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

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

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

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