The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.     

Substrate structural requirements of Schizosaccharomyces pombe RNase P

RNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent Km for two S. pombe tRNA precursors derived from the supS1 and sup3-e tRNA(Ser) genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.     

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.     

Antisuppressor mutations and sulfur-carrying nucleosides in transfer RNAs of Schizosaccharomyces pombe

Antisuppressor mutations reduce the efficiency of nonsense suppressors. A mutation in the gene sin4 of Schizosaccharomyces pombe leads to loss of 5-(methoxycarbonylmethyl) thiouridine (mcm5s2U) from the first anticodon position of tRNAs. This resembles the phenotype of sin3 (Heyer, W. D., Thuriaux, P., Kohli, J., Ebert, P., Kersten, H., Gehrke, C., Kuo, K. C., and Agris, P. F. (1984) J. Biol. Chem. 259, 2856-2862), but the mutations reside in different genes. In vivo 35S-labeled tRNA from the parental suppressor strain sup3, the antisuppressor strains sin3 and sin4, and the double mutant sin3 sin4 has been digested to nucleosides and analyzed with high performance liquid chromatography methods. The major sulfur-carrying nucleoside in wild-type S. pombe tRNA is mcm5s2U. It is reduced in the mutant strains. Two other thiolated nucleosides are also present: 2-thiouridine and a nucleoside of unknown structure. Neither was affected by the antisuppressor mutations. Thiocytidine has not been found. Independent from their effect on suppressors, the two mutations sin3 and sin4 reduce the growth rate of cells, and sin3 also increases cell length. In vivo decoding of the serine codon UCG by the UCA reading serine tRNA is not promoted by the two antisuppressor mutations.     

Temperature sensitive allosuppressor mutants of the fission yeast S. pombe influence cell cycle control over mitosis

Summary A collection of Schizosaccharomyces pombe mutants has been obtained which restore activity to a nonsense suppressing tRNA sup3–5 whose suppressing function has been inactivated by second site mutations within the sup3–5 gene. These mutants were screened for those that were temperature sensitive in suppressing the opal nonsense allele ade6-704. Some of these map within or close to sup3 itself and others define two allosuppressor genes sal2 and sal3. The temperature sensitive mutants fail to efficiently suppress any other opal nonsense alleles although one mutant, sup3–5, r57, rr2, weakly does so at the low temperature. sal2 and sal3 mutants have a pleiotropic effect on the cell cycle causing a transient or complete blockage of mitosis. This blockage and the allosuppressor phenotypes are both eliminated by the presence of wee mutations in wee1 or cdc2. Mutants in sal2 are allelic with cdc25, a gene required for successful completion of mitosis. It is suggested that sal3 and cdc25 influence the mechanism that links the growth rate of the cell with the initiation of mitosis. Mutants in these genes may disturb tRNA biosynthesis or protein synthesis and this disruption may have an effect on both nonsense suppression and the growth rate control over mitosis.     

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.     

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

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-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.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae.

Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number. In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG). In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage. Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function. By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed. Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism. In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast. Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons.     

Suppression of termination mutations caused by defects of the NMD machinery in Saccharomyces cerevisiae

Among a large collection of nonsense (termination) suppressors of Saccharomyces cerevisiae, a few remained obscure for their molecular nature. Of those, a group of weak and recessive suppressors, sup111, sup112 and sup113, is of particular interest because of their dependency on [PSI+], a yeast prion. From the facts that these suppressors map at positions quite similar to the UPF2, UPF3 and UPF1 genes, respectively, and that some mutations in the UPF genes confer termination suppressor activity, we suspected that sup111, sup112 and sup113 would very well be mutant alleles of the UPF genes. We tested our speculation and found that sup113, sup111 and sup112 were in fact complemented with the wild-type alleles of UPF1, UPF2 and UPF3, respectively. We further obtained evidence that the UPF1, UPF2 and UPF3 loci of the strains carrying sup113, sup111 and sup112, respectively, had point mutations. From these results, we conclude that sup111, sup112 and sup113 are mutant alleles of UPF2, UPF3 and UPF1, respectively, and thus attribute suppressor activity of these mutations to defects in the NMD (nonsense-mediated mRNA decay) machinery.