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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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Intrinsic features of mitomiRs versus a control sample of cytosolic-enriched miRNAs.Values for each miRNA subgroup are shown in blue for the mitomiRs and red for the control miRNAs. Length of mature miRNAs (A) and of pre-miRNAs (B), and values of minimal folding free energy (MFE) (C) and adjusted MFE (AMFE) (D) are plotted for each miRNA from the 2 subgroups. Average values are shown as bars in each subgroup. Asterics indicate significant differences between mitomiRs versus control miRNAs (p-value<0.05). NS indicates not significant p-value.
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pone-0020746-g007: Intrinsic features of mitomiRs versus a control sample of cytosolic-enriched miRNAs.Values for each miRNA subgroup are shown in blue for the mitomiRs and red for the control miRNAs. Length of mature miRNAs (A) and of pre-miRNAs (B), and values of minimal folding free energy (MFE) (C) and adjusted MFE (AMFE) (D) are plotted for each miRNA from the 2 subgroups. Average values are shown as bars in each subgroup. Asterics indicate significant differences between mitomiRs versus control miRNAs (p-value<0.05). NS indicates not significant p-value.

Mentions: Secondly, we systematically analyzed the intrinsic features of mitomiRs. We assessed their lengths and thermodynamic features in comparison to the same control sample of 13 cytosolic-enriched miRNAs. Unequivocally, the control sample shared all expected features of miRNAs, in particular an average length of 22-nt for the mature and of 82-nt for the pre-miRNA sequence (Figure 7A). In contrast, the length of the mature mitomiRs varied substantially. Three mitomiRs were smaller than 19 nt (Table S7). In fact, the length distribution of the mature mitomiRs was significantly different from the control (p<0.005) but did not correlate with a difference at the pre-miRNA sequence level, which remained comparable (p = 0.2) (Figure 7A–B). Thus, we assessed the thermodynamic stability of the secondary structures of the mitomiRs by calculating their minimum folding energy (MFE). MFE displayed a significantly different distribution than the control group (p = 0.01) (Figure 7C, Table S7, and Table S8). Because MFE is strongly correlated with the length of the sequence, we also calculated the adjusted MFE (AMFE) (Table S7). Again, the distribution of AMFE values was significantly different from that of the control miRNAs (p<0.05), which revealed that mitochondrial-enriched miRNAs shared specific distinctive features as a group (Figure 7D, Table S7 and Table S8).


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)

Intrinsic features of mitomiRs versus a control sample of cytosolic-enriched miRNAs.Values for each miRNA subgroup are shown in blue for the mitomiRs and red for the control miRNAs. Length of mature miRNAs (A) and of pre-miRNAs (B), and values of minimal folding free energy (MFE) (C) and adjusted MFE (AMFE) (D) are plotted for each miRNA from the 2 subgroups. Average values are shown as bars in each subgroup. Asterics indicate significant differences between mitomiRs versus control miRNAs (p-value<0.05). NS indicates not significant p-value.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020746-g007: Intrinsic features of mitomiRs versus a control sample of cytosolic-enriched miRNAs.Values for each miRNA subgroup are shown in blue for the mitomiRs and red for the control miRNAs. Length of mature miRNAs (A) and of pre-miRNAs (B), and values of minimal folding free energy (MFE) (C) and adjusted MFE (AMFE) (D) are plotted for each miRNA from the 2 subgroups. Average values are shown as bars in each subgroup. Asterics indicate significant differences between mitomiRs versus control miRNAs (p-value<0.05). NS indicates not significant p-value.
Mentions: Secondly, we systematically analyzed the intrinsic features of mitomiRs. We assessed their lengths and thermodynamic features in comparison to the same control sample of 13 cytosolic-enriched miRNAs. Unequivocally, the control sample shared all expected features of miRNAs, in particular an average length of 22-nt for the mature and of 82-nt for the pre-miRNA sequence (Figure 7A). In contrast, the length of the mature mitomiRs varied substantially. Three mitomiRs were smaller than 19 nt (Table S7). In fact, the length distribution of the mature mitomiRs was significantly different from the control (p<0.005) but did not correlate with a difference at the pre-miRNA sequence level, which remained comparable (p = 0.2) (Figure 7A–B). Thus, we assessed the thermodynamic stability of the secondary structures of the mitomiRs by calculating their minimum folding energy (MFE). MFE displayed a significantly different distribution than the control group (p = 0.01) (Figure 7C, Table S7, and Table S8). Because MFE is strongly correlated with the length of the sequence, we also calculated the adjusted MFE (AMFE) (Table S7). Again, the distribution of AMFE values was significantly different from that of the control miRNAs (p<0.05), which revealed that mitochondrial-enriched miRNAs shared specific distinctive features as a group (Figure 7D, Table S7 and Table S8).

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