Newcastle University Eprints
Total Page：16
File Type：pdf, Size：1020Kb
Newcastle University ePrints Bruni F, Gramegna P, Oliveira JM, Lightowlers RN, Chrzanowska-Lightowlers ZMA. REXO2 is an oligoribonuclease active in human mitochondria. PLoS One 2013, 8(5), e64670. Copyright: © 2013 Bruni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The definitive article is available at: http://dx.doi.org/10.1371/journal.pone.0064670 Always use the definitive version when citing. Date deposited: 28th June 2013 This work is licensed under a Creative Commons Attribution 3.0 Unported License ePrints – Newcastle University ePrints http://eprint.ncl.ac.uk REXO2 Is an Oligoribonuclease Active in Human Mitochondria Francesco Bruni1, Pasqua Gramegna1, Jorge M. A. Oliveira3, Robert N. Lightowlers2*, Zofia M. A. Chrzanowska-Lightowlers1* 1 The Wellcome Trust Centre for Mitochondrial Research, Institute for Ageing and Health, Newcastle University, The Medical School, Newcastle upon Tyne, United Kingdom, 2 The Wellcome Trust Centre for Mitochondrial Research, Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Newcastle upon Tyne, United Kingdom, 3 REQUIMTE, Department of Drug Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal Abstract The Escherichia coli oligoribonuclease, ORN, has a 39 to 59 exonuclease activity specific for small oligomers that is essential for cell viability. The human homologue, REXO2, has hitherto been incompletely characterized, with only its in vitro ability to degrade small single-stranded RNA and DNA fragments documented. Here we show that the human enzyme has clear dual cellular localization being present both in cytosolic and mitochondrial fractions. Interestingly, the mitochondrial form localizes to both the intermembrane space and the matrix. Depletion of REXO2 by RNA interference causes a strong morphological phenotype in human cells, which show a disorganized network of punctate and granular mitochondria. Lack of REXO2 protein also causes a substantial decrease of mitochondrial nucleic acid content and impaired de novo mitochondrial protein synthesis. Our data constitute the first in vivo evidence for an oligoribonuclease activity in human mitochondria. Citation: Bruni F, Gramegna P, Oliveira JMA, Lightowlers RN, Chrzanowska-Lightowlers ZMA (2013) REXO2 Is an Oligoribonuclease Active in Human Mitochondria. PLoS ONE 8(5): e64670. doi:10.1371/journal.pone.0064670 Editor: Paul A. Cobine, Auburn University, United States of America Received February 5, 2013; Accepted April 17, 2013; Published May 31, 2013 Copyright: ß 2013 Bruni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: RNL and ZMACL would like to thank The Wellcome Trust [096919/Z/11/Z], BBSRC [BB/F011520/1], http://www.wellcome.ac.uk/, http://www.bbsrc.ac. uk/home/home.aspx. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (RNL); [email protected] (ZCL) Introduction that the enzyme performs a critical role in cell metabolism [7]. Depletion of ORN in P. aeruginosa also promotes accumulation of Ribonucleases are enzymes capable of cleaving the phospho- short RNA species, which are able to prime transcription, such diester bonds between the nucleotide subunits of RNA molecules. that depletion results in global alterations in gene expression [8]. Both endo- and exoribonucleases play important roles in the These examples demonstrate that oligoribonucleases perform regulation of RNA levels, performing functions in both RNA crucial functions in the final steps of RNA decay and can processing and degradation [1]. All the known exoribonucleases influence the transcription initiation process in bacteria. have been divided into six superfamilies based on their sequence, The ORN homologue in S. cerevisiae, named YNT20 [9] or structure and catalytic activity, with the vast majority exhibiting 39 REX2p, has been shown to localize to mitochondria and to have a to 59 degrading activity [2]. The largest is the superfamily DnaQ- function in the mitochondrial DNA escape pathway that is like or DEDD named for the four conserved acidic residues that mediated by YME1, a protease tightly associated with the inner cluster around the catalytic site of all family members. This mitochondrial membrane [10]. In contrast to its previous distinctive core is essential for binding the divalent metal ions that characterization as exclusively mitochondrial, YNT20 is required are required for catalysis [3]. The DnaQ-like domain contains for the processing of U4, U5L, U5S snRNAs as well as for the three conserved sequence motifs, namely ExoI, ExoII and ExoIII maturation of RNA components of both RNase P and 5.8S rRNA [4,5], each containing this distinctive core although the conserva- in yeast [11] all processes that occur within the nucleus. tion patterns of the three motifs may vary between different Very few human mitochondrial RNases have been fully subfamilies. characterized and none appear to be responsible for degrading One group of the DEDD superfamily contains oligoribonu- very short oligoribonucleotides into monoribonucleotides as the cleases (oligoRNases) that are essentially specific for small single- final step of mitochondrial RNA decay process [12]. Here we stranded RNA fragments of less than five nucleotides in length, demonstrate that REXO2 is indeed an active mitochondrial although some including human also display DNase activity. An protein, which is also localised to the cytosol. We provide evidence intriguing feature of this group is their high conservation and that this enzyme is essential for cell turnover and whose depletion distribution among the different phyla: they are represented in affects mitochondrial structure and function. Surprisingly, we vertebrates, invertebrates (insects and worms), yeast and some found the majority of REXO2 to be localized in the intermem- plants [6]. Functional studies of the orn gene in E. coli have brane space (IMS) and comment on this observation in our suggested that inactivation gives rise to the deleterious accumu- discussion. lation of short oligoribonucleotides causing cell death, confirming PLOS ONE | www.plosone.org 1 May 2013 | Volume 8 | Issue 5 | e64670 REXO2 Is a Human Mitochondrial Oligoribonuclease Materials and Methods proteinase K (4 mg/100 mg protein) for 30 min at 4uC and either lysed (1% final v/v Triton X-100) or treated with 1 mM PMSF Tissue Culture, and Transfections before separation through 15% PAG and transfer to a PVDF Human HeLa cells were cultured (37uC, humidified 5% CO2) membrane. For sub-mitochondrial fractionation, 300mg of mito- in Eagle’s MEM (Sigma) supplemented with 10% (v/v) foetal calf chondria prepared as described above were then treated with serum (FCS), 16non-essential amino acids (NEAA) and 2 mM L- proteinase K (1.5mg) on ice for 30 mins followed by addition of glutamine. 143B.206 rho0 osteosarcoma cells [13] were propagat- PMSF (5 mM) to generate shaved mitochondria. This was pelleted ed in Dulbecco’s modified Eagle’s medium supplemented with at 11 kg, 10 mins, 4uC and resuspended in HB (75ml), with 15ml 10% (v/v) FCS, 50 mg/ml uridine and 16 NEAA. Flp-InT-Rex- removed as shaved mitochondria. The remainder was pelleted and 293 cells (HEK293T; Invitrogen) were grown in identical media resuspended in 9 volumes of 10 mM Tris-HCl pH 7.4, divided in supplemented with 10 mg/ml BlasticidinS and 100 mg/ml Zeocin half and proteinase K (0.6mg) added to one sample. Both samples (Invitrogen) and transfected as previously described [14]. Post were left on ice for 30 mins before addition of PMSF (5 mM) and transfection selection was performed with HygromycinB (100 mg/ an equal volume of 1.2 M Mannitol, 2 mM EGTA, 10 mM Tris- ml). HCl pH 7.4. After centrifugation at 12 kg, 10 mins, 4uC, both Stable transfections were performed using constructs described pellets were resuspended in HB (30ml), and 15ml of each were below and Superfect (Qiagen) using manufacturer’s recommen- retained as shaved and unshaved mitoplasts. To prepare the inner dations. membrane fraction, HB (435ml) was added to the proteinase K SiRNA transfections were performed with 60% confluent HeLa treated mitoplasts, which were pelleted at 12 kg, pellet was cells using Lipofectamine RNAiMAX (Invitrogen) in Optimem-I dissolved in 100 mM Na2CO3 and incubated for 10 mins at 4uC. medium (Gibco) with 25 nM siRNA. Reverse transfections were This was centrifuged at 100 kg for 15 mins at 4uC after which the performed as described by Ovcharenko et al (2005) [15] using pellet was resuspended in 26dissociation buffer (20% (v/v) approximately 12 000 HEK293 Tcell per cm2 using Lipofecta- glycerol, 4% (w/v) SDS, 250 mM Tris-HCl pH 6.8, 100 mM mine RNAiMAX in Optimem-I medium (25 nM siRNA). Custom DTT). and control non-targetting (NT, Eurogentec ref OR-0030-neg05) duplex siRNAs were purchased pre-annealed from Eurogentec Imaging (REXO2 sense 59-GCAGGCAGAGUAUGAAUUUdTdT; anti- For nucleoid and mitochondria morphology imaging, cells were sense 59- AAAUUCAUACUCUGCCUGCdTdT.).