Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

Plasmid pFSeGFP-N2 expresses a fusion protein comprising N-terminal U 6 A-derived À2 FS peptide and a C-terminal eGFP tag. 293 T cells (7 Â 10 6 per dish) were seeded onto 15 Â 10 cm dishes and transfected with pFSeGFP-N2 (15 mg per dish) while in suspension using TransIT (Mirus Bio LLC). After 68 h, the proteasome inhibitor MG132 was added to the growth medium (to 20 mM), the cells were harvested 4 h later and lysed in 1 ml lysis buffer containing 50 mM Tris-HCl, pH 8, 5% glycerol, 0.5% IGEPAL CA-630, 200 mM NaCl, 1 mM DTT, 1 mM PMSF and 1 Â complete protease inhibitor tablet (Roche). After clarification, the supernatant was mixed with 50 ml glutathione sepharose beads and incubated for 30 min at 4 C with rotation to pre-clear the lysate. Subsequently, the supernatant was incubated with 20 ml GFP-TRAP_A (ChromoTek) for 2 h at 4 C with rotation to bind GFP-tagged proteins. The beads were washed three times with wash buffer (10 mM Tris-HCl pH 8, 0.1% IGEPAL CA-630, 150 mM NaCl, 0.5 mM EDTA, 1 mM PMSF and 1Â complete protease inhibitor tablet), transferred to an Ultrafree-MC spin column (0.22 mm; Millipore) and any remaining wash buffer removed by centrifugation. The beads were resuspended in 20 ml of 4Â Laemmli's buffer (250 mM Tris pH 6 Nucleic Acid Facility (PNAC). The excised band was subjected to reduction with 5 mM tris(2-carboxyethyl)phosphine and alkylation by addition of iodoacetamide (to 25 mM), followed by liquid removal and washes with 200 ml 100 mM ammonium bicarbonate with 50% acetonitrile. The gel pieces were dried in vacuo for 10 min and 25 ml of 100 mM ammonium bicarbonate containing 10 mg/ml modified trypsin (Promega) was added for trypsin digestion for 17 h at 32 C. Peptides were recovered and desalted using mC18 ZipTip (Millipore) and eluted to a maldi target plate using 2 ml alpha-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% acetonitrile, 0.1% trifluoroacetic acid. Peptide mass was determined using a Maldi micro MX MS (Waters) in reflectron mode and analysed with Masslynx software. For tandem ms/ms analysis (ESIMS/ MS), desalted peptides in 70% methanol, 0.2% formic acid were delivered to a ThermoFinnigan LCQ Classic ion-trap MS using a static nanospray needle (Thermo Proxeon). Peptide masses of interest were manually selected for fragmentation using manufacturer-recommended settings. Fragment ions were matched to possible sequence interpretations using MS-Product (http://prospector.ucsf.edu/).