Interference probing of rRNA with snoRNPs: A novel approach for functional mapping of RNA in vivo

Synthesis of eukaryotic ribosomal RNAs (rRNAs) includes methylation of scores of nucleotides at the 2'-OLribose position (Nm) by small nucleolar RNP complexes (snoRNPs). Sequence specificity is provided by the snoRNA component through base-pairing of a guide sequence with rRNA. Here, we report that methylation snoRNPs can be targeted to many new sites in yeast rRNA, by providing the snoRNA with a novel guide sequence, and that in some cases growth and translation activity are strongly impaired. Novel snoRNAs can be expressed individually or by a unique library strategy that yields guide sequences specific for a large target region. Interference effects were observed for sites in both the small and large subunits, including the reaction center region. Targeting guide RNAs to nucleotides flanking the sensitive sites caused little or no defect, indicating that methylation is responsible for the interference rather than a simple antisense effect or mis-guided chaperone function. To our knowledge, this is the only approach that has been used to mutagenize the backbone of rRNA in vivo.