biostudies-literatureUnknown28(12)Wang JBox C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The defining feature of methylation guide RNAs is two sets of box C and D motifs that form kink-turn structures specifically recognized by L7Ae family proteins. Here, we engineered a new type of methylation guide that lacks C/D motifs and requires no L7Ae for assembly and function. We determined a crystal structure of a bipartite C/D-free guide RNA in complex with Nop5, fibrillarin and substrate in the active form at 2.2 Å resolution. The stems of new guide RNAs functionally replace C/D motifs in Nop5 binding, precisely placing the substrate for site-specific modification. We also found that the bipartite architecture and association of L7Ae with C/D motifs enhance modification when association of guide RNRNA (New York, N.Y.)1597-1605https://www.ebi.ac.uk/biostudies/studies/S-EPMC9670817biostudies-literatureMethylation guide RNAs without box C/D motifs.PMC9670817Yang ZWang JYe KfalseMethylation guide RNAs without box C/D motifs.Box C/D RNAs guide site-specific 2'-O-methylation of RNAs in archaea and eukaryotes. The defining feature of methylation guide RNAs is two sets of box C and D motifs that form kink-turn structures specifically recognized by L7Ae family proteins. Here, we engineered a new type of methylation guide that lacks C/D motifs and requires no L7Ae for assembly and function. We determined a crystal structure of a bipartite C/D-free guide RNA in complex with Nop5, fibrillarin and substrate in the active form at 2.2 Å resolution. The stems of new guide RNAs functionally replace C/D motifs in Nop5 binding, precisely placing the substrate for site-specific modification. We also found that the bipartite architecture and association of L7Ae with C/D motifs enhance modification when association of guide RN2022-01-01T00:00:00Z2022 Dec2025-05-29T21:47:14.735Z2025-05-29T21:47:14.735ZS-EPMC96708173612712510.1261/rna.079379.122