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A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

Wilson WC, Hornig-Do HT, Bruni F, Chang JH, Jourdain AA, Martinou JC, Falkenberg M, Spåhr H, Larsson NG, Lewis RJ, Hewitt L, Baslé A, Cross HE, Tong L, Lebel RR, Crosby AH, Chrzanowska-Lightowlers ZM, Lightowlers RN - Hum. Mol. Genet. (2014)

Bottom Line: The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length.The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure.We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health.

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The p.N478D mutation in mtPAP affects steady-state levels and translatability of mtDNA-encoded transcripts. (A) RNA was isolated from control and patient fibroblasts, and steady-state levels of mitochondrial rRNA and mRNA were assessed by northern blotting (4 µg), using the probes indicated. A probe against the 18S rRNA transcript was used as a loading control. The blot is representative of data from three independent RNA isolations. (B) De novo protein synthesis of mitochondrially encoded proteins was analysed in control and patient fibroblasts. Cells were incubated with [35S]-methionine/cysteine for 1 h in the presence of the cytosolic translation inhibitor emetine dihydrochloride (100 µg/ml). Equal amounts of whole cell lysate (50 µg) were separated via 15–20% denaturing SDS–PAGE and translation products visualized by autoradiography. Individual polypeptides were designated by their mobility (21). The protein profiles are representative of data derived from three independent experiments.
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DDU352F2: The p.N478D mutation in mtPAP affects steady-state levels and translatability of mtDNA-encoded transcripts. (A) RNA was isolated from control and patient fibroblasts, and steady-state levels of mitochondrial rRNA and mRNA were assessed by northern blotting (4 µg), using the probes indicated. A probe against the 18S rRNA transcript was used as a loading control. The blot is representative of data from three independent RNA isolations. (B) De novo protein synthesis of mitochondrially encoded proteins was analysed in control and patient fibroblasts. Cells were incubated with [35S]-methionine/cysteine for 1 h in the presence of the cytosolic translation inhibitor emetine dihydrochloride (100 µg/ml). Equal amounts of whole cell lysate (50 µg) were separated via 15–20% denaturing SDS–PAGE and translation products visualized by autoradiography. Individual polypeptides were designated by their mobility (21). The protein profiles are representative of data derived from three independent experiments.

Mentions: Depletion of mtPAP, mediated by siRNAs targeting the MTPAP transcript, has been shown by several groups to result in transcript-specific effects on the steady-state levels of mt-mRNAs in cultured human cell lines (14–16). In the homozygote patient lines (Fig. 2A lanes 2 and 3), we also saw transcript-specific effects, where steady-state levels of RNA14 and all MTCO were decreased, MTND1 increased and MTND3 unaffected. A similar trend had been noted when mt-transcripts lacked polyadenylated termini as a consequence of the poly(A) tails being removed by a cytosolic poly(A)-specific ribonuclease that had been targeted to mitochondria (17). To determine what effect these altered steady-state levels, caused by the mutant mtPAP, had on the translation of these transcripts, de novo metabolic labelling was performed. Autoradiographic data analysis of the homozygote compared with the control indicated that there were varied but reproducible differences in the amount of translation products from different transcripts (Fig. 2B). There was an evident decrease in de novo synthesis of the components of the COX2/3 ATP6 triplet, which was in contrast to the increase in ND5 and ND2. Those transcripts that showed decreased stability, such as MTCO3, showed a concomitant decrease in translation product. However, there was no consistent correlation between transcript level and translation as the dramatic increase in stability of MTND1 (Fig. 2A) did not result in a concomitant increase in ND1 (Fig. 2B).Figure 2.


A human mitochondrial poly(A) polymerase mutation reveals the complexities of post-transcriptional mitochondrial gene expression.

Wilson WC, Hornig-Do HT, Bruni F, Chang JH, Jourdain AA, Martinou JC, Falkenberg M, Spåhr H, Larsson NG, Lewis RJ, Hewitt L, Baslé A, Cross HE, Tong L, Lebel RR, Crosby AH, Chrzanowska-Lightowlers ZM, Lightowlers RN - Hum. Mol. Genet. (2014)

The p.N478D mutation in mtPAP affects steady-state levels and translatability of mtDNA-encoded transcripts. (A) RNA was isolated from control and patient fibroblasts, and steady-state levels of mitochondrial rRNA and mRNA were assessed by northern blotting (4 µg), using the probes indicated. A probe against the 18S rRNA transcript was used as a loading control. The blot is representative of data from three independent RNA isolations. (B) De novo protein synthesis of mitochondrially encoded proteins was analysed in control and patient fibroblasts. Cells were incubated with [35S]-methionine/cysteine for 1 h in the presence of the cytosolic translation inhibitor emetine dihydrochloride (100 µg/ml). Equal amounts of whole cell lysate (50 µg) were separated via 15–20% denaturing SDS–PAGE and translation products visualized by autoradiography. Individual polypeptides were designated by their mobility (21). The protein profiles are representative of data derived from three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222368&req=5

DDU352F2: The p.N478D mutation in mtPAP affects steady-state levels and translatability of mtDNA-encoded transcripts. (A) RNA was isolated from control and patient fibroblasts, and steady-state levels of mitochondrial rRNA and mRNA were assessed by northern blotting (4 µg), using the probes indicated. A probe against the 18S rRNA transcript was used as a loading control. The blot is representative of data from three independent RNA isolations. (B) De novo protein synthesis of mitochondrially encoded proteins was analysed in control and patient fibroblasts. Cells were incubated with [35S]-methionine/cysteine for 1 h in the presence of the cytosolic translation inhibitor emetine dihydrochloride (100 µg/ml). Equal amounts of whole cell lysate (50 µg) were separated via 15–20% denaturing SDS–PAGE and translation products visualized by autoradiography. Individual polypeptides were designated by their mobility (21). The protein profiles are representative of data derived from three independent experiments.
Mentions: Depletion of mtPAP, mediated by siRNAs targeting the MTPAP transcript, has been shown by several groups to result in transcript-specific effects on the steady-state levels of mt-mRNAs in cultured human cell lines (14–16). In the homozygote patient lines (Fig. 2A lanes 2 and 3), we also saw transcript-specific effects, where steady-state levels of RNA14 and all MTCO were decreased, MTND1 increased and MTND3 unaffected. A similar trend had been noted when mt-transcripts lacked polyadenylated termini as a consequence of the poly(A) tails being removed by a cytosolic poly(A)-specific ribonuclease that had been targeted to mitochondria (17). To determine what effect these altered steady-state levels, caused by the mutant mtPAP, had on the translation of these transcripts, de novo metabolic labelling was performed. Autoradiographic data analysis of the homozygote compared with the control indicated that there were varied but reproducible differences in the amount of translation products from different transcripts (Fig. 2B). There was an evident decrease in de novo synthesis of the components of the COX2/3 ATP6 triplet, which was in contrast to the increase in ND5 and ND2. Those transcripts that showed decreased stability, such as MTCO3, showed a concomitant decrease in translation product. However, there was no consistent correlation between transcript level and translation as the dramatic increase in stability of MTND1 (Fig. 2A) did not result in a concomitant increase in ND1 (Fig. 2B).Figure 2.

Bottom Line: The addition of LRPPRC/SLIRP, a mitochondrial RNA-binding complex, enhanced activity of the wild-type mtPAP resulting in increased overall tail length.The LRPPRC/SLIRP effect although present was less marked with mutated mtPAP, independent of RNA secondary structure.We conclude that (i) the polymerase activity of mtPAP can be modulated by the presence of LRPPRC/SLIRP, (ii) N478D mtPAP mutation decreases polymerase activity and (iii) the alteration in poly(A) length is sufficient to cause dysregulation of post-transcriptional expression and the pathogenic lack of respiratory chain complexes.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health.

Show MeSH
Related in: MedlinePlus