We began our search for new snoRNAs by identifying family members that target known 2'-O-ribose methyl sites. Because there is very little information on ribose methyl modifications for S. cerevisiae rRNA, we inferred the position of 42 ribose methyls based on mapping data from S. carlsbergensis [Maden, 1990]. We applied the snoRNA search program to the sequence of S. cerevisiae, and extracted from the output only candidate snoRNAs that could target one of the inferred methyl sites. These candidates were divided based on target methyl site and sorted by score, producing 42 different lists of best-to-worst snoRNA predictions, one for each methyl site. Depending on search parameter cutoffs and the specific target methylation site, the program found dozens to over a hundred predictions for each methylation site. Candidates overlapping predicted protein coding regions were noted and disfavored relative to other strong, non-overlapping candidates. Seven previously published snoRNAs have been predicted to guide methylation at eight of the 42 sites (U14, U18, U24, snR39, snR39b, snR40, snR41; see Table 4.2). Our searches did not show improved snoRNA predictions over the previously identified snoRNAs, so we did not pursue new assignments for these eight sites.
We tested the top scoring snoRNA gene predictions corresponding to the remaining sites by gene disruption [Baudin et al., 1993]. Each snoRNA-disrupted strain was tested for the ability to methylate at the predicted rRNA site by a dNTP concentration-dependent primer extension assay [Maden et al., 1995,Kiss-Laszlo et al., 1996]. Out of 30 gene disruptions, 24 loci were verified as methylation guide snoRNAs. Seven of these had been previously identified as C/D box snoRNAs (Table 4.2: snR13, snR47, snR48, snR74, snR76, snR77, snR79). Seventeen snoRNAs were new (Table 4.2: snR50-snR57, snR60-snR63, snR66, snR68-snR71). Two sample primer extension gels demonstrating typical loss of rRNA methylation sites for snoRNA disruption mutants appear in Figures 4.3 and 4.4. Primer extension assays for two of the snoRNA disruption mutants, snR55 and snR70, showed a noticeable but minor change in the primer extension pattern at the expected sites (Figures 4.5 and 4.6, thus we qualify these assignments as ``inconclusive''. Twenty-three additional primer extension gels for the other verified snoRNAs can be found at [Lowe & Eddy, 1998]. None of these snoRNA gene disruptions was lethal, nor did we observe impaired growth on rich media.
Given the proposed rule of one snoRNA per methylation site, 24 verified guide snoRNAs implies assignments to 24 of the 34 known but unassigned methyl sites. However, we found that some of these snoRNAs guide modification at more than one methylation site, as previously seen for U24 [Kiss-Laszlo et al., 1996]. The search program predicted and we experimentally verified one additional methylation target site for snR47, snR48, and snR51 (Table 4.2). We also found an additional target site for snR41, a snoRNA previously predicted to guide at a different methyl site [Kiss-Laszlo et al., 1996]. We verified snR41 methylation guide function for both the previously predicted site (SSU-Gm1123) as well as the newly predicted site (SSU-Am541). With these additional site assignments, 28 of 34 known but previously unassigned sites can be attributed to guide snoRNAs.
We have made a tentative methylation assignment to one additional new C/D box snoRNA, snR59 (Table 4.2), whose expression we have verified (see below). We predict snR59 guides methylation at the same site already assigned to snR39, LSU-Am805. Neither snR59 nor snR39 has been checked for functional redundancy. Both snoRNAs are intronic, thus we did not attempt to generate null mutants. The homologous knockout method that we chose uses a large marker gene to replace the target snoRNA, and we have observed that such insertion-based disruptions appear to interfere with host protein intron splicing.
Our searches gave no strong snoRNA candidates for four of the remaining six methylation sites: LSU-Cm648, LSU-Gm1448, LSU-Am2279, and LSU-Gm2919. The one common factor among these sites is that they are all one nucleotide adjacent to methyl sites for which snoRNA assignments or strong predictions have been made. This led us to believe that an unusual snoRNA interaction may be responsible for methylation in these cases. Our disruption of snR13 showed loss of the predicted target LSU-Am2278 (described above), as well as the ribose methyl one nucleotide adjacent, LSU-Am2279 (see Figure 4.4). U24 disruption previously showed loss of LSU-Am1447, as expected, plus the adjacent methyl at LSU-Gm1448 [Kiss-Laszlo et al., 1996]. Taking these data into consideration, we predicted that U18, in addition to modifying LSU-Am647 [Kiss-Laszlo et al., 1996], may also direct methylation at LSU-Cm648. We disrupted the intron-encoded U18 to test our prediction, but could not assay a mutant haploid clone since our deletion was lethal. Because U18 is nonessential [Balakin et al., 1996], we believe we inadvertently disrupted function of the essential host gene, elongation factor-1. We also have a strong candidate snoRNA targeting the site adjacent to Gm2919, snR52. Our disruption of snR52 did not result in loss of either Um2918 or Gm2919, possibly due to functional redundancy (see discussion). We present a model in the discussion that may account for the two observed (snR13, U24) and one hypothesized (U18) methylation assignments.
Out of the 42 previously mapped ribose methyl sites, this leaves three sites for which we could not assign a C/D box snoRNA: SSU-Am436 for which we have no prediction, plus LSU-Um2918 and LSU-Gm2919, for which we could not confirm our snR52 prediction.