GFP), a genomic-based or cDNA-based WT 35S:RTL1 construct (WT genomic and WT cDNA), 35S:RTL1 constructs mutated at cysteine 230 (C230S) and truncated 35S:DCL1 constructs (DCL1-RDD and DCL1-RD) carrying WT DCL1 sequence (WT) or a mutation at cysteine 1742 (C1742S). Note that RNA samples shown in (C) and d were run on a unique gel, so that the control 35S:GU-UG + 35S:GFP for (D) is visible on (C).

Figure 5.

RTL1 dimerizes through the N-terminal domain. (A) Superdex 75 Gel filtration chromatography of His-RTL1 and His-RTL1ΔdsRBD proteins in 150 mM NaCl buffer conditions. The peak positions of conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and ribonuclease A (13.7 kDa) are indicated by arrows (B) His-RTL1 protein untreated (lane 1) or treated (lane 2) with DMP was analyzed by western blot using α-RTL1 antibodies (C) Coomassie Blue staining of His-RTL1, His-RTL1 C230S or His-RTL1 C260S and His-RTL1ΔdsRBD migrated on native gel in absence of DTT or in the presence of increased amount of DTT. In B and C, arrows indicate positions of monomer (m), dimers (d) and higher order structures (hc) according to standard molecular weight markers.

Figure 6.

Glutathionylation of RTL1 affects RNase III cleavage activity. (A) His-RTL1 treated with 5 mM GSSG was trypsin digested and analyzed by nanoLC-MSMS. The panels show fragmentation spectra matching peptides with either unmodified (left) or with glutathionylated C230 (right). (B) Effect of reduced glutathione (GSSG) on cleavage of the 3′UTR sequence by His-RTL1. The protein samples were incubated at room temperature for 30 min with 0–20 mM GSSG before cleavage assay. (C) Effect of oxidized glutathione (GSH) on cleavage of the 3′UTR sequence by His-RTL1 in the presence of H₂O₂. The samples were incubated at room temperature for 30 min with 1 mM H₂O₂ and 0–10 mM GSH. (D) Reactivation of cleavage activity His-RTL1 by deglutathionylation with GRXC1. The samples were incubated at room temperature for 30 min with 10 mM GSSG or buffer only. The samples were further incubated at room temperature for 20 min with or without the GRX system (5 mM NADPH, 2 μM GRXC1, 0.45 units GR). Full system is absent in lanes 1–2 while present in lanes 3–4. GR was omitted in lanes 5–6 and no GRXC1 was added in lanes 7–8. Lane 9 is without RTL1 protein. (E) Schematic representation of the likely process of deglutathionylation of His-RTL1 by GRX based on a classical deglutathionylation model. The thiolate form of the GRX catalytic Cys attacks the disulfide of the glutathionylated Cys residue, releasing the reduced peptide, and becoming glutathionylated. A molecule of GSH reduces the glutathionylated thiol of GRX, releasing the thiolate catalytic Cys and generating a GSSG molecule. RT-PCR reactions were performed using total RNA from 14 days-old Arabidopsis thaliana plants and primers p1/p9 and U3fw/U3rev.

Figure 7.

Plant GSSG treatment affects RTL1 cleavage activity. (A) Top, proteins from Col0 WT and 35S::RTL1 (#1 and #2) plants were extracted and hybridized with α-Flag and α-RTL1 antibodies. Ponceau staining (pink dye) is shown as protein loading control (lanes 1–3 and 4–6). L, indicates protein ladder. Bottom, sequence of dsRNA substrate-2. The putative rcr motif is underlined and the vertical arrows show DCL3/DCL4 cleavage site. (B) [γ-³²P]-ATP RNA substrate-2 was incubated with either WT (lanes 2), 35S:RTL1-Flag #1 (lane 3) or 35S:RTL1-Flag #2 (lane 5) protein extracts or with WT + 35S:RTL1-Flag #1 (lane 4) or WT + 35S:RTL1-Flag #2 (lane 6) protein extracts. Reactions were performed either with 15 μl, 1.5 μl or 15 μl + 1.5 μl. Lane 1 shows [γ-³²P]-ATP RNA substrate-2 only. (C) Glutathione levels in 2-weeks old WT and 35S:RTL1-Flag #1 plantlets treated or not with 10 mM GSSG. Asterisks indicate a significant difference (P ≤ 0.01) between total glutathione levels of WT and 35S:RTL1 plants. Reduced and oxidized glutathione levels are indicated by white and gray bars, respectively. The percentage reduction state of glutathione is indicated above bars. Error bars represent SD (n = 4). (D) [γ-³²P]-ATP RNA substrate-2 was incubated with WT and 35S:RTL1-Flag #1 protein extracts prepared from treated (lanes 3 and 5) or not (lanes 2 and 4) with 10 mM GSSG respectively. Lane 1, shows [γ-³²P]-ATP RNA substrate-2 only and Lane 6, cleavage reaction using 35S:RTL1-Flag #1 protein extract not submitted to GSSG buffer and is used as a positive control.

Figure 8.

In silico and functional analysis of RTL1 suggest a novel regulatory mechanism for RNase III activity in plants. (A) Modeled RTL1 (residues 50–284) homodimer based on mouse Dicer (3c4b.1.A) (23). The RNase III domain of two RTL1 molecules are shown in orange and green while both dsRBD are shown in white. In the left panel, the residues E89, E92, D96 and E185 (E37, E40, D44 and E110 in Aquifex aeolicus RNase III) located in the RNase III domain and required for RNA cleavage are shown. In the right panel, the RTL1 homodimer was rotated 180° and 45° to show conserved cysteines C230 in each dsRBD. The residues T224, N227, E228 and Q231 (T154, Q15, E158 and Q161 in A. aeolicus RNase III) located near to the C230 are indicated. These residues are required for RNA binding of Aa-RNase III. (B) In the proposed model, the cysteine C230, which is essential for cleavage activity, is kept in its reduced state (-SH). Glutathionylation of C230 (S-SG) in RTL1 sequence does not affect RTL1 dimerization but it might inhibit dsRNA binding and/or cleavage activity. RTL1 activity inhibition is reversible upon treatment with GRXC1 or GRXC2.