Box C/D guide RNAs recognize a maximum of 10 nt of substrates

Box C/D RNAs guide site-specific 2′-O-methylation of RNAs in archaea and eukaryotes. The spacer regions between boxes C to D′ and boxes C′ to D contain the guide sequence that can form a stretch of base pairs with substrate RNAs. The lengths of spacer regions and guide-substrate duplexes are variable among C/D RNAs. In a previously determined structure of C/D ribonucleoprotein (RNP), a 12-nt-long spacer forms 10 bp with the substrate. How spacers and guide–substrate duplexes of other lengths are accommodated remains unknown. Here we analyze how the lengths of spacers and guide-substrate duplexes affect the modification activity and determine three structures of C/D RNPs assembled with different spacers and substrates. We show that the guide can only form a duplex of a maximum of 10 bp with the substrate during modification. Slightly shorter duplexes are tolerated, but longer duplexes must be unwound to fit into a capped protein channel for modification. Spacers with 12 nucleotides, the excessive unpaired sequences near the box C/C′ side are looped out. Our results provide insight into the substrate recognition mechanism of C/D RNA and refute the RNA-swapped model for dimeric C/D RNP.