Small RNA sequencing of wild-type and Mrpp3 knock out mouse heart mitochondria
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE94005 | GEO | 2017/03/23
SECONDARY ACCESSION(S): PRJNA363046
REPOSITORIES: GEO
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Project description:Here we find that circularization of RNA prior to deep sequencing enables the discovery and characterization of unprocessed mitochondrial RNAs. Using this approach we identify the most stable processing intermediates and the presence of intermediate processing products that are partially degraded and polyadenylated. Analysis of libraries constructed using RNA from mice lacking the nuclease subunit of the mitochondrial RNase P reveals the identities of stalled processing intermediates, their order of cleavage, and confirms the importance of RNase P (MRPP3) in generating mature mitochondrial RNAs. Using RNA circularization prior to library preparation should provide a generally useful approach to studying RNA processing in many different biological systems.
2017-03-23 | GSE94030 | GEO
Project description:In animals the organization of the compact mitochondrial genome and lack of introns have necessitated a unique mechanism for RNA processing. To date the regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. To understand the in vivo role of the endoribonuclease component of the RNase P complex, MRPP3, we created conditional knockout mice. Here we show that MRPP3 is essential for life, and heart and skeletal muscle-specific knockout leads to a cardiomyopathy early in life, indicating that it is the only RNase P enzyme in mitochondria. We show that RNA processing is required for the biogenesis of the respiratory chain and mitochondrial function. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-Seq enabled us to identify the in vivo cleavage sites of RNase P. Cleavage of the 5â² tRNA ends precedes 3â² end processing in vivo and is required for the correct biogenesis of the mitochondrial ribosomal subunits and mitoribosomal proteins that are differentially stabilized or degraded in the absence of mature rRNAs. Finally we identify that large mitoribosomal proteins can form a subcomplex on a precursor mt-RNA containing the 16S rRNA indicating that mitoribosomal biogenesis proceeds co-transcriptionally. Taken together our data show that RNA processing links transcription to translation via assembly of the mitoribosome. Total RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), TruSeq libraries produced in triplicate, sequenced and analysed for differential expression. Mitochondrial RNA was isolated from heart tissue from 11 week old control (Mrpp3loxP/loxP) and Mrpp3 knockout mice (Mrpp3loxP/loxP, +/Ckmm), PARE libraries produced in triplicate and sequenced for analysis of mitochondrial RNA processing.
2016-08-04 | E-GEOD-83471 | biostudies-arrayexpress
Project description:Background: Mutations of the desmin gene cause familial and sporadic cardiomyopathies and myopathies. Previous studies showed that both the lack of desmin and expression of mutated desmin negatively impact on number, structure and function mitochondria implying a metabolic dysfunction as disease promoting factor. Here, we exploited desmin knock-out mice to analyse the general metabolic performance of cardiac tissue. Methods: We analysed left ventricular cardiac muscle tissue derived from six-month-old homozygous desmin knock-out mice as well as wild-type siblings. Employing a multi-method approach comprising morphological, clinical chemistry, biochemical, genetic, and proteomic techniques, we specifically addressed energy, fatty acid, glucose, and amino acid metabolism. Results: We demonstrated that the lack of desmin in left ventricular cardiac tissue led to a significant decrease in the number of mitochondria in conjunction with ultrastructural defects of mitochondria. Analysis of mitochondria-related metabolic pathways revealed significantly impaired processes of fatty acid transport, activation, and catabolism associated with increased blood levels of short, medium, and long chain acyl-carnitines. In addition to increased amounts of glucose transporter 1 and hexokinase 1, we detected a significantly increased hexokinase enzyme activity. While the amount of mitochondrial creatine kinase was markedly reduced, fetal creatine kinase was increased. The picture of a highly dysbalanced metabolic state was complemented by our quantitative proteomic analysis that revealed significantly reduced levels of multiple proteins centrally involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Conclusions: Our data denoted a picture of widespread metabolic dysfunction far reaching beyond the level of mitochondria in left ventricular cardiac tissue of desmin knock-out mice. The increase of glucose utilisation along with increased level of fetal creatine kinase likely is a compensatory measure counteracting the severely hampered fatty acid metabolism and oxidative phosphorylation. The degree of metabolic disbalance, already observed in desmin knock-out mice kept under standard ‘sedentary’ housing conditions, renders it likely that acute strenuous physical exercise with its steeply increased energy demand will push the cardiac tissue into severe metabolic crisis.
2022-10-21 | PXD030938 | Pride
Project description:In animals the organization of the compact mitochondrial genome and lack of introns have necessitated a unique mechanism for RNA processing. To date the regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. To understand the in vivo role of the endoribonuclease component of the RNase P complex, MRPP3, we created conditional knockout mice. Here we show that MRPP3 is essential for life, and heart and skeletal muscle-specific knockout leads to a cardiomyopathy early in life, indicating that it is the only RNase P enzyme in mitochondria. We show that RNA processing is required for the biogenesis of the respiratory chain and mitochondrial function. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-Seq enabled us to identify the in vivo cleavage sites of RNase P. Cleavage of the 5′ tRNA ends precedes 3′ end processing in vivo and is required for the correct biogenesis of the mitochondrial ribosomal subunits and mitoribosomal proteins that are differentially stabilized or degraded in the absence of mature rRNAs. Finally we identify that large mitoribosomal proteins can form a subcomplex on a precursor mt-RNA containing the 16S rRNA indicating that mitoribosomal biogenesis proceeds co-transcriptionally. Taken together our data show that RNA processing links transcription to translation via assembly of the mitoribosome.
2016-08-04 | GSE83471 | GEO
Project description:Yeast knockout strains were constructed by the Yeast Deletion Project. Three biological replicates were analyzed for each strain. 5 ml cultures were inoculated at OD600 = 0.2 from saturated overnight cultures and grown to mid-log phase (OD600 = 0.6 - 0.8) in YPD media 37 or in phosphate-depleted media 38 at 30C. Cells were harvested by centrifugation and washed with nuclease-free water (Ambion). Total RNA was isolated immediately after harvest using the Ribopure Yeast RNA Isolation Kit (Ambion). 5 mg of total RNA was used to generate labeled probes with standard Affymetrix protocols.
2010-09-24 | E-MEXP-1305 | biostudies-arrayexpress
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Project description:Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after 120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3-ends that can be used by DNA polymerase to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggests a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.
2018-10-26 | GSE103612 | GEO
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