Activity and assembly of bipartite methylation guide RNAs without C/D motifs. (A) Secondary structure models of sR1c, BMG1, BMG2, and BMG3. Box C, D, C′, and D′ motifs are colored in red and spacers in yellow. The D′ and D substrates (sub) are shown in purple with modification sites marked by circles. The sequences deleted in BMG1 mutants are indicated with lines. (B) Methylation assay. Ss RNPs were assembled with guide RNAs (1 µM), Nop5/Fib complex (2 µM), and L7Ae (3 µM). Ct RNPs were assembled with guide RNAs (2 µM), Nop56/Nop58/Fib complex (∼2 µM), and Snu13 (6 µM). The assembled RNPs were incubated with the D or D′ substrates (30 µM) or mixed premethylated substrates (sub-m) in the presence of 30 µM cold SAM and trace amounts of [methyl-3H] SAM for 20 min at 70°C for Ss RNPs or at 50°C for Ct RNPs. RNAs were resolved in denaturing PAGE and visualized by 3H autoradiography. (C) Activity of Ss RNPs in the presence and absence of L7Ae. (D) EMSA of Ss RNPs. Each RNA (1 µM) was assembled with L7Ae (1 or 2 µM, indicated by a small or large plus sign) and Nop5/Fib complex (2 µM) in various combinations, resolved in 5% native PAGE and strained by SYBR Gold. L7Ae*1 and L7Ae*2 RNPs contain one and two copies of L7Ae. RNP1 and RNP2 refer to monomeric and dimeric species. Misfolded RNAs that migrated slower and bound poorly to L7Ae are marked with asterisk.
