Mapping of rRNA Ribose Methylations by Primer Extension

Reverse transcriptase primer extensions were carried out on total RNA with 22-26 nt mapping primers complementary to ribosomal RNA. The sequences of all mapping primers are available by WWW [Lowe & Eddy, 1998]. Total yeast RNA (0.4 $\mu$g/$\mu$l) was annealed with end-labeled mapping primers (0.15 pmol/$\mu$l) at 60C for 4 min. Primer extensions were carried out in 5 $\mu$l reactions containing 0.8 $\mu$g RNA and 0.3 pmol ³²P end-labeled primer in the presence of 50 mM Tris-Cl pH 8.6, 60 mM NaCl, 9 mM MgCl₂, 10 mM dithiothreitol, 1 mM each dNTP, and 0.2 U/$\mu$l AMV reverse transcriptase for 30 minutes at 37C. Low dNTP concentration reactions were carried out the same except using 0.004 mM of each dNTP and 5 mM MgCl₂. Each reaction was analyzed by electrophoresis next to an RNA sequencing ladder on an 8 % polyacrylamide gel. RNA sequencing ladders were generated by AMV reverse transcription in a similar manner as the above primer extensions. Total yeast RNA (0.2 $\mu$g/$\mu$l) was annealed with end-labeled mapping primers (0.15 pmol/$\mu$l) at 60C for 4 min. Sequencing primer extensions were carried out in 5 $\mu$l reactions containing 0.4 $\mu$g RNA and 0.3 pmol end-labeled primer in the presence of 50 mM Tris-Cl pH 8.6, 60 mM NaCl, 10 mM MgCl₂, 10 mM dithiothreitol, 0.33 mM each dNTP, 0.2 mM one of four (A,C,G,T) dideoxy NTPs, and 0.2 U/$\mu$l AMV reverse transcriptase for 30 minutes at 37C.