Probing RNA in vivo with methylation guide small nucleolar RNAs

Most box C/D small nucleolar RNAs (snoRNAs) direct the formation of 2 ' -O- methytated nucleotides in ribosomal RNA and, apparently, other RNAs present in the nucleolar complex. Sites to be modified are selected by a long (> 1 0-nt) antisense guide sequence in the snoRNA and a distance measurement fro m a box D or D ' element that follows the snoRNA guide sequence. Modificati on of the substrate occurs in the region of complementarity, at a position five nucleotides upstream from box D/D '. Methylation can be targeted to no vel sites by expressing a snoRNA with a new guide sequence. Ln some cases m ethylation impairs the growth rate of the cell, indicating that a functiona lly important nucleotide has been altered. With a View to harnessing snoRNA -directed methylation for functional mapping, we have developed a method fo r constructing libraries of snoRNA genes that, in principle, can introduce methylation point mutations into any rRNA segment of interest. The strategy and procedures are described here, and preliminary results are presented t hat show the feasibility of using this technology to probe a region of the yeast large subunit rRNA that includes the core of the peptidyltransferase center. (C) 2001 Academic Press.