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| Germline stem cell attrition and increased lifespan in dEXD2-deficient flies. a, Significantly increased lifespan in dExd2 EY03872 flies compared with controls, particularly for females. NAC normalizes the lifespan of dEXD2-deficient females. Means (s.d.) were compiled from n = 3 independent experiments, each using 60 flies. ***P < 4.03 × 10 −10 for males and ***P < 2.22 × 10 −16 for females, proportional hazard Cox model. b, qRT-PCR analysis of different Drosophila tissues shows high dExd2 mRNA levels in the ovaries of control flies compared with other tissues. Values from n = 3 independent experiments are plotted with the means (s.d.). c, Fecundity was reduced in dExd2 EY03872 flies and partially rescued by NAC. A total of 60 flies was used for each group and the means (s.d.) shown. d, Age-dependent attrition of GSCs in dExd2 EY03872 flies was rescued by NAC. GSCs were identified by Hts staining and indicated by white arrows. Scale bar, 20 µ m. e, Quantification of GSCs of each genotype for the indicated ages with or without NAC treatment. Means were compiled from n = 15 independent germaria at each time point and the means (s.d.) shown. Statistical analysis was performed using the proportional odds cumulative link model (***P = 1.38E-08 for week 7). f, Immunofluorescence analysis revealed a strong decrease in cytochrome c (Cyt c) levels and disorganized ATP synthase patterns (ATP5A) in germaria of dExd2 EY03872 flies. Scale bar, 20 µ m. Source data and additional statistical analysis are provided in Supplementary Table 1.
Source publication
Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' d...
Citations
... (Extended Data Fig. 2e). The soleus muscle showed higher expression levels of mitochondrial proteins using NADPH/NADP in comparison this method suffers from technical artifacts [10][11][12] . As a result, information regarding the mitochondrial proteome from different tissues at the suborganelle level remains incomplete due to a lack of suitable methodology. ...
Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O -methylation activity of COQ3. Rtn4ip1- knockout myoblasts had markedly decreased CoQ 9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of d Rtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.
... Further, fibroblast cell cultures from long-lived Snell dwarf mice had greater expression of UPR mt markers, including elevated 60 kDa mitochondrial heat shock protein (HSP60 or HSPD1) 29 . Therefore, chronic disruption of mitochondrial translation and induction of the UPR mt may promote both longevity and an improved healthspan via mitohormesis 15,[30][31][32][33][34][35] . ...
... Aged Mrpl54 +/− mice did not show improved metabolic phenotypes. Given that disruption of mitochondrial translation and/or UPR mt induction has been linked to improved healthspan in several animal models 15,[30][31][32][33][34][35] , we examined whether reduced Mrpl54 expression altered whole body metabolism during the aging trajectory. Female and male Mrpl54 +/− and WT mice were aged 24 months (24 M) before performing the same metabolic phenotyping tests used for adult 6 M mice. ...
Maintaining mitochondrial function is critical to an improved healthspan and lifespan. Introducing mild stress by inhibiting mitochondrial translation invokes the mitochondrial unfolded protein response (UPRmt) and increases lifespan in several animal models. Notably, lower mitochondrial ribosomal protein (MRP) expression also correlates with increased lifespan in a reference population of mice. In this study, we tested whether partially reducing the gene expression of a critical MRP, Mrpl54, reduced mitochondrial DNA-encoded protein content, induced the UPRmt, and affected lifespan or metabolic health using germline heterozygous Mrpl54 mice. Despite reduced Mrpl54 expression in multiple organs and a reduction in mitochondrial-encoded protein expression in myoblasts, we identified few significant differences between male or female Mrpl54+/− and wild type mice in initial body composition, respiratory parameters, energy intake and expenditure, or ambulatory motion. We also observed no differences in glucose or insulin tolerance, treadmill endurance, cold tolerance, heart rate, or blood pressure. There were no differences in median life expectancy or maximum lifespan. Overall, we demonstrate that genetic manipulation of Mrpl54 expression reduces mitochondrial-encoded protein content but is not sufficient to improve healthspan in otherwise healthy and unstressed mice.
... Mitochondria own a circular 16.6-kb DNA genome encoding two ribosomal RNAs, 22 transfer RNAs, and 13 open reading frames for electron transport chain complex subunits (1). During energy production, mitochondria participate in numerous metabolic reactions, such as tricarboxylic acid cycle and oxidative phosphorylation, and some intermediate metabolites are indispensable for cell proliferation and survival (2), but some by-products, such as reactive oxygen species, are harmful for most cells (3,4). ...
As a relic of ancient bacterial endosymbionts, mitochondria play a central role in cell metabolism, apoptosis, autophagy, and other processes. However, the function of mitochondria-derived nucleic acids in cellular signal transduction has not been fully elucidated. Here, our work has found that Y-box binding protein 1 (YB1) maintained cellular autophagy at a moderate level to inhibit mitochondrial oxidative phosphorylation. In addition, mitochondrial RNA was leaked into cytosol under starvation, accompanied by YB1 mitochondrial relocation, resulting in YB1-bound RNA replacement. The mRNAs encoded by oxidative phosphorylation (OXPHOS)-associated genes and oncogene HMGA1 (high-mobility group AT-hook 1) were competitively replaced by mitochondria-derived tRNAs. The increase of free OXPHOS mRNAs released from the YB1 complex enhanced mitochondrial activity through facilitating translation, but the stability of HMGA1 mRNA was impaired without the protection of YB1, both contributing to breast cancer cell apoptosis and reactive oxygen species production. Our finding not only provided a new potential target for breast cancer therapy but also shed new light on understanding the global landscape of cellular interactions between RNA-binding proteins and different RNA species.
... Proteins were washed in ammonium bicarbonate and digested with trypsin. Mass spectrometry and data analysis using SAINTexpress was performed as described previously [21,22] and dot plot figures generated using ProHits-viz (https://prohits-viz.org). ...
Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.
... EXD2 has been shown to localize to mitochondrial membranes also in somatic cells. 27,28 Both EXD1 and EXD2 contain a 3′-5′ exonuclease domain (InterPro IPR002562) and they belong to the Ribonuclease H-like superfamily (InterPro IPR012337), but they share very little sequence similarity outside their exonuclease domain ( Figure S1A). The function of EXD2 in spermatogenesis and the piRNA pathway has remained unexplored. ...
... Therefore, the localization of EXD2 is restricted to the mitochondria-associated IMC, consistent with its mitochondrial localization in somatic cells. 27,28 Like EXD2, EXD1 was also shown to localize to the IMC by co-immunostaining with antibodies against PIWIL2 and TDRD1 ( Figure 4A). Interestingly, in contrast to EXD2, EXD1 localization was not restricted to the mitochondria-associated IMC, but co-localization with DDX25 revealed that it is also found in CB precursors in late pachytene spermatocytes ( Figure 4B), as well as in the CB in round spermatids ( Figure 4C). ...
... The Exd2 cDNA used in the interaction study encodes a truncated protein lacking the first 150 amino acids, including the motif required for its exonuclease activity. 28,29 Therefore, the interaction of EXD2 with PIWIL2 is not dependent on its catalytic domain. ...
Background:
Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway.
Aims:
In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis.
Results:
The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility.
Conclusion:
Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.
... 41 This result was not in line with previous findings on EXD2 in the nucleus 42 and mitochondrial matrix. 43 Therefore, we confirmed the subcellular localization of EXD2 via APEX-EM imaging which utilizes 3,3′-diaminobenzidine staining of APEX in the fixed sample. 41 We identified the hydrophobic N-terminus of EXD2 (1−37 aa) as an OMM-specific signal-anchor transmembrane domain for EXD2 localization to the OMM. ...
ConspectusProximity labeling can be defined as an enzymatic "in-cell" chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not "accessible" via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell-cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in "spatiomics," that is, for the extraction of spatially distributed biological information in a cell or organism.Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This "in-cell" reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models.Simultaneously, chemists have also adopted the concept and employed chemical "photocatalysts" as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.
... BioID experiments. Plasmids for expression of N-and C-terminal BirA-Flag fusion constructs 75 were a kind gift of Philip Knobel (Laboratory for Applied Radiobiology, University Zurich). BirA-Flag/PAX3-FOXO1 fusion constructs were generated by amplification of a prevalidated PAX3-FOXO1 cDNA, using primers including restriction sites for AscI (forward) and NotI (reverse) Supplementary Data S5D, and cloned into N-or C-terminal Bira-Flag backbone vectors. ...
Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.
... A connection between life span and the nuclear-encoded gene for EXD2 (exonuclease 3 5 domain-containing 2), which has roles in modulating the aberrant association of mitochondrial messenger RNA with the mitochondrial ribosome was shown by Silva et al. [74]. Loss of EXD2 resulted in developmental delays and premature female germline stem cell attrition, with reduced fecundity. ...
Mitochondrial dysfunction is associated with ageing, but the detailed causal relationship between the two is still unclear. We review the major phenomenological manifestations of mitochondrial age-related dysfunction including biochemical, regulatory and energetic features. We conclude that the complexity of these processes and their inter-relationships are still not fully understood and at this point it seems unlikely that a single linear cause and effect relationship between any specific aspect of mitochondrial biology and ageing can be established in either direction.
... At least 15 proteins were shown or suggested to localize to mitochondria by at least one of the resources ( Figure 2C). Additionally, studies focusing on CDK5RAP1, MIGA1, MTFR2, MYO19, USP30, VPS13A, and exonuclease 3 0 -5 0 domain containing 2 (EXD2) showed that these proteins either localize or tether to mitochondria (Bingol et al., 2014;Kumar et al., 2018;Lu et al., 2019;Quintero et al., 2009;Shneyer et al., 2016;Yamamoto et al., 2019;Zhang et al., 2016;Hensen et al., 2018;Silva et al., 2018). This analysis suggests that at least some of these proteins may be missing from annotation in Mitocarta 2.0 and shows the value of our dataset in the detection of novel mitochondrial proteins. ...
... We wondered whether EXD2 was dually localized in different mitochondrial sub-compartments, an extremely rare occurrence. EXD2 was previously reported to localize to the OMM (Hensen et al., 2018;Park et al., 2019), the mitochondrial matrix where it regulates mitochondrial translation (Silva et al., 2018), as well as the nucleus where it was suggested to play a role in double-strand break resection (Broderick et al., 2016;Nieminuszczy et al., 2019). Two EXD2 isoforms are predicted to result from alternative splicing, producing an isoform 2 lacking the first 125 amino acids ( Figure 6B). ...
We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
... Additionally, studies focusing on CDK5RAP1, MIGA1, MTFR2, MYO19, USP30 or VPS13A showed that these proteins either localize or tether to mitochondria (Bingol et al., 2014;Kumar et al., 2018;Lu et al., 2019;Shneyer et al., 2016;Yamamoto et al., 2019;Zhang et al., 2016). EXD2, an endonuclease, was originally described as a nuclear protein with a role in double-stranded DNA breaks (Broderick et al., 2016), but was later shown to localize to mitochondria (Silva et al., 2018) (Hensen et al., 2018); however, with a discrepancy in its sub-mitochondrial localization, a puzzle that we were able to resolve using BioID (see below). This analysis suggests that at least some of these proteins may be missing from annotation in Mitocarta 2.0, and shows the value of our dataset in the detection of novel mitochondrial proteins. ...
... A possible explanation for this behavior could be that this protein is dually localized in different mitochondrial sub-compartments, an extremely rare occurrence. EXD2, Exonuclease 3'-5' Domain Containing 2, is a protein that has been described to localize to the OMM (Hensen et al., 2018;Park et al., 2019), the mitochondrial matrix where it was described to play a role in mitochondrial translation through its RNA endonuclease activity (Silva et al., 2018), as well as the nucleus where it was suggested to play a role in double-strand break resection and (Broderick et al., 2016;Hensen et al., 2018;Nieminuszczy et al., 2019;Park et al., 2019;Silva et al., 2018). Two EXD2 isoforms are predicted in databases ( Fig. 6B), resulting from an alternative splicing (skipping of exon 2), producing an isoform 2 lacking the first 125 amino acids. ...
... A possible explanation for this behavior could be that this protein is dually localized in different mitochondrial sub-compartments, an extremely rare occurrence. EXD2, Exonuclease 3'-5' Domain Containing 2, is a protein that has been described to localize to the OMM (Hensen et al., 2018;Park et al., 2019), the mitochondrial matrix where it was described to play a role in mitochondrial translation through its RNA endonuclease activity (Silva et al., 2018), as well as the nucleus where it was suggested to play a role in double-strand break resection and (Broderick et al., 2016;Hensen et al., 2018;Nieminuszczy et al., 2019;Park et al., 2019;Silva et al., 2018). Two EXD2 isoforms are predicted in databases ( Fig. 6B), resulting from an alternative splicing (skipping of exon 2), producing an isoform 2 lacking the first 125 amino acids. ...
We used BioID, a proximity-dependent biotinylation assay, to interrogate 100 mitochondrial baits from all mitochondrial sub-compartments to create a high resolution human mitochondrial proximity interaction network. We identified 1465 proteins, producing 15626 unique high confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions, and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments, and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles. This proximity network will be a valuable resource for exploring the biology of uncharacterized mitochondrial proteins, the interactions of mitochondria with other cellular organelles, and will provide a framework to interpret alterations in sub-mitochondrial environments associated with mitochondrial disease.
Bullet points
We created a high resolution human mitochondrial protein proximity map using BioID
Bait-bait analysis showed that the map has sub-compartment resolution and correlation analysis of preys identified functional clusters and assigned proteins to specific modules
We identified isoforms of matrix and IMS proteins with multiple cellular localizations and an endonuclease that localizes to both the matrix and the OMM
OMM baits showed specific interactions with non-mitochondrial proteins reflecting organellar contact sites and protein dual localization
