Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants

• Published in: Nucleic acids research Vol. 39; no. 11; pp. 4728 - 4742
• Main Authors: Iben, James R., Epstein, Jonathan A., Bayfield, Mark A., Bruinsma, Monique W., Hasson, Samuel, Bacikova, Dagmar, Ahmad, Daniel, Rockwell, Denise, Kittler, Ellen L. W., Zapp, Maria L., Maraia, Richard J.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.06.2011
• Subjects: Alleles; Base Sequence; Cell Nucleus - genetics; DNA, Mitochondrial - chemistry; Gene Duplication; genes; Genes, Fungal; Genome, Fungal; Genomics; mitochondria; mitochondrial DNA; Molecular Sequence Data; mutants; Mutation; Phenotype; Polymorphism, Single Nucleotide; RNA, Transfer, Ser - chemistry; RNA, Transfer, Ser - genetics; RNA, Transfer, Ser - metabolism; RNA-Binding Proteins - genetics; Schizosaccharomyces - genetics; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - genetics; sequence analysis; Sequence Analysis, DNA; single nucleotide polymorphism; Suppression, Genetic; transfer RNA
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkr066

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNASerUCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNASerUCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNASerUCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNASerUCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a 'reference' mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing.