Relocation of urf a from the mitochondrion to the nucleus cures the mitochondrial mutator phenotype in the fission yeast Schizosaccharomyces pombe

Abstract

In previous papers we have reported the characterisation of mitochondrial mutator mutants of Schizosaccharomzyces pombe. In contrast to nuclear mutator mutants known from other eucaryotes, this mutator phenotype correlates with mutations in an unassigned open reading frame (urf a) in the mitochondrial genome. Since an efficient biolistic transformation system for fission yeast mitochondria is not yet available, we relocated the mitochondrial urf a gene to the nucleus. As host strain for the ectopic expression, we used the nonsense mutant ana(r)-6, which carries a premature stop codon in the urf a gene. The phenotype of this mutant is characterised by continuous segregation of progeny giving rise to fully respiration competent colonies, colonies that show moderate growth on glycerol and a fraction of colonies that are unable to grow on glycerol. The phenotype of this mutant provides an excellent tool with which to study the effects on the mutator phenotype of ectopic expression of the urf a gene. Since a UGA codon encoding tryptophan is present in the original mitochondrial gene, we constructed two types of expression cassettes containing either the mitochondrial version of the urf a gene (mt-urf a) or a standard genetic code version (nc-urf a; UGA replaced by UGG) fused to the N-terminal import leader sequence of the cox4 gene of Saccharomyces cerevisiae. We show that the expression of the mt-urf a gene in its new location is able to cure, at least in part, the phenotype of mutant ana(r)-6, whereas the expression of the nc-urf a gene completely restores the wild-type (non-mutator) phenotype. The significant similarity of the urf a gene to the mitochondrial var1 gene of S. cerevisiae and homologous genes in other yeasts suggests that the urf a gene product might be a ribosomal protein with a dual function in protein synthesis and maintenance of mitochondrial DNA integrity.

SMRT Sequencing Revealed Mitogenome Characteristics and Mitogenome-Wide DNA Modification Pattern in Ophiocordyceps sinensis

“…In addition, the dN/dS values that were > 1 in Hypocreales in relation to the outgroup P. nordicum and the 36 explored positive selection sites suggest that rps3 is under positive selection in Hypocreales fungi, which is in accordance with the results of Lin et al (2015). rps3 is involved in DNA repair and potentially has endonuclease activities in Schizosaccharomyces pombe and nuclear versions of rps3 in human and Drosophila melanogaster (Neu et al, 1998; Lyamouri et al, 2002; Jang et al, 2004). Except for these model organisms, no functional studies confirm that mtDNA-encoded rps3 genes are actually functional (Kim et al, 2009).…”

Forces maintaining organellar genomes: is any as strong as genetic code disparity or hydrophobicity?

“…Additional fungal taxa exist that also possess only this one code disparity but have not transferred A9; however, this cannot be considered contrary to the code disparity hypothesis because A9 is evidently rather hard to transfer for some other reason (such as hydrophobicity, addressed below), being mtDNA-encoded in all species that retain more than 15 assigned, non-endonuclease, proteincoding genes and having resisted functional transfer in S. cerevisiae in the laboratory. (28) RpS3 was apparently the (29) and S. pombe (30) after code correction, so should be absent from the mtDNA in any fungal lineage that (for whatever reason) loses this codedisparity barrier. Evidence available to date is consistent with the hypothesis.…”

Schizosaccharomyces pombe as a fundamental model for research on mitochondrial gene expression: Progress, achievements and outlooks

“…Consequently, upon a relocation of the mitochondrial urfa gene to the nucleus, the TGA codon must be replaced by TGG to produce a functional protein. 69 However, TGA only occurs in the urfa and intronic transcripts, that is, rarely compared to the other tryptophan codon (TGG). 70 The sole mitochondrial trnW(cca) has been proposed to recognise both TGA and TGG, albeit at varying efficiency.…”

“…translation remains ambiguous, though its contribution to mitogenome maintenance has been inferred. 69,71,72 A single Urfa residue substitution enhanced the occurrence of respiratory-deficient progeny, which contained mtDNA point mutations and/or deletions (the 'mutator' phenotype). Whilst, a non-sense urfa mutation produced offspring with varying degrees of respiratory deficiency, ranging from normal to no respiratory growth.…”

“…Whilst, a non-sense urfa mutation produced offspring with varying degrees of respiratory deficiency, ranging from normal to no respiratory growth. 69,71 Such segregation was recorded across generations (i.e. stably inherited), and could be cured by expressing from a nuclear locus the wild-type, mitochondrial-targeted Urfa.…”