Typhoon 9000

Binding of EhRNaseIIIs, including EhRNaseIII256 (the full-length EhRNaseIII), EhRNaseIII229, EhRNaseIII194, and chimeric protein EA256, to the dsRNA substrates was monitored using electrophoretic mobility shift assays (EMSA). Five microliters of EhRNaseIII, 3 μL dsRNA, and 2 μL 5 × binding buffer (150 mM Tris-HCl pH 7.5, 150 mM NaCl, 25 mM MgCl₂, 5 mM DTT, 0.5 mM EDTA, 25% glycerol) were mixed in a thin-wall Eppendorf tube. The final concentrations of EhRNaseIIIs and dsRNAs are indicated on the figures. The reaction mixtures were incubated at room temperature for 10 min followed by incubation on ice for an additional 20 min. Samples were loaded onto a pre-cooled 6% native polyacrylamide gel. Gels were run at 160 V for 3–4 h at 4 °C in 0.5× TBE buffer supplemented with 5 mM MgCl₂. RNAs were visualized by phosphorimaging using a Typhoon 9000 (GE Healthcare) (for Fig. 4) or by staining with Gelred (Biotium) (for Figs 5 and 6).

Both LSP (171-470) and HSP (491-790) were cloned with NcoI and HindIII into the pET-22 vector. For the run-off transcription assay, the LSP or HSP vector was linearized using NcoI or HindIII, respectively. All transcription factors were pre-mixed as a 1:1:1 molar ratio of TFAM: TFB2m: POLRMT. Each reaction mixture (20 μl) contained transcription buffer (20 mM HEPES pH 8.0, 40 mM KCl, 5 mM DTT, 1 mM EDTA and 10 mM MgCl₂), 20 ng of linearized LSP or HSP, 0.02 μM protein mixture, 0.3 μCi [P³²]-αUTP and 3 μl rNTP mixture (0.4 mM ATP, 0.15 mM CTP and GTP, 0.01 mM UTP). The mixture was incubated at 32°C for 30 min, and then terminated by the addition of an equal volume of 20 mM EDTA, 1% SDS, 300 mM sodium acetate and 20 μg calf thymus DNA. The transcription product was ethanol-precipitated and resuspended in 20 μl of loading buffer. The samples were resolved on 10% TBE-Urea gel. The gel was dried for 2 h and exposed to a phosphor screen (GE Healthcare) overnight. The transcription products were visualized using a Typhoon 9000 and analyzed using ImageQuant (GE healthcare). The quantified data were presented as mean ± SEM (standard error of the mean) from three independent experiments. The statistical significance was calculated with a two-tailed unpaired t test.

Two µg of in vitro transcribed snRNA were incubated with 25 µl of the purified SMN complex with 4.5 mM ATP, 3 mM MgCl₂ for 30 min at 37 °C. After the incubation, the samples were briefly pelleted by centrifugation and the supernatant was used for immunoprecipitation. Immunoprecipitation was performed as previously described^{88 (link)} using the mouse anti-Sm Y12 antibody. RNA was extracted using phenol/chloroform, precipitated and resolved in polyacrylamide gel containing 7 M urea and radioactivity detected by imaging phosphor screen (GE Healthcare) and developed by Typhoon 9000 (GE Healthcare).