Micro RNA expression in human skeletal muscle mitochondria II
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE220557 | GEO | 2025/07/02
REPOSITORIES: GEO
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Project description:Mitochondria import over 1000 proteins encoded by the nuclear genome, but the RNA contained within the organelle is generally thought to be only that which is transcribed from the mitochondrial genome. Recent evidence however suggests the presence of specific nuclear-encoded small- and long-non-coding RNAs (ncRNAs) in the mitochondrial matrix, hinting at a previously underappreciated level of subcellular, ncRNA-mediated regulation on mitochondrial function. The aim of this study was to characterise the population of RNAs contained within mitochondria isolated from rat skeletal muscle, and to investigate whether it was altered in response to endurance training, a potent metabolic stimulus that induces mitochondrial adaptations. Rats underwent four weeks of moderate intensity treadmill exercise training that induced typical adaptive responses at the transcriptome and functional levels in heart and skeletal muscle. Mitochondria from gastrocnemius skeletal muscle were isolated by immunoprecipitation, further purified, then sequenced to assess the mitochondrial transcriptome. Approximately 24 nuclear-encoded, coding or non-coding ‘long’ RNAs were detected in purified mitochondria, in addition to ~50 miRNAs with >100 normalised read counts. None were differentially expressed in the exercise vs control group at FDR < 0.05, but exploratory analyses (p<0.05) pointed towards 3 differentially regulated miRNAs in the mitochondria isolated from trained compared with sedentary muscle. In conclusion, we report the presence of a small population of nuclear-encoded long- and small RNAs in the mitochondria isolated from rat muscle tissue. Further research is required to investigate how these are imported into the mitochondria, and their role(s) in regulating exercise adaptations and mitochondrial biology
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Project description:Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein 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. 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’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.
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