RNA analysis

Total RNA was extracted from cells using the Trizol reagent (Life Technologies) under manufacturer's recommended conditions. RNA was fractionated on 12% polyacrylamide (PAGE)-7 M urea/TBE gels, electroblotted to Zeta-Probe or Zeta-probe GT membranes (BioRad), and hybridized to gene-specific oligoprobes as described previously (34,35) or using Rapid-hyb buffer (GE Healthcare). Signals were visualized by sensitive X-ray film or, where indicated in legends, by phosphorimaging (Typhoon™ imaging system, GE Healthcare). For aminoacylation analyses, total RNA was dissolved on ice in 0.1 M sodium acetate, pH 5.2 and fractionated on acidic 6.5% PAGE-7 M urea gels essentially as described previously (36,37). Samples were deacylated by heating for 10 min at 75°C followed by 30 min at 37°C in 1.5 volumes of 0.5 M Tris–HCl, pH 9.0. To analyze the primary structure of tRNA products in EtBr-treated cells, total RNA was isolated, tRNAs deacylated and circularized by T4 RNA ligase (MBI Fermentas) as described (38), reverse-transcribed with oligonucleotides cser1 or cleu1 and PCR-amplified using oligonucleotides cser1 and cser2 for analysis of tRNA^Ser(UCN) or cleu1 and cleu2 for analysis of tRNA^Leu(UUR), essentially as described previously (31). The high-molecular weight tRNA^Leu(UUR) products induced by doxycycline treatment were gel-extracted, circularized and amplified similarly. PCR products were cloned (TOPO TA cloning kit, Invitrogen) and inserts Sanger-sequenced using M13 forward primer and standard dye-terminator technology as described previously (39).