Determination of Thiolation Status in mt-tRNAs

Determination of the thiolation status of mt-tRNAs was carried out as previously described [18 (link), 79 (link)]. Briefly, a volume of approximately 500 μl of worm pellet obtained from mixed-stage populations of liquid-cultured worms was processed using the “Isolation of small and large RNA” NucleoSpin miRNA kit (Macherey-Nagel) and the manufacturer’s instructions. 10 to 20 μg of total small RNA were run on 10% polyacrylamide/8 M urea gels with or without 0.01 mg/ml APM ([p-(N-acrylamino)-phenyl]mercuric chloride). APM was synthesized and kindly provided by Prof. Stephane Vincent [30 (link)]. Gels were run at 200 V for 3 h at 4°C and electroblotted onto positively charged nylon membranes. The RNA was crosslinked to the membrane by exposure to UV light for 2 min and baking at 80°C for 45 min. Pre-hybridization and hybridization were performed with Dig Easy Hyb (Roche) according to the manufacturer’s instructions. mt-tRNAs were detected with specific DIG-labeled synthetic oligodeoxynucleotides (S2 Table) used at 10 nM final concentration. The blots were detected using the DIG luminescent detection CDP-Star kit (Roche) according to the manufacturer´s instructions.