Mitochondrial gene expression is required for platelet function and blood clotting

Summary: Platelets are anucleate blood cells that contain mitochondria and regulate blood clotting in response to injury. Mitochondria contain their own gene expression machinery that relies on nuclear-encoded factors for the biogenesis of the oxidative phosphorylation system to produce energy required for thrombosis. The autonomy of the mitochondrial gene expression machinery from the nucleus is unclear, and platelets provide a valuable model to understand its importance in anucleate cells. Here, we conditionally delete Elac2, Ptcd1, or Mtif3 in platelets, which are essential for mitochondrial gene expression at the level of RNA processing, stability, or translation, respectively. Loss of ELAC2, PTCD1, or MTIF3 leads to increased megakaryocyte ploidy, elevated circulating levels of reticulated platelets, thrombocytopenia, and consequent extended bleeding time. Impaired mitochondrial gene expression reduces agonist-induced platelet activation. Transcriptomic and proteomic analyses show that mitochondrial gene expression is required for fibrinolysis, hemostasis, and blood coagulation in response to injury

Murine cytomegalovirus infection exacerbates complex IV deficiency in a model of mitochondrial disease

The influence of environmental insults on the onset and progression of mitochondrial diseases is unknown. To evaluate the effects of infection on mitochondrial disease we used a mouse model of Leigh Syndrome, where a missense mutation in the Taco1 gene results in the loss of the translation activator of cytochrome c oxidase subunit I (TACO1) protein. The mutation leads to an isolated complex IV deficiency that mimics the disease pathology observed in human patients with TACO1 mutations. We infected Taco1 mutant and wild-type mice with a murine cytomegalovirus and show that a common viral infection exacerbates the complex IV deficiency in a tissue-specific manner. We identified changes in neuromuscular morphology and tissue-specific regulation of the mammalian target of rapamycin pathway in response to viral infection. Taken together, we report for the first time that a common stress condition, such as viral infection, can exacerbate mitochondrial dysfunction in a genetic model of mitochondrial disease

Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

<div><p>The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The <i>Mrps34</i> mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The <i>Mrps34</i> mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.</p></div

Hierarchical RNA Processing Is Required for Mitochondrial Ribosome Assembly

The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5′ tRNA cleavage precedes 3′ tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome

The <i>Mrps34</i> mutation causes reduced oxygen consumption and respiratory complex activities in heart and liver mitochondria.

<p>The activities of the five mitochondrial respiratory complexes were measured in mitochondria isolated from heart (A) and liver (B) of young <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice and aged mice (C and D), respectively. The respiratory complex activities were normalized relative to citrate synthase activity. Data are means ± SEM of three-four separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test. State 3 and 4 respiration was measured in mitochondria isolated from hearts (E) and livers (F) of aged <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice using an OROBOROS oxygen electrode. Data are means ± SEM of three-four separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

Decreased MRPS34 affects the stability of the 12S rRNA and specific mitochondrial mRNAs.

<p>(A) The abundance of mature mitochondrial transcripts in mitochondria isolated from young <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} livers and hearts was analyzed by northern blotting. (B) The abundance of mature mitochondrial transcripts in aged liver and heart was analyzed by northern blotting. 18S rRNA was used as a loading control. The data are representative of results obtained from at least 8 mice from each strain. Data are means ± SEM of three separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

<i>Mrps34</i>^{<i>mut/mut</i>} mice have hypertrophic hearts and increased lipid accumulation in their livers.

<p>(A) Echocardiographic parameters of <i>Mrps34</i>^<i>wt/wt</i> (n = 5) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 5) mice. LVEDD, left ventricular end diastolic diameter; LVESD, left ventricular end systolic diameter; FS, fractional shortening; LVDPW, left ventricular posterior wall in diastole; LVSPW, left ventricular posterior wall in systole; IVDS, intraventricular septum in diastole; IVSS, intraventricular septum in systole. Values are means ± standard error. *p<0.05 compared with <i>Mrps34</i>^<i>wt/wt</i>. Liver sections cut at 8–12 μm thickness were stained with Haematoxylin and Eosin, oil red O and Haematoxylin or Gomori trichrome from young (B) and aged (C) <i>Mrps34</i>^<i>wt/wt</i> (n = 9) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 9) mice and visualized at 40X magnification. (D) Quantitative measurement of oil red staining using Image J. Data are means ± SEM of four different mice; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test. Serum ALT levels in young (E) and aged (F) <i>Mrps34</i>^<i>wt/wt</i> (n = 12) and <i>Mrps34</i>^{<i>mut/mut</i>} (n = 12) mice.</p

Reduced MRPS34 affects the abundance of mitochondrial respiratory complexes.

<p>BN-PAGE was performed on 75 μg of mitochondria isolated from liver and heart of young (A) and aged (B) <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice. Mitochondria lysates (75 μg) isolated from liver and heart of <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} young mice (C) and aged mice (D) were analyzed by BN-PAGE and the gels used for immunoblotting. Specific antibodies representing proteins of each of the mitochondrial complexes were used to compare abundance of protein in the wild type and mutant mice. Data are means ± SEM of five separate experiments; *, <i>p</i> < 0.05 compared with control treatments by a 2-tailed paired Student’s <i>t</i> test.</p

MRPS34 is required for the small ribosomal subunit and the stability of the mitoribosome.

<p>(A) Mitochondrial ribosomal protein levels in mitochondria (50 μg) isolated from liver and heart of <i>Mrps34</i>^<i>wt/wt</i> compared to <i>Mrps34</i>^{<i>mut/mut</i>} mice was determined by immunoblotting. (B) Distribution of the MRPS34 protein in 10–30% sucrose gradients of heart (0.8 mg) and liver (1.2 mg) mitochondria from young and aged <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} mice. Mitochondrial protein lysates from heart (0.8 mg) and liver (1.2 mg) of young (C) and aged (D) <i>Mrps34</i>^<i>wt/wt</i> and <i>Mrps34</i>^{<i>mut/mut</i>} were fractionated on 10–30% sucrose gradients. The distribution of ribosomal proteins was analyzed by immunoblotting against antibodies that are markers of the small and large ribosomal subunits. The data are typical of results from at least three independent biological experiments.</p