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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 MTPAP 1432A>G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. (A) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C, unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A0) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. (B) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. (C) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. (D) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t-test (ns *P < 0.05; **P < 0.01; ***P < 0.001).
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DDU352F1: The MTPAP 1432A>G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. (A) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C, unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A0) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. (B) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. (C) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. (D) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t-test (ns *P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: Our initial investigations were limited to the analysis of blood samples from members of a large Old Order Amish family with multiple children suffering from a progressive and autosomal recessive neurodegenerative disorder. Genomic analysis revealed a candidate pathogenic mutation in MTPAP, the gene encoding mtPAP. We therefore performed mitochondrial poly(A) tail assays of representative mitochondrially encoded transcripts from whole blood. These confirmed an mt-mRNA polyadenylation defect associated with the c.1432A>G (p.N478D) mutation in clinically affected members, but no further studies were possible at that time (19). Here, we present investigations on skin fibroblast cell lines from three family members, two affected individuals homozygous for the mutation (P1 and P2) and one unaffected heterozygote sibling (Het). Initially, it was important to confirm that the polyadenylation defect was recapitulated in the cultured fibroblasts. RNA was isolated, and four mt-mRNAs were subjected to the mitochondrial poly(A) tail length assays as described previously (20). For each mt-mRNA analysed, a lack of polyadenylation was apparent in the pathogenic homozygote lines (P1 and P2), concomitant with an increase in oligoadenylated species (Fig. 1A lanes 2 and 3). The heterozygote behaved similarly to the control line, although there was evidence of a mild increase in oligoadenylated mRNA (Fig. 1A lane 1 cf lane 4). Homopolymeric oligoadenylation was confirmed by sequence analysis of MPAT-derived species for MTCO1 (data not shown). We then assessed whether the marked loss of polyadenylation caused any phenotypic consequences. Cell growth rate was examined first, using either glucose- or galactose-based media. Galactose is a carbon source that forces cells to use oxidative phosphorylation (OXPHOS). The growth defect seen in the homozygote mutant lines was more pronounced on this substrate than on glucose (Supplementary Material, Fig. S1). Cell lysates from the two patient cell lines, the unaffected sibling (Het) and one unrelated control (C) were then prepared and subjected to western blot (Fig. 1B). No observable difference in the steady-state levels of mtPAP could be detected, showing the p.N478D mutation did not affect the stability of the protein. In contrast, in the homozygote p.N478D lines, a substantial decrease was apparent in the mitochondrially encoded proteins of the respiratory chain, ND1, a component of complex I and COX1–3, members of complex IV. NDUFB8, although nuclear encoded, was also present at decreased steady-state levels as it is sensitive to complex I assembly. As expected, no decrease in complex II (SDHA) was observed consistent with all its components being nuclear encoded. Analysis of the fully assembled OXPHOS complexes by blue native gels (Fig. 1C) reflected the severe decrease seen in components of complexes I and IV, and this was further mirrored in the enzymatic activities (Fig. 1D). Intriguingly, complexes III and V appeared unaffected. Taken together, these data are consistent with a selective defect of mitochondrial gene expression.Figure 1.


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 MTPAP 1432A>G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. (A) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C, unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A0) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. (B) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. (C) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. (D) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t-test (ns *P < 0.05; **P < 0.01; ***P < 0.001).
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DDU352F1: The MTPAP 1432A>G mutation causes defective mt-mRNA polyadenylation and a respiratory chain deficiency. (A) RNA was isolated from patient (P1 and P2) and control (Het, unaffected heterozygote; C, unrelated control) fibroblasts before (lanes 1–4) or after (lanes 5–8) transduction with a wild-type MTPAP transgene (±LVMTPAP). The length of the mRNA poly(A) tail was assessed by MPAT in the four transcripts indicated. Representative gels depict fluorescently-labelled products separated through a 10% denaturing polyacrylamide gel and visualized by laser scanning. Zero extension (A0) is the position of migration predicted post-3′ processing of the transcript prior to any addition. A50 indicates the position of a poly(A) of 50 nt. Densitometric profiles of the signal from the RNA14 MPAT are presented in the far right panel. (B) Cell lysates (40 µg) isolated from patient and control fibroblasts were separated via 12% denaturing SDS–PAGE, and immunoblotting was performed. The images are representative of data using antibodies targeting mtPAP and OXPHOS subunits (listed in materials and methods). Detection was by ECL+ and Biorad ChemiDoc MP imaging system. (C) Mitochondria (40 µg) isolated from patient and control fibroblasts were analysed by Blue Native PAGE (4.5–16%). Each of the OXPHOS complexes was decorated using primary antibodies targeted to NDUFA9 (CI), Core 2 subunit (CIII), α-subunit (CV), the holoenzyme (CIV) and SDHA (CII). Sizes of the detected complexes are indicated to the left of panels, and the complex identities are shown on the right. (D) The activities of OXPHOS complexes I, IV and V were determined in mitochondria isolated from patient (black) and control (white) fibroblasts. Activities are expressed as nmol rotenone-sensitive NADH oxidized/min/mg mt-protein (CI), nmol reduced cytochrome c oxidized/min/mg mt-protein (CIV) and NADH oxidized/min/mg mt-protein (CV). N = 4, errors bars indicate ±SD. Student t-test (ns *P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: Our initial investigations were limited to the analysis of blood samples from members of a large Old Order Amish family with multiple children suffering from a progressive and autosomal recessive neurodegenerative disorder. Genomic analysis revealed a candidate pathogenic mutation in MTPAP, the gene encoding mtPAP. We therefore performed mitochondrial poly(A) tail assays of representative mitochondrially encoded transcripts from whole blood. These confirmed an mt-mRNA polyadenylation defect associated with the c.1432A>G (p.N478D) mutation in clinically affected members, but no further studies were possible at that time (19). Here, we present investigations on skin fibroblast cell lines from three family members, two affected individuals homozygous for the mutation (P1 and P2) and one unaffected heterozygote sibling (Het). Initially, it was important to confirm that the polyadenylation defect was recapitulated in the cultured fibroblasts. RNA was isolated, and four mt-mRNAs were subjected to the mitochondrial poly(A) tail length assays as described previously (20). For each mt-mRNA analysed, a lack of polyadenylation was apparent in the pathogenic homozygote lines (P1 and P2), concomitant with an increase in oligoadenylated species (Fig. 1A lanes 2 and 3). The heterozygote behaved similarly to the control line, although there was evidence of a mild increase in oligoadenylated mRNA (Fig. 1A lane 1 cf lane 4). Homopolymeric oligoadenylation was confirmed by sequence analysis of MPAT-derived species for MTCO1 (data not shown). We then assessed whether the marked loss of polyadenylation caused any phenotypic consequences. Cell growth rate was examined first, using either glucose- or galactose-based media. Galactose is a carbon source that forces cells to use oxidative phosphorylation (OXPHOS). The growth defect seen in the homozygote mutant lines was more pronounced on this substrate than on glucose (Supplementary Material, Fig. S1). Cell lysates from the two patient cell lines, the unaffected sibling (Het) and one unrelated control (C) were then prepared and subjected to western blot (Fig. 1B). No observable difference in the steady-state levels of mtPAP could be detected, showing the p.N478D mutation did not affect the stability of the protein. In contrast, in the homozygote p.N478D lines, a substantial decrease was apparent in the mitochondrially encoded proteins of the respiratory chain, ND1, a component of complex I and COX1–3, members of complex IV. NDUFB8, although nuclear encoded, was also present at decreased steady-state levels as it is sensitive to complex I assembly. As expected, no decrease in complex II (SDHA) was observed consistent with all its components being nuclear encoded. Analysis of the fully assembled OXPHOS complexes by blue native gels (Fig. 1C) reflected the severe decrease seen in components of complexes I and IV, and this was further mirrored in the enzymatic activities (Fig. 1D). Intriguingly, complexes III and V appeared unaffected. Taken together, these data are consistent with a selective defect of mitochondrial gene expression.Figure 1.

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