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Nuclear and mitochondrial genome responses in HeLa cells treated with inhibitors of mitochondrial DNA expression

100%

Piechota J., Szczęsny R., Wolanin K., Chlebowski A., Bartnik E.

Acta Biochimica Polonica

2006

vol. 53

issue 3

485-495

The influence of mutations in the mitochondrial DNA (mtDNA) on the bioenergetic metabolism of the cell is still poorly understood. Many of the mutations in the mtDNA affect the expression of the mitochondrial genome. Investigations on cells from patients are not easy, especially as the mitochondrial DNA is heteroplasmic and this state is changed in culture. Moreover, the nuclear background and the mitochondrial haplotype may affect the behaviour of cells. Transfer of patient mitochondria to rho zero cell lines is also not optimal as these cells in general have many nuclear changes which may also affect cell behaviour. Thus, we decided to use inhibitors of mitochondrial genome expression, such as thiamphenicol, ethidium bromide and dideoxycytidine to investigate the bioenergetic metabolism of HeLa cells. We found that oxidative phosphorylation and glycolysis participate equally in ATP production in HeLa cells and that decreased activity of the respiratory chain leads to increased glycolysis and the reduction of cell growth. Insufficient ATP production in the oxidative phosphorylation process was not compensated by increased proliferation of the mitochondria. However, we were able to show that there are some mechanisms compensating limited expression of the mitochondrial genome within the mitochondria. Experiments with dideoxycytidine revealed that 10-fold decrease of the mtDNA copy number resulted in almost normal activity of cytochrome c oxidase. We found that mtDNA depletion is compensated mostly on the level of RNA metabolism in the mitochondria. Thus, our results are in agreement with the hypothesis that transcription initiation rather than mtDNA copy number is a rate limiting factor for expression of the mitochondrial genome.

Differential stability of mitochondrial mRNA in HeLa cells

73%

Piechota J., Tomecki R., Gewartowski K., Szczęsny R., Dmochowska A., Kudła M., Dybczyńska L., Stepien P. P., Bartnik E.

Acta Biochimica Polonica

2006

vol. 53

issue 1

157-168

The physiological significance and metabolism of oligoadenylated and polyadenylated human mitochondrial mRNAs are not known to date. After study of eight mitochondrial transcripts (ND1, ND2, ND3, ND5, CO1, CO2, ATP6/8 and Cyt. b) we found a direct correlation between the half-lives of mitochondrial mRNAs and their steady-state levels. Investigation of the mt-mRNA decay after thiamphenicol treatment indicated that three transcripts (ND2, ND3 and Cyt. b) are significantly stabilized after inhibition of mitochondrial translation. Careful analysis one of them, ND3, showed that inaccurate processing of the H-strand RNA precursor may occasionally occur between the ND3 and tRNAArg locus leading to synthesis of ND3 mRNAs lacking the STOP codon. However, analysis of the oligo(A) fraction observed in case of the ND3 indicates that partially polyadenylated mRNAs are linked rather to the transcription process than to the translation-dependent deadenylation. Analysis of ND3 mRNA turnover in cells with siRNA-mediated knock-down of the mitochondrial poly(A) polymerase shows that strongly decreased polyadenylation does not markedly affect the decay of this transcript. We present a model where oligoadenylated mitochondrial transcripts are precursors of molecules containing full length poly(A) tails.

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2 Acta Biochimica Polonica

2 2006

2 Bartnik E.

2 Piechota J.

2 Szczęsny R.

1 Chlebowski A.

1 Dmochowska A.

1 Dybczyńska L.

1 Gewartowski K.

1 Kudła M.

1 Stepien P. P.

1 Tomecki R.

1 Wolanin K.

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Nuclear and mitochondrial genome responses in HeLa cells treated with inhibitors of mitochondrial DNA expression

Differential stability of mitochondrial mRNA in HeLa cells