Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Using mitoribosomal profiling to investigate human mitochondrial translation

ABSTRACT: Human 143B.206 Rho+ cells (depleted mitochondrial DNA) and cytoplasts (derived from mitochondrial patient fibroblast containing C1624T mutation in mitochondrial tRNAVal ) were previously used to generated transmitochondrial cybrid lines. Hek293T cells,143B cells and cybrid cells were cultured in DMEM supplemented with 10% FCS in 5% CO2 at 37 °C. Cells were treated with Chloramphenicol (100 μg/ ml) for 15 min before harvest. Cells were then lysed and RNase I digested. Lysates were then loaded on a sucrose gradient (7%-47%). Fractions containing mito-monosomes were used to isolated RNA which were used to generate the library for deep sequencing. Mitoribosomal profiling were generated for both control (143B and Hek293T) and cybrid cells to reveal aspects of mitoribosome behaviour.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Fei Gao

PROVIDER: E-MTAB-6284 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).

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Using mitoribosomal profiling to investigate human mitochondrial translation

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