MTPAP is the only mitochondrial adenylase in flies and is required to protect the 3' termini of mRNAs.

<p>(<b>A</b>) Body size comparison in control (wt and FM7,Tb) and DmMTPAP KO larvae (<i>DmMTPAP</i>^KO) at 4 days ael. (<b>B</b>) qRT-PCR analysis of <i>DmMTPAP</i> transcript levels in 1 day heterozygous <i>DmMTPAP</i>^KO flies (<i>DmMTPAP</i>^KO/FM7,Tb) and 4-day-old hemyzygous <i>DmMTPAP</i>^KO larvae (<i>DmMTPAP</i>^KO) and their corresponding age-matched controls (wt, FM7,Tb). Histone 2B transcript was used as endogenous control. Data is represented as mean ± SEM (*P < 0.05, ***P < 0.001, n = 5). (<b>C</b>) mRNA and poly(A) tail length in individually sequenced clones after transcript circularisation (<i>MTATP6/8</i>, <i>MTND4/4L</i>, <i>MTND1</i> and <i>MTND5</i>) or 3' RACE (<i>MTCOX1</i> and <i>MTCYTB</i>) in <i>DmMTPAP</i>^KO (red, n = 14–26) and control larvae (grey, n = 11–25) at 4 days ael. The annotated 3' termini of the indicated transcripts was set to zero to determine poly(A) tail length. (<b>D</b>) rRNA and poly(A) tail length in individually sequenced clones after transcript circularisation in <i>DmMTPAP</i>^KO (red, n = 17–25) and control larvae (grey, n = 24–29) at 4 days ael. Data are represented as mean ± SEM. (***P < 0,001), using a Mann-Whitney test.</p

Polyadenylation is not required for mitochondrial translation.

<p>(<b>A</b>) <i>In organello</i> labelling of mitochondrial translation products on isolated mitochondria from <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1) and control (w;;daGAL4/+ and w;UAS-mtPAPRNAi#1/+) 5 days ael larvae. Labelling was performed for 60min (pulse), followed by a 15 or 45 min chase with cold methionine. Loading was normalised to VDAC levels. (<b>B</b>) <i>In organello</i> labelling of mitochondrial translation products on isolated mitochondria from <i>DmMTPAP</i> KO (<i>DmMTPAP</i>^KO) and control (wt and FM7,Tb) 4-day-old larvae. Coomassie staining of the gels and VDAC Western blotting of the input samples were performed to ensure equal loading of the samples. Western blot analysis (<b>C</b>) and quantification (<b>D</b>) of nuclear-encoded subunit of Complex I (NDUFS3) in isolated mitochondria from control (daGAL4 control, RNAi #1 control and RNAi#2 control) and <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1 and <i>DmMTPAP</i> RNAi #2) 5-day-old larvae. VDAC was used as a loading control. Western blot analysis (<b>E</b>) and quantification (<b>F</b>) of the steady-state levels of a nuclear-encoded subunit of Complex I (NDUFS3) and an mtDNA-encoded subunit of complex IV (COX3) in mitochondria of control (wt and FM7,Tb) and <i>DmMTPAP</i>^KO 4-day-old larvae. VDAC was used as a loading control. Data are represented as mean ± SD.</p

Impaired 3' termini do not affect the stability of most mtDNA-encoded transcripts.

<p>(<b>A</b>) Relative steady-state level of mitochondrial transcripts were determined by Northern Blot in 5 day ael control (white and grey bars) and <i>DmMTPAP</i> KD larvae (black bar) larvae (n = 5). Expression levels were quantified using a Typhoon phosphorimager and normalised to histone 2B mRNA. All data are represented as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0,001). (<b>B</b>) Northern blot analysis and (<b>C</b>) quantification of steady-state levels of mitochondrial transcripts in control (wt and FM7,Tb) and <i>DmMTPAP</i> KO larvae (<i>DmMTPAP</i>^KO) at 4 days ael. Histone 2B transcript was used as loading control. (<b>D</b>) <i>De novo</i> mitochondrial transcription in isolated mitochondria of control and <i>DmMTPAP</i> KO larvae at 4 days ael in the presence of radioactively labelled [³²P]-UTP. Loading of the gels and absence of RNA degradation was confirmed by Northern blotting against COX1 and 16S RNAs. Western blotting of VDAC in the input samples was used as a loading control. (<b>E</b>) qPCR of mtDNA steady-state levels <i>DmMTPAP</i> KO and control larvae at 4 days ael. Primers against the cytosolic ribosomal protein 49 (RP49) were used to normalise to nuclear DNA content of the samples.</p

Mitochondrial respiration is affected due to incomplete OXPHOS assembly.

<p>(<b>A</b>) BN-PAGE and in gel staining of Complex I and Complex IV activities in mitochondrial protein extracts from control (wt and FM7,Tb) and DmMTPAP KO (<i>DmMTPAP</i>^KO) 4-day-old larvae. Coomassie staining of the gel and VDAC western blot of the input samples was performed to ensure equal loading of the gel. (<b>B</b>) Complex V assembly was assessed in <i>DmMTPAP</i> KD (<i>DmMTPAP</i> RNAi #1) 5-day-old larvae by BN-PAGE, followed by Western blot analysis against the F1 subunit of Complex V. Coomassie staining was used to ensure equal loading. (<b>C</b>) Oxygen consumption rates in permeabilised 4-day-old control (wt) and <i>DmMTPAP</i>^KO larvae, using glutamate, malate and pyruvate (GMP + ADP), succinate (GMP + ADP + succ) or TMPD and ascorbate (TMP + asc) as electron donors. Data are normalized to the protein content in each sample and are represented as mean ± SEM (***P < 0,001, n = 8). (<b>D</b>) Relative enzyme activities of respiratory chain complexes in 4-day-old control (wt) and <i>DmMTPAP</i>^KO larvae. Data are represented as mean ± SD (*P<0.05, **P < 0.01, ***P < 0,001, n = 3). (<b>E</b>) Relative enzyme activities of respiratory chain complexes (Complex I-IV) from control (white, grey and striped bars) and <i>DmMTPAP</i> KD (checked and black bars) 5-day-old larvae. Data is represented as mean ± SEM (**P < 0.01, ***P < 0,001, n = 5).</p

Additional file 3: Figure S2. of Respiratory chain complex III deficiency due to mutated BCS1L: a novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model

Coronal section at the level of the left amygdala. The bulk of the white matter is reduced and shows discoloration in the temporal lobe. The corpus callosum is thin and there is moderate lateral and third ventricular dilation. Cortical laminar necrosis is seen in the cingulate gyrus, the superior frontal gyrus, the precentral gyrus, the inferior temporal gyrus and the lateral occipitotemporal gyrus (arrows). (PDF 5698 kb

Creation and characterization of <i>DmMterf3</i> knockout larvae.

<p>(A) The <i>DmMterf3</i> locus and generation of a <i>DmMterf3</i> null mutant. <i>DmMterf3</i> is located on the third chromosome at cytological position 77C6. The construct (ko <i>DmMterf3</i>) used for ends-out homologous recombination is indicated by grey boxes, coding sequences in exons are indicated by black boxes and non-coding sequences in exons by white boxes. The gap between grey boxes represents the genomic region replaced by attP and a <i>white</i> marker gene. The white marker gene was subsequently removed by crossing to <i>cre</i>-recombinase expressing flies. (B) PCR analysis of wild-type and knockout alleles for <i>DmMterf3</i>. (C) Body size comparison of <i>DmMterf3</i> knockout larvae 6 days after egg-laying (ael) showing reduced size. (D) QRT-PCR analysis of <i>DmMterf3</i> transcript levels in control (white and grey bars) and <i>DmMterf3</i> KO larvae (black bars) at 3 and 6 days ael. (E) Q-PCR analysis of mtDNA levels in larvae at 3 and 6 days ael. (F) QRT-PCR analysis of steady-state levels of mitochondrial mRNAs normalized to the nuclear ribosomal protein 49 transcript levels in larvae at 3 and 6 days ael. Error bars indicate mean ± SEM (^*p<0.05; ^**p<0.01; ^***p<0.001; n = 5).</p

Biochemical measurement of respiratory chain function.

<p>(A) Oxygen consumption normalized to protein content in permeabilized control (white and grey bars) and <i>DmMterf3</i> KD (black bars) larvae at 3, 5 and 6 days ael. Substrates that are metabolized to deliver electrons entering at the level of complex I (CPI), complex II (SUCC) and/or glycerol-3-phosphate dehydrogenase, (G3P) were used (n = 3–7). (B) Relative enzyme activities of respiratory chain enzyme complex I (NADH coenzyme Q reductase), complex I+III (NADH cytochrome c reductase), complex II (succinate dehydrogenase), complex II+III (succinate cytochrome c reductase) and complex IV (cytochrome c oxidase) in control (white and grey bars) and <i>DmMterf3</i> KD larvae at 6 days ael (black bars) (n = 6–7). (C) BN-PAGE analysis and in-gel activity of complex I and complex IV in mitochondrial protein extracts from control and <i>DmMterf3</i> KD larvae 6 days ael. (D) Western blot analysis using antibodies against the nuclear-encoded NDUFS3 subunit of complex I and the α-subunit of ATP synthase (complex V) in control larvae at 5 days ael and in <i>DmMterf3</i> KD larvae at 5, 6 and 10 days ael. Error bars indicate mean ± SEM (^*p<0.05; ^**p<0.01; ^***p<0.001).</p

Phenotype and molecular characterization of <i>DmMterf3</i> knockdown larvae.

<p>(A) QRT-PCR analysis of DmMterf3 transcript levels in control (white and grey bars) and DmMterf3 KD larvae at 3, 5 and 6 days ael (black bars). (B) Body weight of control and <i>DmMterf3</i> KD larvae at 6 days ael. (C) QRT-PCR analysis of <i>DmMterf3</i> and <i>DpMterf3</i> transcript levels in <i>DmMterf3</i> KD and rescue larvae at 6 days ael (black bars). (D) Body size comparison of larvae that are <i>DmMterf3</i> KD or <i>DmMterf3</i> KD with <i>DpMterf3</i> rescue. The rescued larvae are indistinguishable from controls. Error bars indicate mean ± SEM (*p<0.05; **p<0.01; ***p<0.001; n = 5).</p

Mitochondrial <i>de novo</i> transcription, ribosome assembly, and <i>de novo</i> translation.

<p>(A) <i>In organello</i> transcription assays in isolated crude mitochondrial preparations from larvae at 3, 4 and 5 days ael. The upper panels show the <i>de novo</i> transcription of mtDNA and the lower panels show Northern blot analysis to detect COX I transcript as loading controls. <i>De novo</i> transcription was quantified for each lane and normalized to COX I mRNA steady-state levels, each value correspond to the average of each replicate. (B) Sedimentation analysis of ribosome assembly in control and <i>DmMterf3</i> KD larvae at 3 days ael. The small (28S) ribosomal subunit, the large (39S) ribosomal subunit and the assembled (55S) ribosome are indicated by arrows. Fractions were collected with continuous monitoring of absorbance at 260 nm (upper panels) and subsequently analyzed by qRT-PCR to detect 12S (red) and 16S (blue) rRNA transcripts (lower panels). (C) Sedimentation analysis of ribosome assembly in control and <i>DmMterf3</i> KD larvae at 5 days ael analyzed as in (B). (D) Analysis of mitochondrial <i>de novo</i> translation as determined by S³⁵-methionine incorporation into newly synthesized proteins <i>in organello</i> in control and <i>DmMterf3</i> KD larvae at 3, 4 and 5 days ael. Equal loading was assessed by analyzing an aliquot of the mitochondrial protein extract to be used for incubation with S³⁵-methionine on a SDS-PAGE gel followed Western blotting to detect Porin.</p

Binding of MTERF3 to mitochondrial 16S rRNA and model of MTERF3 action.

<p>(A) Identification of mitochondrial transcripts interacting with MTERF3 in mice was performed by co-immuno-precipitation using a mouse anti-MTERF3 antibody. Transcripts bound to mouse MTERF3 were quantified using qRT-PCR and their abundance is shown as percentage of levels in control IPs. (B) Identification of mitochondrial transcripts interacting with MTERF3 in flies was performed by co-immuno-precipitation using an anti-FLAG antibody and extracts from flies expressing DmMTERF3-linker-Flag. Transcripts bound to fly MTERF3 were quantified using qRT-PCR and their abundance is shown as percentage of levels in control IPs. Error bars correspond to standard deviation. (C) Model of the physiological role of MTERF3 in control of mtDNA expression (left panel) and model of the phenotypes caused by loss of MTERF3 (right panel).</p