A Genetic Code Alteration Is a Phenotype Diversity Generator in the Human Pathogen Candida albicans

BACKGROUND: The discovery of genetic code alterations and expansions in both prokaryotes and eukaryotes abolished the hypothesis of a frozen and universal genetic code and exposed unanticipated flexibility in codon and amino acid assignments. It is now clear that codon identity alterations involve sense and non-sense codons and can occur in organisms with complex genomes and proteomes. However, the biological functions, the molecular mechanisms of evolution and the diversity of genetic code alterations remain largely unknown. In various species of the genus Candida, the leucine CUG codon is decoded as serine by a unique serine tRNA that contains a leucine 5'-CAG-3'anticodon (tRNA(CAG)(Ser)). We are using this codon identity redefinition as a model system to elucidate the evolution of genetic code alterations. METHODOLOGY/PRINCIPAL FINDINGS: We have reconstructed the early stages of the Candida genetic code alteration by engineering tRNAs that partially reverted the identity of serine CUG codons back to their standard leucine meaning. Such genetic code manipulation had profound cellular consequences as it exposed important morphological variation, altered gene expression, re-arranged the karyotype, increased cell-cell adhesion and secretion of hydrolytic enzymes. CONCLUSION/SIGNIFICANCE: Our study provides the first experimental evidence for an important role of genetic code alterations as generators of phenotypic diversity of high selective potential and supports the hypothesis that they speed up evolution of new phenotypes

The Yeast PNC1 Longevity Gene Is Up-Regulated by mRNA Mistranslation

Translation fidelity is critical for protein synthesis and to ensure correct cell functioning. Mutations in the protein synthesis machinery or environmental factors that increase synthesis of mistranslated proteins result in cell death and degeneration and are associated with neurodegenerative diseases, cancer and with an increasing number of mitochondrial disorders. Remarkably, mRNA mistranslation plays critical roles in the evolution of the genetic code, can be beneficial under stress conditions in yeast and in Escherichia coli and is an important source of peptides for MHC class I complex in dendritic cells. Despite this, its biology has been overlooked over the years due to technical difficulties in its detection and quantification. In order to shed new light on the biological relevance of mistranslation we have generated codon misreading in Saccharomyces cerevisiae using drugs and tRNA engineering methodologies. Surprisingly, such mistranslation up-regulated the longevity gene PNC1. Similar results were also obtained in cells grown in the presence of amino acid analogues that promote protein misfolding. The overall data showed that PNC1 is a biomarker of mRNA mistranslation and protein misfolding and that PNC1-GFP fusions can be used to monitor these two important biological phenomena in vivo in an easy manner, thus opening new avenues to understand their biological relevance

Molecular reconstruction of a fungal genetic code alteration

Fungi of the CTG clade translate the Leu CUG codon as Ser. This genetic code alteration is the only eukaryotic sense-to-sense codon reassignment known to date, is mediated by an ambiguous serine tRNA (tRNACAG(Ser)), exposes unanticipated flexibility of the genetic code and raises major questions about its selection and fixation in this fungal lineage. In particular, the origin of the tRNACAG(Ser) and the evolutionary mechanism of CUG reassignment from Leu to Ser remain poorly understood. In this study, we have traced the origin of the tDNACAG(Ser) gene and studied critical mutations in the tRNACAG(Ser) anticodon-loop that modulated CUG reassignment. Our data show that the tRNACAG(Ser) emerged from insertion of an adenosine in the middle position of the 5'-CGA-3'anticodon of a tRNACGA(Ser) ancestor, producing the 5'-CAG-3' anticodon of the tRNACAG(Ser), without altering its aminoacylation properties. This mutation initiated CUG reassignment while two additional mutations in the anticodon-loop resolved a structural conflict produced by incorporation of the Leu 5'-CAG-3'anticodon in the anticodon-arm of a tRNA(Ser). Expression of the mutant tRNACAG(Ser) in yeast showed that it cannot be expressed at physiological levels and we postulate that such downregulation was essential to maintain Ser misincorporation at sub-lethal levels during the initial stages of CUG reassignment. We demonstrate here that such low level CUG ambiguity is advantageous in specific ecological niches and we propose that misreading tRNAs are targeted for degradation by an unidentified tRNA quality control pathway.D.D.M. was financially supported by FCT (PhD grant SFRH/BD/2006/27867). We thank Rita Rocha for her help with SerRS purification. This study was funded by FEDER/ FCT projects PTDC/BIA-MIC/099826/2008 and PTDC/ BIA-GEN/110383/2009.publishe

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/E-cadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin- deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset

Expression of <i>S. cerevisiae</i> tRNA^leu in <i>C. albicans.</i>

<p>A) Aminoacylation <i>in vivo</i> in <i>C. albicans</i> of <i>S. cerevisiae</i> tRNA_UAG/CAG^Leu and tRNA_AGA^Ser was monitored by Acidic Page and Northern Blot analysis. For this, total tRNAs were extracted under acidic conditions from pUA13, pUA14, pUA15, and pUA16 clones and fractionated on an acidic polyacrylamide gel, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000996#s4" target="_blank">materials and methods</a>. These gels separated deacylated (-AA) from aminoacylated tRNAs (+AA), which were detected using a tDNA_Leu/Ser probe labeled with [³²P]. B) Transformation efficiencies of plasmids encoding tRNA^Leu, which decoded the <i>C. albicans</i> serine CUG codons as leucine, was significantly lower that that of control plasmids (pUA12 and pUA16), indicating that the leucine tRNAs were slightly toxic. C) However, clones that survived the transformation procedure adapted readily to CUG ambiguity and showed growth rates similar to control clones (pUA12).</p

Reconstruction model for the <i>Candida</i> genetic code alteration.

<p>The ancestor of <i>Candida</i> decoded the CUG codon as leucine using a single leucine tRNA (tRNA^Leu). This situation changed dramatically with appearance 272±25My of a mutant serine tRNA that acquired a 5′-CAG-3′anticodon (tRNA_CAG^Ser). The latter competed with the tRNA^Leu for decoding of CUG codons, inserting both leucine and serine, at high level, at CUG positions, on a proteome wide scale. Such ambiguity decreased over time due to disappearance of the tRNA^Leu gene, however charging of the tRNA_CAG^Ser with leucine and serine prevented complete change of identity of the CUG codon from leucine to serine. In order to elucidate why CUG ambiguity was preserved in <i>C. albicans</i> and clarify whether CUG identity could be partially reverted from serine back to leucine, we have reconstructed the early stages of CUG identity change (high level of ambiguity) in <i>C. albicans</i> using <i>S. cerevisiae</i> tRNAs that decode CUG codons as leucine.</p

Ambiguous CUG decoding induced karyotype rearrangements and ploidy-shift.

<p>A) In ambiguous cell lines (pUA15) polyploidy was predominant and very high ploidy was often detected (>32N). Aneuploidy was also observed (6N). B) However, after plating cells several consecutive times on fresh agar chromosome numbers were reduced indicating that most cells returned to low ploidy (2N or 4N). Ploidy reduction after mating normally occurs by chromosome loss in <i>C. albicans</i> and it is likely that such mechanism also played a role in ploidy reduction in pUA15 transformed cells. C) CUG ambiguity also promoted extensive rearrangements of the R-chromosome (highlighted in white circles). Chromosomes were separated by PFGE on 0.6% agarose gels under the following conditions: 120–300 s for 24h at 80 V, then 420–900 s for 48 h at 80 V. The numbers 1–7 and R identify <i>C. albicans</i> chromosomes.</p

Increased CUG ambiguity up-regulated morphogenesis genes.

<p>Cells of pUA15 clones showed significant up-regulation of the <i>WOR1</i> (7.0±2.5) gene and hyphal-specific genes <i>CaHWP1</i> (41.76±9.96) and <i>HGC1</i> (2.64±1.12). Since <i>WOR1</i> increases the frequency of the white-opaque transition the very high percentage of opaque cells found in transformed clones may be a consequence of <i>WOR1</i> up-regulation. On the other hand, expression of the hypha-specific genes, <i>CaHWP1</i> and <i>CaHGC1,</i> supported the hypothesis that morphogenesis and hyphal growth triggered by CUG ambiguity resulted from expression of morphogenesis regulators. Induction of the <i>CaHWP1</i> gene was accompanied by repression of the <i>CaMCM1</i> (−1.84±0.44) gene, which controls cell morphology through the recruitment of other morphogenesis regulatory factors.</p

Ambiguous CUG decoding triggered morphogenesis and phenotypic switching.

<p>A) Smooth colony morphology of control clones growing on MM-uri-phloxin B (50µg/ml) agar plates. B) Ambiguous pUA15 clones formed long hyphae, even in absence of external inducers, just growing in MM-uri agar plates at 30°C. Similar results were obtained for pUA13 and pUA14 ambiguous clones (data not shown). D). Expression of <i>S. cerevisiae</i> tRNA^Leu in <i>C. albicans</i> also induced phenotypic switching, which is characterized by transition between different cell-phase forms, namely white-opaque and myceliated-unmyceliated, giving rise to sectored colonies. D) Phenotypic switching was quantified by counting sectored colonies grown in MM-uri, after 7 days of incubation at 30°C. For each plasmid, up to 10 clones were plated and 3000 colonies were screened.</p