FIG. 2.

Mapping of the modified nucleotides in SSU by a dNTP starvation assay and alkaline hydrolysis. (a) Total RNA was prepared from NOP1-silenced cells without induction (−Tet) or after 3 days of induction (+Tet). Total RNA (8 μg) was subjected to primer extension with radiolabeled oligonucleotide 1923, specified in Fig. S-7 in the supplemental material. Primer extension was performed using different concentrations of dNTPs (0.05, 0.5, and 5 mM) to detect the methylations. Extension products were analyzed on a 6% polyacrylamide-7 M urea gel, and DNA sequencing was performed with the same oligonucleotides used in the primer extension assay. Partial sequences are given; open arrows indicate structural stops. To control for the level of RNA in each sample, primer extension was performed with primers specific to U4 snRNA. The percentages of reduction in the levels of the stops and the standard deviations based on three independent experiments are given on the right. (b) Locations of the Nms on rRNA. Nm modifications in T. brucei (T), S. cerevisiae (Y), the plant A. thaliana (P), and humans (H) are indicated. (c) Mapping of the Nms by alkaline hydrolysis. RNA (50 μg) was subjected to alkaline hydrolysis. The same primer used for panel a was used for primer extension of the partially hydrolyzed RNA (L). The sequence of the DNA using the same primer is given, and mapping with different dNTP levels, as in panel a, is shown in the four right lanes. Arrows mark the positions of the methylated nucleotides, and their identities are given.