A new strategy for RNA structure probing in vivo: Targeting methylation small nucleolar RNPs to new sites in ribosomal RNA
Eukaryotic ribosomal RNAs (rRNAs) contain scores of 2′-O-methylated nucleotides that are formed by a large family of small RNA:protein machines called snoRNPs. The snoRNPs contain a small nucleolar RNA (snoRNA) and several proteins. Nucleotides to be modified are selected by the snoRNA component through base pairing with a long (10–21 nucleotides) guide sequence, and methylation is catalyzed by one of the snoRNP proteins. Modifications can be targeted to new sites by outfitting a snoRNA with a new guide sequence. In this thesis, targeting ribosome methylation to new sites in vivo was used to interfere with yeast rRNA function for the purpose of functional mapping. New snoRNAs were: (1) targeted to individual nucleotides known or predicted to have important roles, or (2) used in a gene library format to target all nucleotides in the reaction center of the ribosome.
For several sites examined in detail, interference was shown not to be due to a simple anti-sense phenomenon. The results indicate that this new strategy is, indeed, effective for identifying important rRNA regions. The deleterious effects of novel methylations on rRNA function were analyzed at different levels for sensitive nucleotides located in the peptidyl transferase center (PTC). Detailed analyses were conducted on: cell growth, global translation, rDNA transcription, pre-rRNA processing, and ribosome production. The results show that the interfering snoRNPs can cause severe growth defects by two general means: (1) disrupting ribosome production, causing a low yield or; (2) creating ribosomes that are functionally defective. This novel approach should also be useful for functional mapping of other RNAs in Saccharomyces cerevisiae, and for use in other organisms as well including humans.