Maintaining a conserved methylation in plant and insect U2 snRNA through compensatory mutation by nucleotide...

The extensive post-transcriptional modification of U2 snRNA is required for spliceosome assembly and pre-mRNA splicing in vertebrates. However, the rare modification of U2 snRNA in yeast implies a different mechanism for regulating spliceosome biogenesis in single-celled eukaryotes. To understand the evolutionary pattern of U2 snRNA methylation, we determined for the first time, the 2'-O-methylations of U2 snRNA in Oryza sativa, Arabidopsis thaliana and Drosophila melanogaster, and revealed two methylations which are conserved in a crucial region of U2 snRNA in plants. Interestingly, one of the methylations, U2-Cm29 is also methylated in D. melanogaster, but not in vertebrates. According to the methylation of U2-C29, computational analysis of databases identified three canonical box C/D snoRNAs, named OsmgU2-29, AtmgU2-29 and DmmgU2-28, as small methylation guides of U2 snRNA from O. sativa, A. thaliana and D. melanogaster, respectively. Although very divergent in their sequence, the three snoRNAs exhibit in common an 11 nucleotide-long sequence complementarity to corresponding U2 snRNA, implying a functional constraint on the modification during evolution. Interestingly, a nucleotide is found to be inserted both in U2 snRNA and DmmgU2-28 and maintains a perfect match of duplex specifying the methylation of C28 in Drosophila U2 snRNA. This is the first time a new model is being provided for compensatory mutations between a small guide RNA and its target by nucleotide insertion, instead of the known nucleotide substitution. In contrast to small Cajal body-specific RNAs (scaRNAs), the snoRNAs are similar to the reported singlet guide RNAs and are known to localize in nucleolus.