Dissecting the role of conserved box C/D sRNA sequences in di-sRNP assembly and function

In all three kingdoms of life, nucleotides in riboso-mal RNA (rRNA) are post-transcriptionally modified. One type of chemical modification is 20-O-ribose methylation, which is, in eukaryotes and archaea, performed by box C/D small ribonucleoproteins (box C/D sRNPs in archaea) and box C/D small nu-cleolar ribonucleoproteins (box C/D snoRNPs in eu-karyotes), respectively. Recently, the first structure of any catalytically active box C/D s(no)RNP determined by electron microscopy and single particle analysis surprisingly demonstrated that they are dimeric RNPs. Mutational analyses of the Nop5 protein interface suggested that di-sRNP for-mation is also required for the in vitro catalytic activity. We have now analyzed the functional rele-vance of the second interface, the sRNA interface, within the box C/D di-sRNP. Mutations in conserved sequence elements of the sRNA, which allow sRNP assembly but which severely interfere with the cata-lytic activity of box C/D sRNPs, prevent formation of the di-sRNP. In addition, we can observe the dimeric box C/D sRNP architecture with a different box C/D sRNP, suggesting that this architecture is conserved. Together, these results provide further support for the functional relevance of the di-sRNP architecture and also provide a structural explan-ation for the observed defects in catalysis of 20-O-ribose methylation