Figure 1. Increased RNMT (RNA guanine-7-methyltransferase) expression increases cellular mRNA cap methyltransferase activity.

a) Whole cell extracts from the IMEC (Tert-immortalised human mammary epithelial cell) lines indicated were analysed by Western Blot for expression of RNMT, RNMT-GFP, c-MYC and β-Tubulin. The migration of endogenous RNMT and RNMT-GFP is indicated. Cells were lysed in Triton lysis buffer (10 mM Tris, pH 7.05, 50 mM NaCl, 30 mM Na₄ pyrophosphate, 50 mM NaF, 5 μM ZnCl₂, 10% glycerol, 0.5% Triton X-100, 10μM Leupeptin, 1μM Pepstatin and 0.1mg/ml Aprotinin). Polyclonal anti-RNMT antibodies were raised against full length recombinant GST-RNMT in sheep and affinity-purified and other antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). b) The same extracts were analysed for their cap methyltransferase activity. Assay was adapted from (Pillutla et al., 1998). A 55 base strand of in vitro transcribed RNA was capped using Vaccina Virus capping enzyme and ³²P alpha-GTP according to manufacturers instructions (Epicentre Biotechnologies, Madison, WI, USA). 10-40ng G³²P-RNA was incubated with 2μg cell extract in 10μl 50mM Tris pH 8, 6mM KCl, 1.25mM MgCl₂, 100nM S-adenosyl methionine at 37°C for 10mins. RNA was purified, precipitated and resuspended in 4μl 50mM NaAcetate and 0.25units P1 nuclease for 30mins at 37°C. Cap (GpppG) and methyl-cap (m7GpppG) were resolved in 0.4M Ammonium Sulphate thin layer chromatography using PEI cellulose plates. Standards were visualised by UV light to establish correct migration. Labelled spots were visualised and quantified by autoradiography, and percentage conversion of GpppG to m7GppG was calculated. Average results for three independent experiments are shown and error bars indicate the standard deviation. A representative assay is also shown including a negative control (-), in which no extract was added and a positive control (+), in which recombinant RNMT was added. c) IMEC cells were transfected with two independent siRNAs directed against RNMT or Cyclophilin B (negative control), according to manufacturer’s instructions (Dharmacon Inc, Lafayette, CO, USA). After 24hrs, cell extracts were prepared and analysed for their cap methyltransferase activity, as above, except 4μg of cell extract was used.