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

Iben JR, Epstein JA, Bayfield MA, Bruinsma MW, Hasson S, Bacikova D, Ahmad D, Rockwell D, Kittler EL, Zapp ML, Maraia RJ - Nucleic Acids Res. (2011)

Bottom Line: Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype.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.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT
We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-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 tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1- 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.

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The Sla1p and suppressor-tRNASerUCA-C47:6U components of the yMWB3-15 Strain. (A) Precursor (left) and mature (right) suppressor tRNASerUCA-C47:6U harbors a C47:6U mutation on the variable arm indicated by a blue circle and arrow. The three anticodon residues are boxed. The intron in the precursor is demarcated by the solid lines at the intron junctions. (B) Structure of the LAM (gold) and RRM1 (green) of La protein bound to a 10-mer RNA ending in UUU-3′OH (orange) modified from PDB 2VON (71). The highly conserved β-sheet surface residues that extend from the RRM1 binding surface of Sla1p, Y157 and F201 mutated for this study, are highlighted in blue. Loop-3 which comprises part of the canonical RNA-binding site of RRM1 (47) is also indicated.
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Figure 1: The Sla1p and suppressor-tRNASerUCA-C47:6U components of the yMWB3-15 Strain. (A) Precursor (left) and mature (right) suppressor tRNASerUCA-C47:6U harbors a C47:6U mutation on the variable arm indicated by a blue circle and arrow. The three anticodon residues are boxed. The intron in the precursor is demarcated by the solid lines at the intron junctions. (B) Structure of the LAM (gold) and RRM1 (green) of La protein bound to a 10-mer RNA ending in UUU-3′OH (orange) modified from PDB 2VON (71). The highly conserved β-sheet surface residues that extend from the RRM1 binding surface of Sla1p, Y157 and F201 mutated for this study, are highlighted in blue. Loop-3 which comprises part of the canonical RNA-binding site of RRM1 (47) is also indicated.

Mentions: To try to better understand this, we developed a genetic screen in S. pombe that relies on accumulation of a functional suppressor tRNASerUCA-C47:6U despite a debilitating substitution of a G:C to a G:U basepair in its variable arm (Figure 1A). In cells carrying wild-type Sla1p, the pre-tRNASerUCA-C47:6U from this allele is matured and functionally suppresses a nonsense codon in ade6-704, alleviating accumulation of red pigment. However, this allele does not produce tRNA in the sla1-rrm1 mutant carrying the point mutations in the RRM1 β-sheet surface at levels sufficient to mediate TMS (32). We isolated spontaneous revertants that restore tRNASer-mediated suppression in sla1-rrm cells and mapped the phenotypic mutation by whole genome sequencing. We establish that this approach is applicable to S. pombe and report that the phenotypic mutation is duplication of the suppressor tRNASer gene. We show that a surprising disproportionate increase in suppressor-tRNA levels that occurs with tRNA gene duplication requires sla1-rrm, provoking a model in which activation by La of tRNA maturation is dependent on the concentration of the pre-tRNA.Figure 1.


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

Iben JR, Epstein JA, Bayfield MA, Bruinsma MW, Hasson S, Bacikova D, Ahmad D, Rockwell D, Kittler EL, Zapp ML, Maraia RJ - Nucleic Acids Res. (2011)

The Sla1p and suppressor-tRNASerUCA-C47:6U components of the yMWB3-15 Strain. (A) Precursor (left) and mature (right) suppressor tRNASerUCA-C47:6U harbors a C47:6U mutation on the variable arm indicated by a blue circle and arrow. The three anticodon residues are boxed. The intron in the precursor is demarcated by the solid lines at the intron junctions. (B) Structure of the LAM (gold) and RRM1 (green) of La protein bound to a 10-mer RNA ending in UUU-3′OH (orange) modified from PDB 2VON (71). The highly conserved β-sheet surface residues that extend from the RRM1 binding surface of Sla1p, Y157 and F201 mutated for this study, are highlighted in blue. Loop-3 which comprises part of the canonical RNA-binding site of RRM1 (47) is also indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113579&req=5

Figure 1: The Sla1p and suppressor-tRNASerUCA-C47:6U components of the yMWB3-15 Strain. (A) Precursor (left) and mature (right) suppressor tRNASerUCA-C47:6U harbors a C47:6U mutation on the variable arm indicated by a blue circle and arrow. The three anticodon residues are boxed. The intron in the precursor is demarcated by the solid lines at the intron junctions. (B) Structure of the LAM (gold) and RRM1 (green) of La protein bound to a 10-mer RNA ending in UUU-3′OH (orange) modified from PDB 2VON (71). The highly conserved β-sheet surface residues that extend from the RRM1 binding surface of Sla1p, Y157 and F201 mutated for this study, are highlighted in blue. Loop-3 which comprises part of the canonical RNA-binding site of RRM1 (47) is also indicated.
Mentions: To try to better understand this, we developed a genetic screen in S. pombe that relies on accumulation of a functional suppressor tRNASerUCA-C47:6U despite a debilitating substitution of a G:C to a G:U basepair in its variable arm (Figure 1A). In cells carrying wild-type Sla1p, the pre-tRNASerUCA-C47:6U from this allele is matured and functionally suppresses a nonsense codon in ade6-704, alleviating accumulation of red pigment. However, this allele does not produce tRNA in the sla1-rrm1 mutant carrying the point mutations in the RRM1 β-sheet surface at levels sufficient to mediate TMS (32). We isolated spontaneous revertants that restore tRNASer-mediated suppression in sla1-rrm cells and mapped the phenotypic mutation by whole genome sequencing. We establish that this approach is applicable to S. pombe and report that the phenotypic mutation is duplication of the suppressor tRNASer gene. We show that a surprising disproportionate increase in suppressor-tRNA levels that occurs with tRNA gene duplication requires sla1-rrm, provoking a model in which activation by La of tRNA maturation is dependent on the concentration of the pre-tRNA.Figure 1.

Bottom Line: Further sequence analyses found a duplication of the tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype.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.

View Article: PubMed Central - PubMed

Affiliation: Intramural Research Program on Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, University of Massachusetts Medical School, Worcester, MA, USA.

ABSTRACT
We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNA(Ser)UCA-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 tRNA(Ser)UCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNA(Ser)UCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNA(Ser)UCA-C47:6U levels in sla1-rrm but not sla1- 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.

Show MeSH
Related in: MedlinePlus