Fig 2. tRNA^His interacting with BCDIN3D is phospho-methylated.

A. Synthetic pre-miR-145 with either [5’-OH], [5’-P], [5’-Pme1] or [5’-Pme2] ends was treated with Antarctic Phosphatase. Please note that pre-miR-145-5’-OH (cyan *) migrates faster than pre-miR-145 with [5’-P], [5’-Pme1] or [5’-Pme2] ends (red *) on a denaturing 15% polyacrylamide/urea gel. B. Synthetic pre-miR-145 [5’-P] was in vitro methylated with BCDIN3D using radioactive [³H]-SAM as methyl group donor prior to treatment with Antarctic Phosphatase. The left panel shows the ethidium bromide stained gel that was used for the autoradiography in the right panel, which shows only the in vitro methylated RNAs. Please note that only a small fraction of 5’P pre-miR-145 is dimethylated by BCDIN3D in vitro, explaining why AP treatment induces a gel shift in the left panel, which shows the total pre-miR-145, but not in right panel, which shows only the methylated pre-miR-145. C. Schematic showing the 5’ ends and the effect of Antarctic Phosphatase (AP) and Polynucleotide Kinase (PNK). D. Synthetic tRNA^His-5’-P was in vitro methylated with BCDIN3D using radioactive [³H]-SAM as methyl group donor prior to treatment with Antarctic Phosphatase. The left panel shows the ethidium bromide stained gel that was used for the autoradiography in the right panel. Please note that tRNA^His-5’-OH (cyan *) migrates slower than tRNA^His with [5’-P], [5’-Pme1] or [5’-Pme2] (red *) ends. E. RNAs purified from HeLa-S3-Flp-In and HeLa-S3-Flp-In-BCDIN3Df FLAG eluates were treated with mock, AP or PNK, and separated on a denaturing 15% polyacrylamide/urea gel and probed with the tRNA^His northern blot probe #1. Neither the treatment with AP nor T4 PNK shifted the migration of tRNA^His (Fig 1C). The treatment with PNK is a control to verify that the 5’ end is not 5’OH in BCDIN3Df-interacting tRNA^His.