signature=76b9f12384ebc9da0fe851d513c586a3,Nuclear Outsourcing of RNA Interference Components to Hum...

Nuclear Outsourcing of RNA Interference Components to Human Mitochondria

Simonetta Bandiera1,Silvia Ru¨berg2,Muriel Girard1,Nicolas Cagnard3,Sylvain Hanein1,Dominique Chre′tien1,Arnold Munnich1,Stanislas Lyonnet1,Alexandra Henrion-Caude1*

1INSERM U781Ho?pital Necker–Enfants Malades,Paris,France,2Miltenyi Biotec GmbH,Bergisch Gladbach,Germany,3Paris-Descartes Bioinformatics Platform,Faculte′de Me′decine,Site Necker–Enfants Malades,Paris,France

Abstract

MicroRNAs(miRNAs)are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm.However,recent studies have reported that some miRNAs localize to and function in other cellular compartments.Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation.We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria.To enable identification of mitochondrial-enriched miRNAs,we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis.Mitochondria were purified using a combination of cell fractionation and immunoisolation,and assessed for the lack of protein and RNA contaminants.We found57miRNAs differentially expressed in HeLa mitochondria and cytosol.Of these57,a signature of 13nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275and hsa-miR-1974.The significance of their mitochondrial localization was investigated by characterizing their genomic context,cross-species conservation and instrinsic features such as their size and thermodynamic parameters.Interestingly,the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic http://www.doczj.com/doc/63f79568af1ffc4ffe47acd6.htmlputational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets.The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute2localization to mitochondria revealed by immunoblotting and confocal microscopy,and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3.This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria.Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed‘mitomiRs’.

Citation:Bandiera S,Ru¨berg S,Girard M,Cagnard N,Hanein S,et al.(2011)Nuclear Outsourcing of RNA Interference Components to Human Mitochondria.PLoS ONE6(6):e20746.doi:10.1371/journal.pone.0020746

Editor:Sebastien Pfeffer,French National Center for Scientific Research-Institut de biologie mole′culaire et cellulaire,France

Received December6,2010;Accepted May12,2011;Published June13,2011

Copyright:?2011Bandiera 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:The authors have no support or funding to report.

Competing Interests:One author,SR,is employed by a commercial company(Miltenyi Biotec GmbH).This does not alter the authors’adherence to all the PLoS ONE policies on sharing data and materials,as detailed online in the guide for authors.

*E-mail:alexandra.caude@inserm.fr

Introduction

Mitochondria are eukaryotic organelles that maintain and express their own genome,known as the mitochondrial DNA (mtDNA).The transcription and translation of the mtDNA as well as the processing of mitochondrial transcripts requires the involvement of several types of non-coding RNAs(ncRNA), which can be either mitochondrially encoded or transcribed within the nucleus and subsequently localized to mitochondria[1].In human mitochondria,the full set of mitochondrial transfer RNAs (tRNAs)and two ribosomal RNAs(rRNAs),namely the12S and 16S rRNAs,are transcribed from the mtDNA[2],while the RNA moiety of the RNase MRP enzymes[3,4,5],the5S rRNA[6,7], and two species of tRNA Gln[8]are all RNAs delivered into mitochondria from the nucleus.

Among ncRNAs,microRNAs(miRNAs)have emerged as an important class of post-transcriptional regulators of gene expression in virtually all fundamental cellular processes[9]. MiRNAs are transcribed within the nucleus and are then extensively processed and matured in the cytosol as,22-bp double-stranded RNA.Mature miRNAs associate with Argo-naute(AGO)proteins to form the core of a ribonucleoprotein complex named RNA-induced silcencing complex(RISC),which exerts RNA interference(RNAi)[10,11].RNAi occurs upon pairing one of the two miRNA strands,embedded in an AGO protein,with target sites in an mRNA,thereby affecting the stability/translation of this mRNA[11].In mammals,there are four AGO proteins,AGO1through AGO4,which were all shown to function in translational repression[12],but only AGO2can catalyze the cleavage of the targeted transcript [13,14].Furthermore,knockdown and knockout of AGO2, respectively in human cells and in mice,suggest that the protein may have specific functions that may not be complemented by the other AGOs[13,15].

Initially,mature miRNAs and AGO2were believed to accumulate and function exclusively in the cytosol and/or into unstructured cytosolic foci,such as P-bodies and stress granules [16,17].However,mounting evidence suggests that they can also