A Computational Screen for Methylation Guide snoRNAs in Yeast

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Random sequences were generated by a fifth-order Markov chain based on 6-mer frequencies within the yeast genome.

snoRNA disruptions were generated by homologous gene replacement in S. cerevisiae haploid strain yM4585 (Mat a his3Δ200 lys2-801 leu2-3,2-112 trp1-901 tyr1-501 URA3+ ADE2+ CAN^S) and diploid strain yM4587 (Mat a/Mat α his3Δ200/his3Δ200 lys2-801/ lys2-801 leu2-3,2-112/ leu2-3,2-112 trp1-901/ trp1-901 tyr1-501/ tyr1-501 URA3+/ URA3+ ADE2+/ ADE2+ CAN^S/ can^r). A polymerase chain reaction (PCR)–based HIS3 insertion scheme was used as described by A. Baudin, O. Ozier-Kalogeropoulos, A. Denouel, F. Lacroute, and C. Cullin [Nucleic Acids Res. 21, 3329 (1993)], with a protocol provided by L. Riles. Transformants growing on yeast extract, peptone, and dextrose His plates were picked and assayed by PCR for correct integration of the HIS3 marker gene replacing the target snoRNA.

Mapping of rRNA 2′-O-methyl sites was based on previously described methods from B. E. H. Maden, M. E. Corbett, P. A. Heeney, K. Pugh, and P. M. Ajuh [Biochemie 77, 22 (1995)] and (6). The sequences of all mapping primers are available on the World Wide Web (WWW) (28). Total yeast RNA (0.4 μg/μl) was annealed with end-labeled mapping primers (0.15 pmol/μl) at 60°C for 4 min. Primer extensions were carried out in 5-μl reactions containing 0.8 μg of RNA and 0.3 of pmol ³²P–end-labeled primer in the presence of 50 mM tris-Cl (pH 8.6), 60 mM NaCl, 9 mM MgCl₂, 10 mM dithiothreitol, 1 mM concentrations of each dNTP, and avian myeloblastosis virus reverse transcriptase (0.2 U/μl) for 30 min at 37°C. Low dNTP concentration reactions were carried out in the same manner except with 0.004 mM concentrations of each dNTP and 5 mM MgCl₂. Each reaction was analyzed by electrophoresis next to an RNA sequencing ladder on an 8% polyacrylamide gel.

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All computer codes, snoRNA search results, primer sequences, gel images, and other referenced data can be accessed on the WWW at rna.wustl.edu/snoRNAdb/

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The S. cerevisiae database is available at: genome-www.stanford.edu/Saccharomyces/

We thank L. Lutfiyya and L. Riles from the M. Johnston lab for protocols and guidance in all aspects of yeast handling, gene disruptions, and colony PCR and RNA preparations; M. Fournier, J. Ni, D. Samarsky, and B. E. H. Maden for helpful discussions and sharing of unpublished observations; S. Johnson and L. Lutfiyya for careful reading of the manuscript; and M. Johnston for providing the haploid strain yM4585 and diploid strain yM4587. Supported by NIH grant R01-HG01363 and by a gift from Eli Lilly.