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Pre-microRNA and mature microRNA in human mitochondria.

Barrey E, Saint-Auret G, Bonnamy B, Damas D, Boyer O, Gidrol X - PLoS ONE (2011)

Bottom Line: Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test.Forty six miRNA were significantly expressed (2(nd) derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration.A set of miRNA were significantly detected in mitochondria fraction.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie Intégrative des Adaptations à l'Exercice-INSERM U902, Genopole Evry, France. eric.barrey@inserm.fr

ABSTRACT

Background: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells.

Methodology/principal findings: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test. In situ hybridization of pre-mir-302a, pre-let-7b and mir-365, using specific labelled locked nucleic acids and confocal microscopy, demonstrated that these miRNA were localized in mitochondria of human myoblasts. Total RNA was extracted from enriched mitochondria isolated by an immunomagnetic method from a culture of human myotubes. The detection of 742 human miRNA (miRBase) were monitored by RT-qPCR at three increasing mtRNA inputs. Forty six miRNA were significantly expressed (2(nd) derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05).

Conclusions/significance: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria.

Show MeSH
In situ hybridization pattern of digoxigenin-labeled Locked Nucleic Acid (LNA) for specific miR and pre-miR in human skeletal muscle myoblasts cells observed in classic optic microscopy.Using locked nucleic acid (LNA) probes digoxigenin labelled, we determined the in situ hybridization pattern of mir and pre-mir. Hoechst 33342 staining of nuclei (lane 1), specific signal of scramble miRNA (A; negative control), U6 small nuclear RNA (B; positive control), let-7b (C), mir-365 (D), pre-let-7b (E) and pre-mir-302a (F) probes (lane 2) and MitoTracker® Red CM-H2XRos staining of respiring mitochondria (lane 3) are represented in gray scale. All these images were acquired using an Olympus BX61 straight microscope controlled with Metamorph software (Molecular Devices, Downington, PA19335) using a 100× oil-immersion objective. In the overlays (lane 4) provided by Image J software, positive in situ hybridization signals are visualized in green, respiring mitochondria signal in red and nuclei staining in blue. Yellow staining suggests co-localization of LNA probes (green fluorescence) and MitoTracker® Red CM-H2Ros (red fluorescence). Scale bars = 10 µm. Images are not scaled to the same intensity range. Positive in situ hybridization signals were normalized by scramble miR signal intensity (negative control).
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pone-0020220-g003: In situ hybridization pattern of digoxigenin-labeled Locked Nucleic Acid (LNA) for specific miR and pre-miR in human skeletal muscle myoblasts cells observed in classic optic microscopy.Using locked nucleic acid (LNA) probes digoxigenin labelled, we determined the in situ hybridization pattern of mir and pre-mir. Hoechst 33342 staining of nuclei (lane 1), specific signal of scramble miRNA (A; negative control), U6 small nuclear RNA (B; positive control), let-7b (C), mir-365 (D), pre-let-7b (E) and pre-mir-302a (F) probes (lane 2) and MitoTracker® Red CM-H2XRos staining of respiring mitochondria (lane 3) are represented in gray scale. All these images were acquired using an Olympus BX61 straight microscope controlled with Metamorph software (Molecular Devices, Downington, PA19335) using a 100× oil-immersion objective. In the overlays (lane 4) provided by Image J software, positive in situ hybridization signals are visualized in green, respiring mitochondria signal in red and nuclei staining in blue. Yellow staining suggests co-localization of LNA probes (green fluorescence) and MitoTracker® Red CM-H2Ros (red fluorescence). Scale bars = 10 µm. Images are not scaled to the same intensity range. Positive in situ hybridization signals were normalized by scramble miR signal intensity (negative control).

Mentions: To demonstrate that these miRNA were localized in mitochondria, we performed in situ hybridization using specific labelled locked nucleic acid sequences that specifically hybridized with corresponding miRNA. The mitochondria were labelled with a selective fluorescent probe which passively diffused across the plasma membrane and accumulate in active mitochondria (Red MitoTracker®). Thus, the staining of the mitochondria by the MitoTracker® was a proof of their integrity and functionality. The images were then analyzed by fluorescent conventional microscopy (Figure 3) and then confirmed by confocal microscopy (see below). As positive control we used a LNA targeting the small nuclear RNA, RNU6B, (Figure 3A). A LNA targeting scramble miRNA, was used as negative control and did not generate any signal (Figure 3B). Mir-365, pre-mir-302a and pre-let7b specific fluorescent LNA were clearly co-localized in perinuclear mitochondria, as demonstrated by the yellow signal one could observed in that area (Figure 3D, E, F). Mir 365 exhibited a strong signal in the mitochondria but also in some areas of the nucleus (Figure 3D). The same observation was made for pre-let-7b (Figure 3E). Surprisingly, let-7b localized only in some points of the nucleus (Figure 3C). To further evaluate the specificity of the labels, we chased fluorescent LNA with a 10× excess of unlabelled LNA. As demonstrated in Figure 4, in situ hybridization was no longer observed in presence of unlabelled LNA, thus demonstrating the specificity of the in situ hybridization. Although specific, the co-localization of miRNA and mitochondria that we observed could be biased by the conventional microscopy. As a final demonstration of miRNA co-localization in the mitochondria, we analyzed LNA-mediated specific miRNA in situ hybridization by fluorescent confocal microscopy (Figure 5). We confirmed the intense co-localization of mir-365 in the mitochondria and some local areas in the nucleus (Figure 5 D). Strikingly, we also observed that two pre-miRNAs, pre-mir302a and pre-let-7b were located within mitochondria surrounding the nucleus (Figures 5E, F). As in coventional microscopy, some traces of pre-let-7b were observed in the nucleus (Figure 5E). On the opposite, the mature form of let-7b was poorly detected in the mitochondria (co-localization in 1% of the pixels) but mainly in the nucleus (Figure 5C). Since in situ hybridization experiments clearly established the presence of at least one miRNA and two pre-miRNA in human mitochondria, we next analyzed the small RNA content in highly purified mitochondria isolated from human myotubes.


Pre-microRNA and mature microRNA in human mitochondria.

Barrey E, Saint-Auret G, Bonnamy B, Damas D, Boyer O, Gidrol X - PLoS ONE (2011)

In situ hybridization pattern of digoxigenin-labeled Locked Nucleic Acid (LNA) for specific miR and pre-miR in human skeletal muscle myoblasts cells observed in classic optic microscopy.Using locked nucleic acid (LNA) probes digoxigenin labelled, we determined the in situ hybridization pattern of mir and pre-mir. Hoechst 33342 staining of nuclei (lane 1), specific signal of scramble miRNA (A; negative control), U6 small nuclear RNA (B; positive control), let-7b (C), mir-365 (D), pre-let-7b (E) and pre-mir-302a (F) probes (lane 2) and MitoTracker® Red CM-H2XRos staining of respiring mitochondria (lane 3) are represented in gray scale. All these images were acquired using an Olympus BX61 straight microscope controlled with Metamorph software (Molecular Devices, Downington, PA19335) using a 100× oil-immersion objective. In the overlays (lane 4) provided by Image J software, positive in situ hybridization signals are visualized in green, respiring mitochondria signal in red and nuclei staining in blue. Yellow staining suggests co-localization of LNA probes (green fluorescence) and MitoTracker® Red CM-H2Ros (red fluorescence). Scale bars = 10 µm. Images are not scaled to the same intensity range. Positive in situ hybridization signals were normalized by scramble miR signal intensity (negative control).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102686&req=5

pone-0020220-g003: In situ hybridization pattern of digoxigenin-labeled Locked Nucleic Acid (LNA) for specific miR and pre-miR in human skeletal muscle myoblasts cells observed in classic optic microscopy.Using locked nucleic acid (LNA) probes digoxigenin labelled, we determined the in situ hybridization pattern of mir and pre-mir. Hoechst 33342 staining of nuclei (lane 1), specific signal of scramble miRNA (A; negative control), U6 small nuclear RNA (B; positive control), let-7b (C), mir-365 (D), pre-let-7b (E) and pre-mir-302a (F) probes (lane 2) and MitoTracker® Red CM-H2XRos staining of respiring mitochondria (lane 3) are represented in gray scale. All these images were acquired using an Olympus BX61 straight microscope controlled with Metamorph software (Molecular Devices, Downington, PA19335) using a 100× oil-immersion objective. In the overlays (lane 4) provided by Image J software, positive in situ hybridization signals are visualized in green, respiring mitochondria signal in red and nuclei staining in blue. Yellow staining suggests co-localization of LNA probes (green fluorescence) and MitoTracker® Red CM-H2Ros (red fluorescence). Scale bars = 10 µm. Images are not scaled to the same intensity range. Positive in situ hybridization signals were normalized by scramble miR signal intensity (negative control).
Mentions: To demonstrate that these miRNA were localized in mitochondria, we performed in situ hybridization using specific labelled locked nucleic acid sequences that specifically hybridized with corresponding miRNA. The mitochondria were labelled with a selective fluorescent probe which passively diffused across the plasma membrane and accumulate in active mitochondria (Red MitoTracker®). Thus, the staining of the mitochondria by the MitoTracker® was a proof of their integrity and functionality. The images were then analyzed by fluorescent conventional microscopy (Figure 3) and then confirmed by confocal microscopy (see below). As positive control we used a LNA targeting the small nuclear RNA, RNU6B, (Figure 3A). A LNA targeting scramble miRNA, was used as negative control and did not generate any signal (Figure 3B). Mir-365, pre-mir-302a and pre-let7b specific fluorescent LNA were clearly co-localized in perinuclear mitochondria, as demonstrated by the yellow signal one could observed in that area (Figure 3D, E, F). Mir 365 exhibited a strong signal in the mitochondria but also in some areas of the nucleus (Figure 3D). The same observation was made for pre-let-7b (Figure 3E). Surprisingly, let-7b localized only in some points of the nucleus (Figure 3C). To further evaluate the specificity of the labels, we chased fluorescent LNA with a 10× excess of unlabelled LNA. As demonstrated in Figure 4, in situ hybridization was no longer observed in presence of unlabelled LNA, thus demonstrating the specificity of the in situ hybridization. Although specific, the co-localization of miRNA and mitochondria that we observed could be biased by the conventional microscopy. As a final demonstration of miRNA co-localization in the mitochondria, we analyzed LNA-mediated specific miRNA in situ hybridization by fluorescent confocal microscopy (Figure 5). We confirmed the intense co-localization of mir-365 in the mitochondria and some local areas in the nucleus (Figure 5 D). Strikingly, we also observed that two pre-miRNAs, pre-mir302a and pre-let-7b were located within mitochondria surrounding the nucleus (Figures 5E, F). As in coventional microscopy, some traces of pre-let-7b were observed in the nucleus (Figure 5E). On the opposite, the mature form of let-7b was poorly detected in the mitochondria (co-localization in 1% of the pixels) but mainly in the nucleus (Figure 5C). Since in situ hybridization experiments clearly established the presence of at least one miRNA and two pre-miRNA in human mitochondria, we next analyzed the small RNA content in highly purified mitochondria isolated from human myotubes.

Bottom Line: Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test.Forty six miRNA were significantly expressed (2(nd) derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration.A set of miRNA were significantly detected in mitochondria fraction.

View Article: PubMed Central - PubMed

Affiliation: Unité de Biologie Intégrative des Adaptations à l'Exercice-INSERM U902, Genopole Evry, France. eric.barrey@inserm.fr

ABSTRACT

Background: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells.

Methodology/principal findings: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value<0.1) were found in the reference mitochondrial sequence and some of the best candidates were chosen for a co-localization test. In situ hybridization of pre-mir-302a, pre-let-7b and mir-365, using specific labelled locked nucleic acids and confocal microscopy, demonstrated that these miRNA were localized in mitochondria of human myoblasts. Total RNA was extracted from enriched mitochondria isolated by an immunomagnetic method from a culture of human myotubes. The detection of 742 human miRNA (miRBase) were monitored by RT-qPCR at three increasing mtRNA inputs. Forty six miRNA were significantly expressed (2(nd) derivative method Cp>35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05).

Conclusions/significance: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria.

Show MeSH