2.7. MBP-MS2 RNA Affinity Purification

To generate lysate for MBP-MS2 RNA affinity, HeLa cell monolayers were infected with PV1-3′MS2 or wild type PV1 at an MOI of 5. After 30 min adsorption at room temperature, DMEM-8% NCS was added and cells were incubated at 37 °C for 4 h (wild type PV1) or 4 to 6 h (PV1-3′MS2). At indicated times post-infection, cells were washed twice with warm PBS, followed by cross linking with 0.4% formaldehyde in PBS for 10 min at room temperature with shaking. Formaldehyde was quenched with 0.25 M glycine for 5 min at room temperature. Cells were washed twice with ice-cold PBS, scraped, and pelleted at ~1000 rcf for 5 min at 4 °C. Cells were lysed in 100 μL NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% NP-40) per 100 cm² plate of HeLa cells for 25 min on ice. Cell debris was pelleted, supernatant was collected, and protein concentration was determined via Bradford assay.

RNA affinity resin was generated by incubating 40 μg recombinant MBP-MS2 protein per 10 μL washed magnetic amylose resin (NEB) in buffer B (20 mM Tris HCl, pH 7.5, 100 mM KCl, 2.5 mM MgCl₂, 2 mM DTT, 0.5 mM Pefabloc SC (Roche, Basel, Switzerland), 20 U/mL RNasin (Promega, Madison, WI, USA)) for 1 h with rotation at 4 °C. Unbound MBP-MS2 was removed, and the resin was blocked in buffer B with 1 μg BSA and 1 μg tRNA per μL magnetic amylose resin. After 1 h at 4 °C, the resin was washed 3 times in buffer B and then incubated with lysates from infected cells. NP-40 lysates from wild type PV1 or PV1-3′MS2 infected cells were pre-blocked with equal volume amylose resin for 1 h at 4 °C prior to incubation with MBP-MS2 amylose (1 μL resin per 10 μg lysate). Samples were incubated for 4 h at 4 °C with rotation. The resin was washed 3 times with buffer B and complexes were eluted with 30 mM maltose in buffer B for 30 min at 4 °C. Purified samples were either heated for 1 h at 70 °C for mass spectrometry analysis or LSB was added to samples which were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were analyzed by SYPRO Ruby staining (Lonza, Basel, Switzerland) or proteins were transferred to a PVDF membrane and analyzed by Western blot. For Western blot analysis, antibodies directed against PABP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), hnRNP C1 + C2 (Abcam, Cambridge, MA, USA), or AUF1 (Millipore, Temecula, CA, USA) were used. For mass spectrometry analysis, eluted samples were digested with trypsin and subjected to nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). For purification of eluted RNA, 10% of final elution volume was subjected to phenol/chloroform extraction and ethanol precipitation.