RNA electrophoretic mobility shift assay

Recombinant RFL79 and RFL29a proteins were expressed as maltose-binding protein-fusions and purified by affinity chromatography as described previously for other PPR proteins⁸⁷. The coding sequences of RL79 and RFL29a were amplified from genomic DNA using the PrimeStar HS DNA polymerase (Takara Bio) with primers containing attB sites for Gateway cloning technology (Invitrogen) (Supplementary Table ⁶). The obtained PCR products were subcloned into pDONR207 vector (Invitrogen) with Gateway BP Clonase II enzyme mix (Invitrogen). The positive clones were identified by sequencing at Macrogen and cloned into the expression vector pETG-41K (EMBL, Heidelberg, Germany) with Gateway LR Clonase II enzyme mix (Invitrogen). The pETG-41K vector allows an addition of 6xhistidine (His) tag that can be used for Ni-NTA purification and an MBP (maltose-binding protein) tag at the N-terminus of the recombinant protein. For protein expression, the chemically competent cells of E. coli C41(DE3) strain (MilliporeSigma) were used. Transformed cells were grown in 500 mL LB (1× Luria-Bertani and 50 mM Tris-HCl, pH 8.0) medium at 37 °C and 220 rpm until the OD₆₀₀ reached 0.4. Glass flasks with the cultures were then transferred on ice for 15 min and protein expression was initiated by addition of isopropyl β-d-1-thiogalactopyranoside (Promega) to a final concentration of 0.1 mM. The cultures were grown at 16 °C and 220 rpm overnight and harvested by centrifugation at 3000 × g for 15 min in an Avanti J-26XP centrifuge (Beckman Coulter). Bacterial pellets were dissolved in 35 mL of lysis buffer (0.5 M NaCl, 50 mM HEPES-KOH pH 8, 10 mM imidazole, and 7 mM β-mercaptoethanol) and cells were disrupted by homogenization with Avestin Emulsiflex C5 (Avestin, Ottawa, Ontario, Canada). Soluble protein fractions were cleared from cell debris by centrifugation for 15 min at 13,000 × g at 4 °C, incubated with Profinity IMAC Ni-charged resin (Bio-Rad) on a rotating wheel for 1 h and packed into empty Econo-Pac gravity columns (Bio-Rad). After three washes with 1x wash buffer (0.5 M NaCl, 50 mM HEPES-KOH pH 8, 7 mM β-mercaptoethanol, 20 mM imidazole) proteins were eluted from the Ni-charged resin with elution buffer (0.5 M NaCl, 50 mM Tris-HCl, pH 8, 250 mM imidazole) and dialysed overnight at 4 °C in dialysis buffer (0.5 M NaCl, 50 mM Tris-HCl, pH 8, 50% glycerol, 2 mM EDTA, 7 mM β-mercaptoethanol) with slow stirring on a magnetic mixer. The concentrations of the dialysed proteins were measured at ND-1000 NanoDrop spectrophotometer (Thermo Fisher Scientific). More details on protein purification and analysis are given in Supplementary Fig. ⁷. The sequences of fluorescein-labelled oligonucleotides used in the REMSAs performed following the previously published protocol⁸⁷ are listed in Supplementary Table ⁶. Briefly, 10 μL of binding buffer consisting of 1× THE (34 mM Tris, 66 mM HEPES, 0.1 mM EDTA pH 8), 0.2 M NaCl, 5 mM dithiothreitol, 5 mg/mL heparin, 0.1 mg/mL BSA, and 8 units of RNaseOUT (Invitrogen) were mixed with 5 μL of dialysed-protein dilution and incubated at room temperature for 10 min. The 5′-fluorescein-labelled probes (MilliporeSigma) were heated for 2 min at 94 °C and incubated on ice for at least 4 min. 10 μL of denatured probes were added to the 15 μL binding reaction for a total reaction volume of 25 μL and incubated at 25 °C for 15 min. 15 μL of the binding reaction were loaded onto a pre-run 5% native polyacrylamide gel using the 1× THE as a running buffer in a cold room. After the run gels were imaged with a Typhoon Biomolecular Imager (Cytiva). Fluorescein-labelled probes were excited by a 488 nm laser and detected through a 520 nm band-pass emission filter.