title: Nuclear outsourcing of RNA interference components to human mitochondria.
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 found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and 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 parameters. Computational 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 Argonaute 2 localization 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'.
authors.affiliation: INSERM U781 Hôpital Necker-Enfants Malades, Paris, France.
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bandiera
authors.forename: Silvia
authors.initials: S
authors.lastname: Rüberg
authors.forename: Muriel
authors.initials: M
authors.lastname: Girard
authors.forename: Nicolas
authors.initials: N
authors.lastname: Cagnard
authors.forename: Sylvain
authors.initials: S
authors.lastname: Hanein
authors.forename: Dominique
authors.initials: D
authors.lastname: Chrétien
authors.forename: Arnold
authors.initials: A
authors.lastname: Munnich
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.forename: Alexandra
authors.initials: A
authors.lastname: Henrion-Caude
mesh_terms: D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics*; D002877:Chromosomes, Human / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D054629:Genome, Mitochondrial / Q000235:genetics; D023281:Genomics; D006801:Humans; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000302:isolation & purification; D008928:Mitochondria / Q000235:genetics*; D008954:Models, Biological; D021381:Protein Transport; D034622:RNA Interference; D034443:RNA Transport / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D012333:RNA, Messenger