Systematic mapping of 2'-O-methylation sites in rRNAs implicates new strategy for ribose methylation in Arabidopsis [FIB CLIP]

2'-O-methylation (2'-O-me) is one of the most abundant covalent modifications in ribosomal RNAs (rRNAs) vital for the functionality of the ribosome, in particular, translational regulation. Aberrant 2'-O-me causes developmental defects in animals and is associated with human cancer. However, a lack of systematic delineation of 2'-O-me sites significantly impedes our understanding of 2'-O-me in plants. Here we report the global profiling of 2'-O-me in Arabidopsis rRNAs, leading to the identification of 221 2'-O-me sites, including 137 novel ones compared to the Arabidopsis database that documents predicted rRNA 2-O-me sites. We show that 65% of yeast and 53% of human 2'-O-me sites are conserved in Arabidopsis. We discover not only 2'-O-me guided by box C/D snoRNAs, but also unprecedentedly a prevalent snoRNA-independent mechanism in Arabidopsis: 48% of the 2'-O-me sites are devoid of snoRNAs, including some conserved sites where snoRNAs are indispensable in human and yeast. Through HITS-CLIP (high-throughput sequencing of RNAs isolated by cross-linking immunoprecipitation), we show that Fibrillarin (FIB1) and FIB2 predominantly associate with box C/D snoRNAs in vivo and uncover previously unknown snoRNAs. By generating and analyzing mutants in the FIB genes, we demonstrate that they are responsible for the ribose methylation at many rRNA sites regardless of their snoRNA-dependent or independent nature. We report, for the first time, extensive 2'-O-me of chloroplast rRNAs and, intriguingly, the dependence of FIB1/2 at some sites. Our studies reveal the landscape of 2'-O-me in nuclear and chloroplast rRNAs and provide novel insights into the deposition of rRNA 2'-O-me. Overall design: RNAs associated FIB1/2 were immunoprecipitated and extracted from 14-day-old Arabidopsis seedlings expressing GFP-FIB1 and CFP-FIB2. GFP was used as negative control. Four replicates were included.