ABSTRACT
Mitochondrial RNAs are modified posttranscriptionally. These modifications are required for proper functioning of RNA molecules, and thereby contribute to essential mitochondrial processes. Herein, we describe our latest mass spectrometry-based platform for analysis of posttranscriptional modifications of mitochondrial tRNAs, and measuring the in vitro activity of mitochondrial RNA-modifying enzymes.
Subject(s)
Mass Spectrometry/methods , Mitochondria/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Mitochondrial/chemistry , RNA, Mitochondrial/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Biocatalysis , Humans , Nucleic Acid Conformation , Nucleosides/chemistry , RNA, Mitochondrial/isolation & purification , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/isolation & purification , tRNA Methyltransferases/metabolismABSTRACT
Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.
Subject(s)
RNA Processing, Post-Transcriptional , RNA, Mitochondrial/chemistry , RNA, Transfer/chemistry , Female , Genetic Code , HEK293 Cells , HeLa Cells , Humans , Mass Spectrometry , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Molecular Structure , Nucleoside Q/biosynthesis , Nucleoside Q/chemistry , Oxidative Phosphorylation , Placenta , Pregnancy , RNA, Mitochondrial/isolation & purification , RNA, Mitochondrial/metabolism , RNA, Transfer/isolation & purification , RNA, Transfer/metabolism , RNA-SeqABSTRACT
Our study purpose was to evaluate mitochondrial (mt)DNA and RNA in peripheral blood mononuclear cells (PBMCs) and body shape changes (BSC) in HBV-infected patients. mtDNA and mtRNA were measured in PBMCs. The presence of BSC was evaluated through a questionnaire and clinical evaluation. A total of 157 subjects were enrolled, of these 107 were HBV-infected patients, 54 receiving nucleoside analogues (NAs, Group A), 53 naive to antivirals (Group B) and 50 age-sex matched controls (Group C). All HBV-treated patients had negative HBV-DNA. Twenty (37,0%) received lamivudine + adefovir, 20 (37.0%) tenofovir, 2 (3.7%) lamivudine and 12 (22.2%) entecavir. Therapy median duration was 38 months (IQR 20-60) in NA-treated patients. Group A showed significantly higher mtDNA/nuclear (n) DNA ratio (p = 0.000008) compared to Group C and Group B (p = 0.002). Group B showed significantly higher mtDNA/nDNA ratio compared to Group C (p = 0.017). Group A and B had significantly lower mtRNA/nRNA ratio compared to Group C (p = 0.00003 and p = 0.00006, respectively). Tenofovir and entecavir showed less impact compared to lamivudine + adefovir. mtDNA/nDNA ratio positively (Rho = 0.34, p < 0.05) and mtRNA/nRNA ratio negatively (Rho = -0.34, p < 0.05) correlated with therapy duration. BSC were significantly more frequent in Group A [10/54 (18.5%)] compared to Group B [3/53 (5.6%, p = 0.04)] and Group C [0/50, (p = 0.0009)]. In conclusion, long-term NA therapy was associated both to mitochondrial toxicity and BSC, showing significant differences in mtDNA and mtRNA levels. Tenofovir and entecavir showed lower impact on alterations, compared to 1st generation NA.
Subject(s)
Adiposity/drug effects , Antiviral Agents/adverse effects , Hepatitis B, Chronic/drug therapy , Mitochondria/drug effects , Adenine/adverse effects , Adenine/analogs & derivatives , Cross-Sectional Studies , DNA, Mitochondrial/isolation & purification , DNA, Viral/isolation & purification , Drug Resistance, Viral , Drug Therapy, Combination/adverse effects , Drug Therapy, Combination/methods , Female , Guanine/adverse effects , Guanine/analogs & derivatives , Hepatitis B virus/genetics , Hepatitis B virus/isolation & purification , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/virology , Humans , Lamivudine/adverse effects , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Male , Middle Aged , Mitochondria/genetics , Organophosphonates/adverse effects , RNA, Mitochondrial/isolation & purification , Tenofovir/adverse effectsABSTRACT
The genus Pneumocystis comprises potential pathogens that reside normally in the lungs of a wide range of mammals. Although they generally behave as transient or permanent commensals, they can occasionally cause life-threatening pneumonia (Pneumocystis pneumonia; PCP) in immunosuppressed individuals. Several decades ago, the presence of Pneumocystis morphotypes (trophic forms and cysts) was described in the lungs of normal cats and cats with experimentally induced symptomatic PCP (after immunosuppression by corticosteroids); yet to date spontaneous or drug-induced PCP has not been described in the clinical feline literature, despite immunosuppression of cats by long-standing retrovirus infections or after kidney transplantation. In this study, we describe the presence of Pneumocystis DNA in the lungs of normal cats (that died of various unrelated causes; n = 84) using polymerase chain reactions (PCRs) targeting the mitochondrial small and large subunit ribosomal RNA gene (mtSSU rRNA and mtLSU rRNA). The presence of Pneumocystis DNA was confirmed by sequencing in 24/84 (29%) cats, with evidence of two different sequence types (or lineages). Phylogenetically, lineage1 (L1; 19 cats) and lineage 2 (L2; 5 cats) formed separate clades, clustering with Pneumocystis from domestic pigs (L1) and carnivores (L2), respectively. Results of the present study support the notion that cats can be colonized or subclinically infected by Pneumocystis, without histological evidence of damage to the pulmonary parenchyma referable to pneumocystosis. Pneumocystis seems most likely an innocuous pathogen of cats' lungs, but its possible role in the exacerbation of chronic pulmonary disorders or viral/bacterial coinfections should be considered further in a clinical setting.
Subject(s)
Cat Diseases/diagnosis , DNA, Fungal/isolation & purification , Lung/microbiology , Pneumocystis/isolation & purification , Pneumonia, Pneumocystis/veterinary , Animals , Cat Diseases/microbiology , Cats , Female , Male , Phylogeny , Pneumocystis/genetics , Pneumonia, Pneumocystis/diagnosis , RNA, Mitochondrial/isolation & purification , RNA, Ribosomal/isolation & purificationABSTRACT
The presence of noncoding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by Percoll gradient purification and RNA extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by qPCR following synthesis of cDNA. After qPCR-based mitomiR-profiling, the Normfinder algorithm is applied to identify the suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.
