Figure 6. In vitro methylation activity assay results using co-expressed and purified sR3 sRNP and U14 snoRNP.

A). Sequences of the target RNAs complementary to upstream sequences of box D of sR3 and U14 and box D′ of sR3 are shown. Radiolabeled nucleotide is indicated by “*C” and is the methylation target nucleotide. B). Various target RNA containing α-³²P-cytidine were incubated with co-expressed and purified sR3 RNP, or reconstituted RNP control, phenol/chloroform extracted, ethanol precipitated, and RNase P1 digested and separated on a thin layer chromatography plate. The loading position and those of cytidine monophosphate (unmethylated or methylated) were indicated by “Origin”, “CMP” and “mCMP”, respectively. The target RNA complementary to box D, D′ guide of sR3 RNA, and to box D guide of U14 RNA are indicated by “D”, “D′”, “U14”, respectively. C). Methylation activity analysis of U14 snoRNP at different temperatures (Lanes 1–3). Co-purified sR3 sRNP with target RNA complementary to its box D is included as a positive control (Lane 4).