FIGURE 5.

Target pre-mRNA 2′-O-methylation for splicing regulation. (A) Mutant adenovirus pre-mRNA free of cryptic branch sites is schematized. Three A-to-G substitutions are indicated. (B) Splicing inhibition by artificial box C/D guide RNA targeting the branch point adenosine. The figure legend is essentially the same as that to Figure 3A. The numbers in parentheses indicate the percentage of splicing that occurred (calculated according to the relative ratio of lariat intron/pre-mRNA in lanes 1 and 2, with the ratio in lane 2 being set to 100%). (C) Quantitation of 2′-O-methylation at the branch point adenosine. The figure legend is identical to that to Figure 3B. Note, no 2′-O-methylated adenosine was detected in lane 2. (D) Native gel analysis. Reactions were carried out exactly as described (see Fig. 5B, lanes 1,2; Fig. 3A, lanes 1,2), except that, after in vitro transcribed U2 was injected, oocytes were incubated for only 10 min. Immediately following the 10-min incubation, nuclei were isolated, broken with loading dye, and directly loaded onto a 4% native polyacrylamide gel. (Lane 1) Oocytes with a box C/D guide RNA targeting the branch point adenosine; (lane 2) oocytes with a random box C/D guide RNA. Complexes H, A, B, and C, as well as high-order complexes, are indicated. Asterisks denote mutant adenovirus pre-mRNA (free of cryptic branch sites), mutant lariat intron, and box C/D guide RNA targeting the branch point of the mutant pre-mRNA.