Nuclear Modifier Gene MSS1Modulates The Neomycin-sensitive Phenotypic Expression Of Mitochondrial15S RRNA C1477G Mutation In Saccharomyces Cerevisiae

Mitochondrial function is mainly controled by mitochondrial genes, nuclear genes and environmental factors. The yeast mitochondrial DNA (mtDNA)15S rRNA C1477G mutation is corresponding to human mtDNA12S rRNA A1555G mutation. The A1555G mutation is a primary factor for aminoglycoside-induced and nonsyndromic deafness, but is not sufficient to produce a deafness phenotype, suggesting that nuclear modifier genes and aminoglycosides modulate the phenotypic manifestation of the A1555G mutation. At present the mtDNA mutation phenotypic expression researches are mainly focused on one factor, mtDNA-nuclear gene interaction or mtDNA-environmental factor interaction. However, the interaction among mtDNA, nuclear gene and environmental factor remains poorly understood.This research used yeast S.cerevisiae as a model system, and we constructed four kind of yeast strains, MSS1(PS), mssl(PS), MSS1(PR) and mss1(PR), which carrying with wild-type mtDNA15S rRNA (Ps), mutant mtDNA15S rRNA (PR), wild-type nuclear modifier gene MSS1and mutant nuclear modifier gene mss1. We observed the aminoglycoside sensitivity of the yeast strains in glucose medium (GYP). The results showed in wild-type mtDNA strains, whether MSS1gene mutated or not, yeast growth had no significant difference and the yeast strains were not sensitive to neomycin. Interestingly, only when mtDNA carrying the C1477G mutation, the growth of MSS1(PR) strain was significantly suppressed in GYP medium with neomycin and exhibited neomycin-sensitive phenotype, but mtDNA C1477G mutation coupling with mssl mutant, mssl(PR) strain growth was only partially inhibited and the strain was less sensitive to neomycin. Therefore, we hypothesised that the nuclear modidier gene MSSI mutation suppressed the neomycin sensitivity of yeast strains with mtDNA15S rRNA C1477G mutation. We further studied the interaction among mtDNA15S rRNA C1477G mutation, nuclear modifier gene MSSI and environmental factor neomycin, and uncovered the underlying mechanism about the yeast phenotypic manifestation.(1) The yeast strain which carrying15S rRNA C1477G mutation was sensitive to neomycin. When mtDNA C1477G mutation coexisting with neomycin, the yeast oxygen consumption rate was remarkably reduced, indicating that the mitochondrial function was impaired. Moreover, in GYP medium with neomycin, the transcript abundance of glycolytic key-enzyme genes HXK2and PYK1decreased significantly, suggesting the glycolysis was decreased. Therefore, the mitochondrial dysfunction and decrease of glycolysis could not meet the strain energy needs, and exhibited neomycin-sensitive phenotypic manifestation.(2) MSS1gene mutation suppressed the neomycin sensitivity of yeast carrying with mtDNA C1477G mutation. Although in GYP medium with neomycin, the oxygen consumption rate reduced significantly and mitochondrial genes expressions were also decereased in mssl(PR) strain compared with MSS1(PR) strain. The mRNA expression of glycolytic enzyme genes HXK2, PFK1and PYK1were upregulated significantly mss1(PR) strain. The glycolysis was drove and it produced enough energy to meet yeast strain need, and finally the strain manifested less neomycin-sensitive phenotype. In another hand, the expression of neomycin-resistance genes NEO1, NEO6and NE07were not changed significantly in mssl(PR) strain. Moreover, the neomycin absorption of mssl(PR) strain was not changed obviously, compared with it in other strains GYP medium. These results suggested that expression of neomycin-resistance genes and neomycin taken up was not involved with mssl(PR) strain phenotypic manifestation in GYP medium with neomycin but because of the increase of glycolysis.(3) HAP5, a component of HAP (heme-activated protein) complex, mediated increase of glycolysis in mssl(PR) strain. In GYP medium with neomycin, in contrast to MSS1(PR) strain, in mssl(PR) strain expression of HAP5increased, which then mediated the increase mRNA expression of glycolytic transcription factors such as GCR1. GCR1combined with other glycolytic transcription factors upregulated the expression of glycolytic genes thus promoted the glycolysis. We constructed the HAP5knockdown/overexpression yeast srains and found the yeast neomycin-sensitive phenotype was further enhanced or suppressed in these strains.The study for the first time illuminated the interaction among mtDNA C1477G mutation, nuclear modifier gene MSS1and environmental factor neomycin, providing research evidence for the three factors interaction. Furthermore, our research will provide insights on maternally inherited diseases such as A1555G mutation induced maternally deafness.