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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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Isolation of mitochondrial and cytosolic miRNA.A. Schematic workflow of the experimental design. B. Integrity, quality and purity analyses of RNA fractions. Electrophoretic images of mitochondrial and cytosolic RNAs were retrieved from analysis using the Agilent 2100 Bioanalyzer. Bands corresponding to ribosomal RNAs (rRNAs) 16S, 12S, 28S and 18S are indicated when present. Representative image is shown of three independent experiments. C. Purity assessment of mitochondrial RNA fraction. 16S rRNA and GAPDH were amplified by RT-PCR in each fraction as shown by electrophoretic image. GAPDH was assessed in the mitochondrial fraction to check for cytoplasmic contaminant relatively to mitochondrial 16S ribosomal RNA. Representative image is shown of three independent experiments. 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.
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pone-0020746-g004: Isolation of mitochondrial and cytosolic miRNA.A. Schematic workflow of the experimental design. B. Integrity, quality and purity analyses of RNA fractions. Electrophoretic images of mitochondrial and cytosolic RNAs were retrieved from analysis using the Agilent 2100 Bioanalyzer. Bands corresponding to ribosomal RNAs (rRNAs) 16S, 12S, 28S and 18S are indicated when present. Representative image is shown of three independent experiments. C. Purity assessment of mitochondrial RNA fraction. 16S rRNA and GAPDH were amplified by RT-PCR in each fraction as shown by electrophoretic image. GAPDH was assessed in the mitochondrial fraction to check for cytoplasmic contaminant relatively to mitochondrial 16S ribosomal RNA. Representative image is shown of three independent experiments. 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.

Mentions: We reasoned that differential identification of miRNAs in subcellular compartments from the same cells would provide the most reliable and effective method to investigate localization of miRNAs to mitochondria. Our experimental design enabled us to isolate mitochondria and cytosol fractions from the same cells and to profile differentially expressed miRNAs in each fraction using the miRXplore™ microarrays (Figure 4A). Total RNA was isolated respectively from mitochondrial and cytosolic fractions. Each RNA fraction was examined for its integrity, quality and purity through the Agilent 2100 Bioanalyzer. Electrophoretic gel images were observed for mitochondrial and cytosolic RNA fractions (Figure 4B). Consistently with the electrophoregrams, the 28S rRNA and 18S rRNA, which are located exclusively in the cytoplasm, were not observed in the mitochondrial RNA fraction indicating that mitochondrial and cytoplasmic RNA fractions were distinct. We further applied the RNA Integrity Number (RIN) algorithm to each sample to assign an integrity value [34]. Mean RIN values were respectively of 2.8 and 8.3 for the mitochondrial and cytosolic fractions. The cytosolic value was indicative of RNA of high quality. Since no standard exists as to the mitochondrial fraction, which consists of a distinct RNA population, we considered a value smaller than 6 as an indication of the depletion of cytosolic RNAs that we further ascertained by the depletion of cytosolic 18S and 28S rRNAs from mitochondrial RNA in the Bioanalyzer run. Finally, we assessed the purity of mitochondrial and cytosolic RNA fractions by reverse transcription-polymerase chain reaction (RT-PCR). 16S rRNA, which was chosen as a mitochondrial marker and thereby as a positive control for mitochondrial RNA, was enriched in the mitochondrial RNA fraction whereas it was depleted from the cytosolic RNA (Figure 4C). In contrast, GAPDH, an unambiguous cytosolic marker, could be amplified exclusively in the cytosolic fraction (Figure 4C). Altogether, these data indicated that no cytosolic contaminants could be detected in mitochondrial RNA, while faint signals of mitochondrial RNA were detected in the cytosol. These results are consistent with a high level of purity in either fraction.


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)

Isolation of mitochondrial and cytosolic miRNA.A. Schematic workflow of the experimental design. B. Integrity, quality and purity analyses of RNA fractions. Electrophoretic images of mitochondrial and cytosolic RNAs were retrieved from analysis using the Agilent 2100 Bioanalyzer. Bands corresponding to ribosomal RNAs (rRNAs) 16S, 12S, 28S and 18S are indicated when present. Representative image is shown of three independent experiments. C. Purity assessment of mitochondrial RNA fraction. 16S rRNA and GAPDH were amplified by RT-PCR in each fraction as shown by electrophoretic image. GAPDH was assessed in the mitochondrial fraction to check for cytoplasmic contaminant relatively to mitochondrial 16S ribosomal RNA. Representative image is shown of three independent experiments. 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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113838&req=5

pone-0020746-g004: Isolation of mitochondrial and cytosolic miRNA.A. Schematic workflow of the experimental design. B. Integrity, quality and purity analyses of RNA fractions. Electrophoretic images of mitochondrial and cytosolic RNAs were retrieved from analysis using the Agilent 2100 Bioanalyzer. Bands corresponding to ribosomal RNAs (rRNAs) 16S, 12S, 28S and 18S are indicated when present. Representative image is shown of three independent experiments. C. Purity assessment of mitochondrial RNA fraction. 16S rRNA and GAPDH were amplified by RT-PCR in each fraction as shown by electrophoretic image. GAPDH was assessed in the mitochondrial fraction to check for cytoplasmic contaminant relatively to mitochondrial 16S ribosomal RNA. Representative image is shown of three independent experiments. 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.
Mentions: We reasoned that differential identification of miRNAs in subcellular compartments from the same cells would provide the most reliable and effective method to investigate localization of miRNAs to mitochondria. Our experimental design enabled us to isolate mitochondria and cytosol fractions from the same cells and to profile differentially expressed miRNAs in each fraction using the miRXplore™ microarrays (Figure 4A). Total RNA was isolated respectively from mitochondrial and cytosolic fractions. Each RNA fraction was examined for its integrity, quality and purity through the Agilent 2100 Bioanalyzer. Electrophoretic gel images were observed for mitochondrial and cytosolic RNA fractions (Figure 4B). Consistently with the electrophoregrams, the 28S rRNA and 18S rRNA, which are located exclusively in the cytoplasm, were not observed in the mitochondrial RNA fraction indicating that mitochondrial and cytoplasmic RNA fractions were distinct. We further applied the RNA Integrity Number (RIN) algorithm to each sample to assign an integrity value [34]. Mean RIN values were respectively of 2.8 and 8.3 for the mitochondrial and cytosolic fractions. The cytosolic value was indicative of RNA of high quality. Since no standard exists as to the mitochondrial fraction, which consists of a distinct RNA population, we considered a value smaller than 6 as an indication of the depletion of cytosolic RNAs that we further ascertained by the depletion of cytosolic 18S and 28S rRNAs from mitochondrial RNA in the Bioanalyzer run. Finally, we assessed the purity of mitochondrial and cytosolic RNA fractions by reverse transcription-polymerase chain reaction (RT-PCR). 16S rRNA, which was chosen as a mitochondrial marker and thereby as a positive control for mitochondrial RNA, was enriched in the mitochondrial RNA fraction whereas it was depleted from the cytosolic RNA (Figure 4C). In contrast, GAPDH, an unambiguous cytosolic marker, could be amplified exclusively in the cytosolic fraction (Figure 4C). Altogether, these data indicated that no cytosolic contaminants could be detected in mitochondrial RNA, while faint signals of mitochondrial RNA were detected in the cytosol. These results are consistent with a high level of purity in either fraction.

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