By snoRNA gene disruption, we have verified snoRNA-directed modification for 41 of the 55 total ribose methylation sites in ribosomal RNA. Three additional sites for U24 have previously been demonstrated [Kiss-Laszlo et al., 1996]. Five other sites are strongly predicted to be guided by experimentally isolated C/D box snoRNAs but have not been confirmed yet because the deletion is lethal (U14), or the snoRNA is in an intron (U18 x 2 sites, snR38, snR39/snR59). Two more sites have been tentatively assigned to newly identified, expressed C/D box snoRNAs (snR55, snR70). This leaves four sites for which we could not assign a prediction (SSU-Am436), locate the methyl site (SSU-Gm?), or experimentally verify a prediction (LSU-Um2918, LSU-Gm2919). We believe there are several explanations for the inconclusive or missing snoRNA assignments.
Technical difficulties with the primer extension assay are the most likely cause for the two inconclusive methyl site assignments. The occurrence of other modifications or secondary structures on or near the target rRNA nucleotide can make interpretation of primer extensions difficult. For example, there is a very strong stop on wild-type rRNA at SSU-G1266 that prevents almost all read-through by the reverse transcriptase (Figure 4.5, lanes 5 and 6). This primer extension stop has been previously observed, and hypothesized to be some type of base modification [Bakin & Ofengand, 1995]. For the snR55 disruption mutant, a noticeable but far from complete loss of the stop at G1266 is visible (Figure 4.5, lane 10), as well as improved read-through of larger products. We believe that the decreased intensity of the stop indicates loss of the Um1265 ribose modification, although we cannot be certain by this assay. We also believe at least one unknown base modification or secondary structure element at SSU-C1640 and/or SSU-G1641 is responsible for difficulty in visualizing the small, faint ribose methyl stop for SSU-Cm1637 (Figure 4.6, lanes 5 and 6). The uncharacterized, strong stops just downstream of Cm1637 make loss of the weak stop in the snR70 disruption mutant (lanes 7 and 8) somewhat less convincing. Use of an alkaline hydrolysis primer extension assay [Kiss-Laszlo et al., 1996], or another apparently more sensitive methyl assay [Yu et al., 1997] may give clearer results in these cases.
Incomplete or undetected methyl site loss could also be due to functional redundancy of snoRNAs. The snR52 disruption mutant showed no change at one of its two predicted methylation sites, LSU-Um2918. However, we believe that snR52 may still be involved in modification at Um2918 based on a perfect 11 bp rRNA complementarity, a high snoRNA score of 34.46 bits, and the fact that snR52 has already been confirmed to guide methylation at another methyl site. In this case, we believe an unidentified, functionally redundant snoRNA may exist.
A more obvious example of functional redundancy appears to exist between snR39, and an apparent homologue, snR59. Both snoRNAs are intron-encoded, one within the ribosomal protein gene YL8A on chromosome VII, and one within YL8B on chromosome XVI. The snoRNAs appear to have been duplicated relatively recently with their host genes [Mizuta et al., 1995]. An alignment of the snoRNAs shows that only a single nucleotide differs between the box C, box D, and rRNA complementary regions. We did not detect any other pairs of close homologues, although less similar but functionally redundant snoRNAs could exist. Multiple disruptions of potentially redundant snoRNAs may be necessary to test for methylation guide function.
It is possible that some sites may be modified by a different mechanism. Prokaryotes contain a fraction of the rRNA modifications found in eukaryotes and do not appear to contain snoRNAs. Thus, a handful of site-specific enzymes may accomplish these modifications without snoRNAs. Conservation of such enzymes in yeast would obviate the need for several specific guide snoRNAs. In yeast, three 2'-O-methyl groups (SSU-Cm1637, LSU-Gm2616, LSU-Um2918) are conserved with homologous modified positions in prokaryotes (E. coli SSU-Cm1402, LSU-Gm2251, and LSU-Um2552, respectively [Raue et al., 1988]). For one of these sites, we have found and confirmed a yeast guide snoRNA (snR67). However, at the other two conserved sites, we have either failed to verify a guide snoRNA (Um2918), or failed to obtain definitive evidence of methyl site loss (Cm1637). Protein methyltransferases targeting these specific sites may account for our difficulty in finding and/or verifying guide snoRNAs in these cases.