The structure of human EXD2 reveals a chimeric 3′ to 5′ exonuclease domain that discriminates substrates via metal coordination

Park, Jumi; Lee, Song-Yi; Jeong, Hanbin; Kang, Minchul; Haute, Lindsey Van; Minczuk, Michal; Seo, Jeong-Kon; Jun, Youngsoo; Myung, Kyungjae; Rhee, Hyun‐Woo; Lee, Chang-Wook

doi:10.1093/nar/gkz454

Cited by 24 publications

(44 citation statements)

References 39 publications

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“…An identical protein would also arise if a downstream methionine, M126, were used for translation initiation, and such alternative translation initiation could explain our BioID data (as the tagged bait is not spliced). The second isoform lacks a large part of the 3'-5' endonuclease which extends from aa 76-295, but still contains the HNH endonuclease domain from aa 408-470 (Park et al, 2019). The presence of several EXD2 isoforms (or products from posttranslational processing) was confirmed using stealth siRNA to knock-down EXD2 in cells ( Supplementary Fig.…”

Section: Sub-compartmental Localization Of Exd2 Isoforms In Mitochondriamentioning

confidence: 87%

“…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.…”

Section: Sub-compartmental Localization Of Exd2 Isoforms In Mitochondriamentioning

confidence: 99%

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A high-density human mitochondrial proximity interaction network

Antonická

Lin

Janer

et al. 2020

Preprint

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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 2013). Mitochondrial diseases due to deficiencies in the oxidative phosphorylation machinery are amongst the most common inherited metabolic disorders (Frazier et al., 2019;Nunnari and Suomalainen, 2012). While nearly 300 causal genes have now been identified, the molecular basis for the extraordinary tissue specificity associated with these diseases remains an enduring mystery.Mitochondria are double-membraned organelles, whose ultrastructure has been investigated for decades. The outer membrane (OMM), which serves as a platform for molecules involved in the regulation of innate immunity and for regulation of apoptosis, is permeable to metabolites and small proteins, and it forms close contacts with the endoplasmic reticulum (ER) for the exchange of lipids and calcium (Csordas et al., 2018). The impermeable inner membrane (IMM) is composed of invaginations called cristae that contain the complexes of the oxidative phosphorylation system. The intermembrane space (IMS) comprises both the space formed...

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Section: Sub-compartmental Localization Of Exd2 Isoforms In Mitochondriamentioning

confidence: 87%

Section: Sub-compartmental Localization Of Exd2 Isoforms In Mitochondriamentioning

confidence: 99%

A high-density human mitochondrial proximity interaction network

Antonická

Lin

Janer

et al. 2020

Preprint

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“…This raises the speculation that the pleiotropic effect of PfCRT mutations may not be the sole cause for changes in antimalarial drug sensitivity. We did find PPQ resistant isolates with exo mutations and single copy of PM2; P. falciparum exonuclease (PF3D7_1362500) has 25.62%, 17.92%, and 17.07% amino acid sequence identity to exosome complex exonuclease RRP6 [51], exonuclease 3′→5′ domain containing protein 2 (EXD2) [52], and exonuclease 3′→5′ domain containing protein 1 (EXD1) [53], respectively. The eukaryotic RNA exosome is a 3′→5′ degradation machinery involved in the processing of coding and noncoding RNAs [54].…”

Section: Discussionmentioning

confidence: 95%

Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

Boonyalai

Vesely

Thamnurak

et al. 2020

Preprint

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Background: High rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. It is essential to establish sensitive and reliable markers of PPQ resistance which can be adopted for monitoring the prevalence of drug resistance and guide drug policy change. Several genetic markers have been proposed such as copy numbers of P. falciparum multidrug resistance 1 ( pfmdr1 ) and plasmepsin 2 (PM2), and mutations in exonuclease (exo-E415G) and P. falciparum chloroquine resistance transporter (PfCRT) genes.Methods: To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia and the presence of exo-E415G and PfCRT mutations as well as PM2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. The efficacy of several drug combinations between standard clones and newly cloned P. falciparum Cambodian isolates was also measured.Results: The characterization of culture-adapted isolates revealed that exo-E415G and novel PfCRT mutations can confer PPQ-resistance, in the absence of PM2 amplification. In vitro testing of PPQ resistant parasites showed bimodal dose-response phenotype as previously reported in both Cambodian isolates and genome-edited Dd2 strains. Our finding is the first PPQ resistant clinical isolate with documented swollen digestive vacuole phenotype. From the field isolates, we were able to clone a new parasite line, 14-B5, which is sensitive to arteminsinin and piperaquine but resistant to chloroquine. Most of the drug combinations tested revealed antagonistic interaction against the 14-B5 line, indicating its different genetic background from other P. falciparum laboratory reference lines.Conclusions: Surveillance for PPQ resistance in regions that rely on DHA-PPQ as the first line treatment should depend on the monitoring of the reflective molecular markers. Bimodal dose response curves and swelling of the food vacuole can be used as PPQ resistant phenotype. The use of currently circulating parasite isolates is necessary for relevant drug combination development against current P. falciparum resistance profiles.

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“…Our group found homology of the N-terminus of ECSIT to the (organellar) RNA-binding pentatricopeptide repeat domains (Elurbe and Huynen, 2016), suggesting an additional RNA-binding function. The 3′−5′ exonuclease domain-containing protein 2 (EXD2), acting on both DNA and RNA, was recently published as a mitochondrial protein (Hensen et al, 2018; Silva et al, 2018; Park et al, 2019). Silva et al reported EXD2 as a mitochondrial matrix or inner membrane ribonuclease involved in among others mitochondrial translation, while Hensen et al and Park et al show a mitochondrial outer membrane localization.…”

Section: Discussionmentioning

confidence: 99%

A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome

Esveld

Cansiz-Arda²,

Hensen³

et al. 2019

Front. Cell Dev. Biol.

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In order to synthesize the 13 oxidative phosphorylation proteins encoded by mammalian mtDNA, a large assortment of nuclear encoded proteins is required. These include mitoribosomal proteins and various RNA processing, modification and degradation enzymes. RNA crosslinking has been successfully applied to identify whole-cell poly(A) RNA-binding proteomes, but this method has not been adapted to identify mitochondrial poly(A) RNA-binding proteomes. Here we developed and compared two related methods that specifically enrich for mitochondrial poly(A) RNA-binding proteins and analyzed bound proteins using mass spectrometry. To obtain a catalog of the mitochondrial poly(A) RNA interacting proteome, we used Bayesian data integration to combine these two mitochondrial-enriched datasets as well as published whole-cell datasets of RNA-binding proteins with various online resources, such as mitochondrial localization from MitoCarta 2.0 and co-expression analyses. Our integrated analyses ranked the complete human proteome for the likelihood of mtRNA interaction. We show that at a specific, inclusive cut-off of the corrected false discovery rate (cFDR) of 69%, we improve the number of predicted proteins from 185 to 211 with our mass spectrometry data as input for the prediction instead of the published whole-cell datasets. The chosen cut-off determines the cFDR: the less proteins included, the lower the cFDR will be. For the top 100 proteins, inclusion of our data instead of the published whole-cell datasets improve the cFDR from 54% to 31%. We show that the mass spectrometry method most specific for mitochondrial RNA-binding proteins involves ex vivo 4-thiouridine labeling followed by mitochondrial isolation with subsequent in organello UV-crosslinking.

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The structure of human EXD2 reveals a chimeric 3′ to 5′ exonuclease domain that discriminates substrates via metal coordination

Cited by 24 publications

References 39 publications

A high-density human mitochondrial proximity interaction network

A high-density human mitochondrial proximity interaction network

Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization

A Combined Mass Spectrometry and Data Integration Approach to Predict the Mitochondrial Poly(A) RNA Interacting Proteome

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