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Nuclear outsourcing of RNA interference components to human mitochondria.

Bandiera S, Rüberg S, Girard M, Cagnard N, Hanein S, Chrétien D, Munnich A, Lyonnet S, Henrion-Caude A - PLoS ONE (2011)

Bottom Line: We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol.Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters.This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria.

View Article: PubMed Central - PubMed

Affiliation: INSERM U781 Hôpital 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 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'.

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Novel localization of Argonaute 2 (AGO2) to human mitochondria.A. Purity assessment of mitochondrial fraction. Cyclin-dependent kinase 2 (CDK2) was assessed in the mitochondrial fraction by immunoblot analyses to check for nuclear and cytoplasmic contaminants relatively to mitochondrial protein ATP synthase subunit α (ATP5A1). The density of bands was measured using the ImageJ software and is represented as a relative intensity. Values are means±SD of three independent experiments. B. Western blot analysis of AGO2 in subcellular mitochondrial fraction from HeLa cells. AGO2 protein was detected using a rabbit polyclonal antibody anti-AGO2 in mitochondrial protein fraction (Mito) and total protein extracts (Lysate) from HeLa cells. ATP5A1 was used as a mitochondrial marker, actin as a cytosolic marker, and CDK2 as a nuclear/cytosolic marker. Representative image is shown of three independent experiments. C. Western blot analysis of AGO2 at mitochondrial membranous and soluble fraction from U2OS cells. Mitochondria were suspended either in isotonic mitochondrial buffer (MB) or in hypo-osmotic mitochondrial buffer (MB/10) alone or supplemented either with NaCl 1 M or Na2CO3 0.1 M, and fragilized by freeze-thaw cycles and sonication when indicated. All samples were centrifuged (150,000× g) to separate the membrane pellet (p) from the soluble protein supernatants (s). 15 µg of protein extracts of each sample were subjected to Western Blot. The following proteins were immunodecorated on blots: voltage-dependent anion channel 1 (VDAC1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9) as markers of mitochondrial membranes and cytochrome c (CYCS), as a intermembrane marker. Immunodetection of AGO2 is shown as the lower panel.
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pone-0020746-g001: Novel localization of Argonaute 2 (AGO2) to human mitochondria.A. Purity assessment of mitochondrial fraction. Cyclin-dependent kinase 2 (CDK2) was assessed in the mitochondrial fraction by immunoblot analyses to check for nuclear and cytoplasmic contaminants relatively to mitochondrial protein ATP synthase subunit α (ATP5A1). The density of bands was measured using the ImageJ software and is represented as a relative intensity. Values are means±SD of three independent experiments. B. Western blot analysis of AGO2 in subcellular mitochondrial fraction from HeLa cells. AGO2 protein was detected using a rabbit polyclonal antibody anti-AGO2 in mitochondrial protein fraction (Mito) and total protein extracts (Lysate) from HeLa cells. ATP5A1 was used as a mitochondrial marker, actin as a cytosolic marker, and CDK2 as a nuclear/cytosolic marker. Representative image is shown of three independent experiments. C. Western blot analysis of AGO2 at mitochondrial membranous and soluble fraction from U2OS cells. Mitochondria were suspended either in isotonic mitochondrial buffer (MB) or in hypo-osmotic mitochondrial buffer (MB/10) alone or supplemented either with NaCl 1 M or Na2CO3 0.1 M, and fragilized by freeze-thaw cycles and sonication when indicated. All samples were centrifuged (150,000× g) to separate the membrane pellet (p) from the soluble protein supernatants (s). 15 µg of protein extracts of each sample were subjected to Western Blot. The following proteins were immunodecorated on blots: voltage-dependent anion channel 1 (VDAC1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9) as markers of mitochondrial membranes and cytochrome c (CYCS), as a intermembrane marker. Immunodetection of AGO2 is shown as the lower panel.

Mentions: We first addressed the possibility that endogenous AGO2 could localize to mitochondria. To isolate highly-purified mitochondria, we performed cell fractionation combined with subsequent immunoisolation of mitochondria. This isolation procedure adapted from Hornig-Do et al. [25] was checked for efficiency by measuring the activity of mitochondrial and cytosolic marker enzymes (Supporting Information S1). Mitochondrial fraction was analyzed for its purity by immunoblot as assessed by the mitochondrial marker ATP synthase subunit α 1 (ATP5A1) and the nuclear/cytosolic marker cyclin-dependent kinase 2 (CDK2), which indicated the lack of nuclear and cytosolic contaminants reproducibly (Figure 1A). To study the localization of endogenous AGO2, we chose the same polyclonal antibody as that used in the founding studies of AGO2 localization [16], [26], [27]. By immunoblotting, we detected AGO2 as a band of ∼102 kDa in HeLa mitochondrial proteins as well as in the total protein extract from the same cells (Figure 1B). Assessment of actin as a cytosolic marker further validated the lack of cytosolic contaminants (Figure 1B). To further ascertain AGO2 at mitochondria, we extracted the crude membrane pellet and the soluble protein fraction from mitochondria isolated from a U2OS cell line. Mitochondria were incubated in hypo-osmotic buffer alone or supplemented with either 1 M NaCl or 0.1 M Na2CO3, pH 11 prior and after fragilization. Efficacy of treatments was ensured by immunoblotting detection of the following proteins: voltage-dependent anion channel 1 (VDAC1), cytochrome c (CYCS) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9), which all showed patterns consistent with the known location of those markers, respectively as a mitochondrial membrane marker, an intermembrane marker and a mitochondrial membrane marker (Figure 1C). As shown in Figure 1C, AGO2 was detected in the mitochondrial membrane fraction and to a lesser extent in the soluble fraction, suggesting its preferential association to mitochondrial membranes.


Nuclear outsourcing of RNA interference components to human mitochondria.

Bandiera S, Rüberg S, Girard M, Cagnard N, Hanein S, Chrétien D, Munnich A, Lyonnet S, Henrion-Caude A - PLoS ONE (2011)

Novel localization of Argonaute 2 (AGO2) to human mitochondria.A. Purity assessment of mitochondrial fraction. Cyclin-dependent kinase 2 (CDK2) was assessed in the mitochondrial fraction by immunoblot analyses to check for nuclear and cytoplasmic contaminants relatively to mitochondrial protein ATP synthase subunit α (ATP5A1). The density of bands was measured using the ImageJ software and is represented as a relative intensity. Values are means±SD of three independent experiments. B. Western blot analysis of AGO2 in subcellular mitochondrial fraction from HeLa cells. AGO2 protein was detected using a rabbit polyclonal antibody anti-AGO2 in mitochondrial protein fraction (Mito) and total protein extracts (Lysate) from HeLa cells. ATP5A1 was used as a mitochondrial marker, actin as a cytosolic marker, and CDK2 as a nuclear/cytosolic marker. Representative image is shown of three independent experiments. C. Western blot analysis of AGO2 at mitochondrial membranous and soluble fraction from U2OS cells. Mitochondria were suspended either in isotonic mitochondrial buffer (MB) or in hypo-osmotic mitochondrial buffer (MB/10) alone or supplemented either with NaCl 1 M or Na2CO3 0.1 M, and fragilized by freeze-thaw cycles and sonication when indicated. All samples were centrifuged (150,000× g) to separate the membrane pellet (p) from the soluble protein supernatants (s). 15 µg of protein extracts of each sample were subjected to Western Blot. The following proteins were immunodecorated on blots: voltage-dependent anion channel 1 (VDAC1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9) as markers of mitochondrial membranes and cytochrome c (CYCS), as a intermembrane marker. Immunodetection of AGO2 is shown as the lower panel.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113838&req=5

pone-0020746-g001: Novel localization of Argonaute 2 (AGO2) to human mitochondria.A. Purity assessment of mitochondrial fraction. Cyclin-dependent kinase 2 (CDK2) was assessed in the mitochondrial fraction by immunoblot analyses to check for nuclear and cytoplasmic contaminants relatively to mitochondrial protein ATP synthase subunit α (ATP5A1). The density of bands was measured using the ImageJ software and is represented as a relative intensity. Values are means±SD of three independent experiments. B. Western blot analysis of AGO2 in subcellular mitochondrial fraction from HeLa cells. AGO2 protein was detected using a rabbit polyclonal antibody anti-AGO2 in mitochondrial protein fraction (Mito) and total protein extracts (Lysate) from HeLa cells. ATP5A1 was used as a mitochondrial marker, actin as a cytosolic marker, and CDK2 as a nuclear/cytosolic marker. Representative image is shown of three independent experiments. C. Western blot analysis of AGO2 at mitochondrial membranous and soluble fraction from U2OS cells. Mitochondria were suspended either in isotonic mitochondrial buffer (MB) or in hypo-osmotic mitochondrial buffer (MB/10) alone or supplemented either with NaCl 1 M or Na2CO3 0.1 M, and fragilized by freeze-thaw cycles and sonication when indicated. All samples were centrifuged (150,000× g) to separate the membrane pellet (p) from the soluble protein supernatants (s). 15 µg of protein extracts of each sample were subjected to Western Blot. The following proteins were immunodecorated on blots: voltage-dependent anion channel 1 (VDAC1) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9) as markers of mitochondrial membranes and cytochrome c (CYCS), as a intermembrane marker. Immunodetection of AGO2 is shown as the lower panel.
Mentions: We first addressed the possibility that endogenous AGO2 could localize to mitochondria. To isolate highly-purified mitochondria, we performed cell fractionation combined with subsequent immunoisolation of mitochondria. This isolation procedure adapted from Hornig-Do et al. [25] was checked for efficiency by measuring the activity of mitochondrial and cytosolic marker enzymes (Supporting Information S1). Mitochondrial fraction was analyzed for its purity by immunoblot as assessed by the mitochondrial marker ATP synthase subunit α 1 (ATP5A1) and the nuclear/cytosolic marker cyclin-dependent kinase 2 (CDK2), which indicated the lack of nuclear and cytosolic contaminants reproducibly (Figure 1A). To study the localization of endogenous AGO2, we chose the same polyclonal antibody as that used in the founding studies of AGO2 localization [16], [26], [27]. By immunoblotting, we detected AGO2 as a band of ∼102 kDa in HeLa mitochondrial proteins as well as in the total protein extract from the same cells (Figure 1B). Assessment of actin as a cytosolic marker further validated the lack of cytosolic contaminants (Figure 1B). To further ascertain AGO2 at mitochondria, we extracted the crude membrane pellet and the soluble protein fraction from mitochondria isolated from a U2OS cell line. Mitochondria were incubated in hypo-osmotic buffer alone or supplemented with either 1 M NaCl or 0.1 M Na2CO3, pH 11 prior and after fragilization. Efficacy of treatments was ensured by immunoblotting detection of the following proteins: voltage-dependent anion channel 1 (VDAC1), cytochrome c (CYCS) and NADH dehydrogenase (ubiquinone) 1 α subcomplex 9 (NDUFA9), which all showed patterns consistent with the known location of those markers, respectively as a mitochondrial membrane marker, an intermembrane marker and a mitochondrial membrane marker (Figure 1C). As shown in Figure 1C, AGO2 was detected in the mitochondrial membrane fraction and to a lesser extent in the soluble fraction, suggesting its preferential association to mitochondrial membranes.

Bottom Line: We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol.Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters.This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria.

View Article: PubMed Central - PubMed

Affiliation: INSERM U781 Hôpital 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 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'.

Show MeSH
Related in: MedlinePlus