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 are defective, mainly because they cannot load the substrate in the active conformation. For 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.
College of Biological Sciences, China Agricultural University, Beijing 100193, China; National Institute of Biological Sciences, Beijing 102206, China; Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;
Zuxiao
Z
Yang
National Institute of Biological Sciences, Beijing 102206, China;
Jinzhong
J
Lin
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China yekeqiong@ibp.ac.cn.
Keqiong
0000-0001-6169-7049
K
Ye
D000818:Animals; D017360:Arabidopsis / Q000378:metabolism; D001105:Archaea / Q000378:metabolism; D001483:Base Sequence; D006801:Humans; D051379:Mice; D009711:Nucleotides / Q000378:metabolism*; D012275:Oryza / Q000235:genetics; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D000094704:RNA, Guide, CRISPR-Cas Systems
D009711:Nucleotides; D012261:Ribonucleoproteins; D000094704:RNA, Guide, CRISPR-Cas Systems
RNA modification; RNA–protein complex; box C/D snoRNA; crystal structure