Limits...
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'.

Show MeSH

Related in: MedlinePlus

Co-immunoprecipitation of AGO2 and mitochondrial transcripts.Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III (COX3), cytochrome b (cyt b) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are indicative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113838&req=5

pone-0020746-g003: Co-immunoprecipitation of AGO2 and mitochondrial transcripts.Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III (COX3), cytochrome b (cyt b) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are indicative of three independent experiments.

Mentions: To assess the possibility of AGO2 to function at the mitochondria, we then examined whether AGO2 could interact with the mitochondrial transcript cytochrome c oxidase III (COX3) as previously found in HEK293 cells [33]. By co-immunoprecipitating endogenous AGO2 with the associated RNAs in HeLa cell extracts and subsequent RT-PCR, we consistently identified COX3 as reproducibly associated, in comparison to a mitochondrial transcript cytochrome b (cyt b) and a cytosolic transcript glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that were not associated (Figure 3). Those co-immunoprecipitation results were performed along the control immunoprecipitation of a transcriptional factor SLUG, which as expected did not lead to the identification of any targets. Collectively, our data support the novel localization and functioning of AGO2 at the human mitochondria, a finding that prompted us to search for mitochondrial miRNAs.


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)

Co-immunoprecipitation of AGO2 and mitochondrial transcripts.Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III (COX3), cytochrome b (cyt b) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are indicative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020746-g003: Co-immunoprecipitation of AGO2 and mitochondrial transcripts.Either AGO2, or SLUG, which serves as a negative control were co-immunoprecipitated with associated mRNAs in HeLa protein extracts. Coimmunoprecipitated RNA was extracted with Trizol and subjected to RT-PCR amplification with the indicated primers: cytochrome c oxidase III (COX3), cytochrome b (cyt b) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results are indicative of three independent experiments.
Mentions: To assess the possibility of AGO2 to function at the mitochondria, we then examined whether AGO2 could interact with the mitochondrial transcript cytochrome c oxidase III (COX3) as previously found in HEK293 cells [33]. By co-immunoprecipitating endogenous AGO2 with the associated RNAs in HeLa cell extracts and subsequent RT-PCR, we consistently identified COX3 as reproducibly associated, in comparison to a mitochondrial transcript cytochrome b (cyt b) and a cytosolic transcript glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that were not associated (Figure 3). Those co-immunoprecipitation results were performed along the control immunoprecipitation of a transcriptional factor SLUG, which as expected did not lead to the identification of any targets. Collectively, our data support the novel localization and functioning of AGO2 at the human mitochondria, a finding that prompted us to search for mitochondrial miRNAs.

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