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E201800082.Full.Pdf Published Online: 31 August, 2018 | Supp Info: http://doi.org/10.26508/lsa.201800082 Downloaded from life-science-alliance.org on 28 September, 2021 Research Article Mitochondrial ubiquinone–mediated longevity is marked by reduced cytoplasmic mRNA translation Marte Molenaars1, Georges E Janssens1, Toon Santermans2, Marco Lezzerini1, Rob Jelier2, Alyson W MacInnes1, Riekelt H Houtkooper1 Mutations in the clk-1 gene impair mitochondrial ubiquinone addition to lifespan extension and decreased respiration, clk-1 biosynthesis and extend the lifespan in Caenorhabditis elegans. (qm30) mutants are developmentally delayed and have reduced We demonstrate here that this life extension is linked to the progeny (Felkai et al, 1999). Mammalian Clk1, known as Mclk-1 or Coq7, repression of cytoplasmic mRNA translation, independent of the is also involved in the lifespan (Liu et al, 2005; Stepanyan et al, 2006; alleged nuclear form of CLK-1. Clk-1 mutations inhibit poly- Takahashi et al, 2014). Transgenic mice, rescued from embryonic ribosome formation similarly to daf-2 mutations that dampen lethality via the transgenic expression of mouse clk-1, with reduced insulin signaling. Comparisons of total versus polysomal RNAs in Clk1 expression live longer and have smaller bodies than WT mice, clk-1(qm30) mutants reveal a reduction in the translational ef- demonstrating a conserved role for clk-1 in longevity across species ficiencies of mRNAs coding for elements of the translation (Takahashi et al, 2014). However, it is not fully understood how the machinery and an increase in those coding for the oxidative reduction of UQ biosynthesis extends the lifespan of these animals. phosphorylation and autophagy pathways. Knocking down the Until recently, CLK-1 protein was thought to reside exclusively in transcription initiation factor TATA-binding protein-associated the mitochondria. However, a distinct nuclear form of CLK-1 has been factor 4, a protein that becomes sequestered in the cytoplasm reported to independently regulate lifespan by mitochondrial– during early embryogenesis to induce transcriptional silencing, nuclear retrograde signaling (Monaghan et al, 2015). Adding back ameliorates the clk-1 inhibition of polyribosome formation. These acopyoftheclk-1 gene that lacks the mitochondrial targeting signal results underscore a prominent role for the repression of cyto- (MTS) to the clk-1 deletion mutant led to a subtle but significant plasmic protein synthesis in eukaryotic lifespan extension and reduction in the lifespan extension phenotype observed in the full suggest that mutations impairing mitochondrial function are able clk-1 deletion mutant (Monaghan et al, 2015). However, the existence to exploit this repression similarly to reductions of insulin sig- and function of a nuclear form of CLK-1 remains controversial. Other naling. Moreover, this report reveals an unexpected role for TATA- studies fail to detect any nuclear localization of the MCLK1/CLK-1 binding protein-associated factor 4 as a repressor of polyribosome proteins or any biological activity of a C. elegans CLK-1 protein devoid formation when ubiquinone biosynthesis is compromised. of an MTS (Liu et al, 2017). The importance of communication between stressed mitochondria DOI 10.26508/lsa.201800082 | Received 30 April 2018 | Revised 23 August and the rest of the cell is becoming increasingly appreciated (Quiros 2018 | Accepted 23 August 2018 | Published online 31 August 2018 et al, 2016). One such reaction to stress is carried out by the mito- chondrial unfolded protein response (UPRmt)(Durieux et al, 2011; Baker et al, 2012; Houtkooper et al, 2013). GCN-2, an eIF2α kinase that Introduction modulates cytosolic protein synthesis, is phosphorylated in response to the activated UPRmt in clk-1(qm30) mutants in a manner required Slowing down mitochondrial metabolism is a well-known means of for lifespan extension (Baker et al, 2012). Another nonmitochondrial increasing the lifespan of multiple species (Dillin et al, 2002; factor, TATA-binding protein-associated factor 4 (TAF-4), is a tran- Copeland et al, 2009; Houtkooper et al, 2013; Breitenbach et al, 2014). scription factor that has been implicated in the extension of clk-1 In C. elegans, one such model is the clk-1(qm30) mutant that harbors (qm30) mutant lifespan (Khan et al, 2013). TAF-4 was identified in an a deletion in the gene encoding a ubiquinone (UQ) biosynthesis RNAi screen for transcription factorsrequiredforthelifespanextension enzyme (Felkai et al, 1999). UQ is a redox-active lipid that plays of clk-1(qm30) mutants (Khan et al, 2013). Beyond clk-1,TAF-4wasalso a central role in the electron transport chain and mitochondrial required for the lifespan extension phenotype of two mutants of the oxidative phosphorylation (OXPHOS) by carrying electrons from mitochondrial electron transport chain (isp-1 and tpk-1)(Khan et al, complexes I and II to complex III (Stefely & Pagliarini, 2017). In 2013). TAF-4 is a component of the transcription factor II D mRNA 1Laboratory Genetic Metabolic Diseases, Amsterdam University Medical Centers, University of Amsterdam, Amsterdam Gastroenterology and Metabolism, Amsterdam, The Netherlands 2Centre of Microbial and Plant Genetics University of Leuven, Leuven, Belgium Correspondence: [email protected]; [email protected] ©2018Molenaars et al. https://doi.org/10.26508/lsa.201800082 vol 1 | no 5 | e201800082 1of17 transcription complex and is best known for its role in transcriptional clk-1 in clk-1(qm30) resultsinonlysmallphenotypicchangescompared silencing in early embryogenesis by becoming sequestered in the with the clk-1(qm30) mutants (Monaghan et al, 2015; Liu et al, 2017). cytoplasm because of phosphorylation by OMA-1 (Oocyte MAturation defective) (Walker et al, 2001). The loss of TAF-4 in C. elegans suppresses Transcripts encoding the translation machinery are strongly the lifespan extension phenotype induced by clk-1 mutations; however, reduced in clk-1(qm30) the mechanism through which this happens is not known. Reducing protein synthesis is another well-known means by We next identified the biological pathways that are altered in clk-1(qm30) which eukaryotes extend the lifespan (Hansen et al, 2007; Pan et al, worms using The Database for Annotation, Visualization and Integrated 2007). Caloric restriction, amino acid reduction, and knocking down Discovery (DAVID) and Gene Ontology (GO) analysis (Huang da et al, myriad factors involved in mRNA translation (such as ribosomal 2009a; Huang da et al, 2009b). Among the most up-regulated processes in proteins, elongation/initiation factors, or tRNA synthetases) all in- clk-1(qm30) are metabolic pathways (e.g., oxidation–reduction pathway) crease the lifespan (Masoro, 2000; Min & Tatar, 2006; Hansen et al, and ion transport (Fig 1D). A substantial proportion (25%) of significantly 2007; Pan et al, 2007). The genetic or pharmacological inhibition of the down-regulated processes in clk-1(qm30) mutants is involved in re- mechanistic target of rapamycin (mTOR)/nutrient sensing pathway production and development (Fig 1D). Developmental delay and reduced or insulin receptor signaling pathway is also marked by reduced progeny have been extensively reported as linked to the longevity mRNA translation rates and a suppression of polyribosome forma- phenotype of clk-1(qm30) mutants (Larsen et al, 1995; Tissenbaum & tion (Genolet et al, 2008; Stout et al, 2013). Proteomic analysis of the Ruvkun, 1998). Remarkably, another 20% of down-regulated GO terms are daf-2 mutants previously revealed a substantial reduction of ribo- involved in translation and mRNA processing (Fig 1D). In total, 38 somal proteins, translation factors, and protein metabolism com- genes encoding ribosomal proteins, initiation, and elongation factors ponents coupled to a repression of polyribosome formation (Stout are down-regulated in the clk-1(qm30) mutants compared with clk-1 et al, 2013). Moreover, our previous work demonstrated that similarly (qm30)+WT. In addition, 28 genes specifically involved in biogenesis to daf-2 mutants, mutations in clk-1 worms also result in a strong and processing of ribosomes were down-regulated in the clk-1 repression of polyribosome formation (Essers et al, 2015). This led us (qm30) worms. Similar down-regulation of ribosomal proteins and to hypothesize the existence of unexplored regulatory links between initiation/elongation factors was observed in the clk-1(qm30)+nuc dysfunctional mitochondria and cytoplasmic protein synthesis that mutants. These observations suggest that mRNA translation is re- are contributing to eukaryotic lifespan extension. In particular, we pressed in the clk-1(qm30)/+nuc mutants and are in line with the investigated which subsets of mRNAs are preferentially translated in repressed polysome profiles we observed before (Essers et al, 2015). the longer lived C. elegans with impaired UQ biosynthesis. There is a minor role for nuclear clk-1 in transcriptional changes Because the nuclear form of clk-1 was suggested to associate with Results the chromatin in the nucleus (Monaghan et al, 2015), we expected changes in the transcriptome in the mutant expressing exclusively RNAseq of clk-1(qm30) strains shows major changes in the nuclear form of clk-1. However, differentially expressed genes transcription in clk-1(qm30)+nuc were very similar to the differentially expressed genes clk-1(qm30) (Fig 2A). There were 3,720 genes similarly To uncover how the transcriptome of C. elegans changes on deletion up-regulated in the clk-1(qm30)+nuc as in the total clk-1(qm30) of clk-1 and the influence of the clk-1 mitochondrial localization mutants compared with clk-1(qm30)+WT and 4,397 genes similarly signal, we performed next-generation sequencing of total RNAs down-regulated in both strainscomparedwiththeWTcontrol(Fig 2B). (depleted of ribosomal RNA [rRNA]) isolated from worms at the L4 The 292 up-regulated genes and 275 down-regulated genes in the stage that precedes young adulthood. Total RNA pools depleted of clk-1(qm30)+nuc strain that were not up- or down-regulated in the clk- rRNA isolated from clk-1(qm30) mutants were compared with (1) clk-1 1(qm30) strain did not lead to significantly clustered GO terms.
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