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Map of the human mitochondrial genome. A double-stranded circular mitochondrial DNA (mtDNA) molecule includes 37 genes encoding 13 subunits of the OXPHOS system, 2 rRNAs, and 22 tRNAs. Transcription of both mtDNA strands initiates within the non-coding regulatory region (NCR), and the black arrows indicate transcription direction. HSP, LSP-transcription promoter of H-and L-strand, respectively. Open reading frames of ATP8/ATP6 and ND4L/ND4 are marked. Created with BioRender.com.
Source publication
Mitochondria, often referred to as the powerhouses of cells, are vital organelles that are present in almost all eukaryotic organisms, including humans. They are the key energy suppliers as the site of adenosine triphosphate production, and are involved in apoptosis, calcium homeostasis, and regulation of the innate immune response. Abnormalities o...
Contexts in source publication
Context 1
... human mitochondrial genome is a circular double-stranded DNA molecule of 16,569 base pairs, composed of heavy (H) and complementary light (L) strands that can be differentiated by the guanine (G) distribution [1,2]. Mitochondrial DNA (mtDNA) contains 37 genes that encode 13 subunits of the oxidative phosphorylation (OXPHOS) system, two ribosomal RNAs (rRNAs), and 22 transfer RNAs (tRNAs) [2] (Figure 1). ...
Context 2
... all mitochondrial genes, including those that encode 12 subunits of the OXPHOS system, 2 mt-rRNAs (12S and 16S), and 14 mt-tRNAs, are transcribed from the template of the G-rich H-strand under control of the H-strand promoter (HSP). The complementary L-strand serves as a template for the production of only 1 mitochondrial messenger RNA (mt-mRNA) that encodes subunit 6 of NADH dehydrogenase (ND6), 8 mt-tRNAs, and mainly non-coding RNAs (ncRNAs) that are produced under control of the L-strand promoter (LSP) [2] (Figure 1). The transcription of both mtDNA strands spans nearly the entire length of mtDNA, resulting in the formation of long polycistronic transcripts (Figure 2). ...
Context 3
... all mitochondrial genes, including those that encode 12 subunits of the OXPHOS system, 2 mt-rRNAs (12S and 16S), and 14 mt-tRNAs, are transcribed from the template of the G-rich H-strand under control of the H-strand promoter (HSP). The complementary L-strand serves as a template for the production of only 1 mitochondrial messenger RNA (mt-mRNA) that encodes subunit 6 of NADH dehydrogenase (ND6), 8 mt-tRNAs, and mainly non-coding RNAs (ncRNAs) that are produced under control of the L-strand promoter (LSP) [2] (Figure 1). The transcription of both mtDNA strands spans nearly the entire length of mtDNA, resulting in the formation of long polycistronic transcripts (Figure 2). ...
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In eukaryotes, DNA of mitochondria is transferred into the nucleus and forms nuclear mitochondrial DNAs (NUMTs). Taking advantage of the abundant genomic resources for bumblebees, in this study, we de novo generated mitochondrial genomes (mitogenomes) for 11 bumblebee species. Then, we identified and characterized NUMTs in genus-wide bumblebee spec...
Citations
... Mitochondrial transcription is polycistronic, generating two transcripts spanning nearly the entire genome from a single L-strand and a single H-strand promoter, which are subsequently processed. Mitochondrial ribonuclease P (RNase P) and mitochondrial RNase Z (ELAC2) catalyze tRNA-5 0 cleavage and tRNA-3 0 cleavage, respectively, thereby releasing the rRNAs and most mRNAs [37]. It has been proposed that mitoribosome assembly initiates with a subset of 27 mtLSU proteins forming a The main structural features of the mtSSU (head, body, foot, mRNA entry channel) and the mtLSU (polypeptide exit tunnel, L1 stalk, and L7/L12 stalk) are labeled in bold. ...
Mitoribosome biogenesis is a complex process involving RNA elements encoded in the mitochondrial genome and mitoribosomal proteins typically encoded in the nuclear genome. This process is orchestrated by extra‐ribosomal proteins, nucleus‐encoded assembly factors, which play roles across all assembly stages to coordinate ribosomal RNA processing and maturation with the sequential association of ribosomal proteins. Both biochemical studies and recent cryo‐EM structures of mammalian mitoribosomes have provided insights into their assembly process. In this article, we will briefly outline the current understanding of mammalian mitoribosome biogenesis pathways and the factors involved. Special attention is devoted to the recent identification of iron–sulfur clusters as structural components of the mitoribosome and a small subunit assembly factor, the existence of redox‐sensitive cysteines in mitoribosome proteins and assembly factors, and the role they may play as redox sensor units to regulate mitochondrial translation under stress.
... Two promoters on the light (LSP1 and LSP2) and one on the heavy strand (HSP) [5][6][7][8][9] give rise to long, polycistronic transcripts that cover almost the entire length of the genome [2]. The primary transcripts then undergo numerous processing and maturation steps, which include cleavage, modification, and/or polyadenylation to become mature and functional tRNAs, rRNAs, or mRNAs [10]. These steps are critical for correct gene expression and mitochondrial function, and defects in this process are associated with severe mitochondriopathies and other diseases [11][12][13][14]. ...
... The very first step of RNA processing consists of cleavage of the primary transcripts, which separates mRNAs, tRNAs, and rRNAs [10]. This is carried out by dedicated molecular machineries that must recognise the individual gene junctions and catalyse their endonucleolytic cleavage. ...
... This is carried out by dedicated molecular machineries that must recognise the individual gene junctions and catalyse their endonucleolytic cleavage. The mammalian mtDNA encodes for two types of gene junctions that are recognised by two fundamentally different RNA processing mechanisms ( Fig. 1) [10,14]. In a seminal paper, Attardi and colleagues recognized that the rRNAs and most mRNAs are interspersed by tRNAs in the human mitochondrial genome, and proposed a 'tRNA punctuation model' in which the tRNAs act as excision sites to release the individual RNAs [2]. ...
Human mitochondria harbour a circular, polyploid genome (mtDNA) encoding 11 messenger RNAs (mRNAs), two ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs). Mitochondrial transcription produces long, polycistronic transcripts that span almost the entire length of the genome, and hence contain all three types of RNAs. The primary transcripts then undergo a number of processing and maturation steps, which constitute key regulatory points of mitochondrial gene expression. The first step of mitochondrial RNA processing consists of the separation of primary transcripts into individual, functional RNA molecules and can occur by two distinct pathways. Both are carried out by dedicated molecular machineries that substantially differ from RNA processing enzymes found elsewhere. As a result, the underlying molecular mechanisms remain poorly understood. Over the last years, genetic, biochemical and structural studies have identified key players involved in both RNA processing pathways and provided the first insights into the underlying mechanisms. Here, we review our current understanding of RNA processing in mammalian mitochondria and provide an outlook on open questions in the field.
... The "noncoding" probe Dr-MT-ND5-sense-C3 was custom designed to contain sense sequences of the same mitochondrial gene; it binds regions of the coding (antisense) mtDNA strand. Antisense mtRNA transcripts from the noncoding strand of mtDNA are typically degraded (Pietras et al., 2018;Jedynak-Slyvka et al., 2021), so this probe reports primarily mtDNA. ...
... The mitochondrial genome consists of a "heavy" coding strand, and a complementary "light" noncoding strand. During transcription mtRNA is generated from both strands; sense transcripts from the coding strand are processed and translated into proteins, transfer RNAs, and ribosomal RNAs (Pietras et al., 2018;Jedynak-Slyvka et al., 2021). mtRNA transcripts from the noncoding strand are typically degraded, except for a few transfer RNAs and one protein (Chomyn et al., 1986;Mercer et al., 2011). ...
Background
Mitochondrial health has gained attention in a number of diseases, both as an indicator of disease state and as a potential therapeutic target. The quality and amount of mitochondrial DNA (mtDNA) and RNA (mtRNA) can be important indicators of mitochondrial and cell health, but are difficult to measure in complex tissues.
Methods
mtDNA and mtRNA in zebrafish retina samples were fluorescently labeled using RNAscope™ in situ hybridization, then mitochondria were stained using immunohistochemistry. Pretreatment with RNase was used for validation. Confocal images were collected and analyzed, and relative amounts of mtDNA and mtRNA were reported. Findings regarding mtDNA were confirmed using qPCR.
Results
Signals from probes detecting mtDNA and mtRNA were localized to mitochondria, and were differentially sensitive to RNase. This labeling strategy allows for quantification of relative mtDNA and mtRNA levels in individual cells. As a demonstration of the method in a complex tissue, single photoreceptors in zebrafish retina were analyzed for mtDNA and mtRNA content. An increase in mtRNA but not mtDNA coincides with proliferation of mitochondria at night in cones. A similar trend was measured in rods.
Discussion
Mitochondrial gene expression is an important component of cell adaptations to disease, stress, or aging. This method enables the study of mtDNA and mtRNA in single cells of an intact, complex tissue. The protocol presented here uses commercially-available tools, and is adaptable to a range of species and tissue types.
... Firstly, the import processing of nuclear genome encoded circRNAs into mitochondria is not clear. MtDNA encodes limited genes [3], not only the transcription of mtDNA needs the assistance of nuDNA encoded proteins but also the processing of mitochondrial RNA (mtRNA) needs [119]. NuDNA encoded proteins designated to mitochondria were imported into mitochondrial membrane and mitochondrial matrix by several pathways [58]. ...
Mitochondria participate in varieties of cellular events. It is widely accepted that human mitochondrial genome encodes 13 proteins, 2 rRNAs, and 22 tRNAs. Gene variation derived from human nuclear genome cannot completely explain mitochondrial diseases. The advent of high-throughput sequencing coupled with novel bioinformatic analyses decode the complexity of mitochondria-derived transcripts. Recently, circular RNAs (circRNAs) from both human mitochondrial genome and nuclear genome have been found to be located at mitochondria. Studies about the roles and molecular mechanisms underlying trafficking of the nucleus encoded circRNAs to mitochondria and mitochondria encoded circRNAs to the nucleus or cytoplasm in mammals are only beginning to emerge. These circRNAs have been associated with a variety of diseases, especially cancers. Here, we discuss the emerging field of mitochondria-located circRNAs by reviewing their identification, expression patterns, regulatory roles, and functional mechanisms. Mitochondria-located circRNAs have regulatory roles in cellular physiology and pathology. We also highlight future perspectives and challenges in studying mitochondria-located circRNAs, as well as their potential biomedical applications.
... Notably, mitochondrial genes were consistently over-sampled in FFPE samples, which is consistent with a previous study ( 19 ). This phenomenon suggested that RNA in mitochondria may be protected from degradation by the mitochondrial membranes and RNA-binding proteins within the mitochondria ( 40 ). Unexpectedly, almost no gene correlated with the DV200 values of the FFPE samples, indicating that the DV200 values were only suitable to evaluate the probability of successful library construction ( 8 ,9 ). ...
Formalin-fixed paraffin-embedded (FFPE) tissues are widely available specimens for clinical studies. However, RNA degradation in FFPE tissues often restricts their utility. In this study, we determined optimal FFPE preparation conditions, including tissue ischemia at 4°C (<48 h) or 25°C for a short time (0.5 h), 48-h fixation at 25°C and sampling from FFPE scrolls instead of sections. Notably, we observed an increase in intronic reads and a significant change in gene rank based on expression level in the FFPE as opposed to fresh-frozen (FF) samples. Additionally, we found that more reads were mapped to genes associated with chemical stimulus in FFPE samples. Furthermore, we demonstrated that more degraded genes in FFPE samples were enriched in genes with short transcripts and high free energy. Besides, we found 40 housekeeping genes exhibited stable expression in FF and FFPE samples across various tissues. Moreover, our study showed that FFPE samples yielded comparable results to FF samples in dimensionality reduction and pathway analyses between case and control samples. Our study established the optimal conditions for FFPE preparation and identified gene attributes associated with degradation, which would provide useful clues for the utility of FFPE tissues in clinical practice and research.
... Mammalian mitochondrial ribosomes (mitoribosomes), which are responsible for the translation of the 13 protein component genes, are composed of the 28 S mitochondrial small subunit (mt-SSU, containing 12 S rRNA and 30 nucleus-encoded mitoribosomal proteins) and 39 S mitochondrial large subunit (mt-LSU, containing 16 S rRNA and 52 nucleus-encoded mitoribosomal proteins) [7][8][9] . A series of post-transcription modifications have been found in mitochondrial rRNAs, which are located at the functionally critical regions of the mitoribosome and play a crucial role in mitoribosome assembly and efficient translation [10][11][12] . Abnormal expression of mt-rRNA modification enzymes directly affects modification levels of mt-rRNA and mitoribosome assembly, which damages mitochondria function and leads to a series of mitochondrial diseases 13,14 . ...
... Methyltransferase-like (METTL) family proteins are characterized by a conserved Rossman-like fold S-adenosyl methionine (SAM)-binding domain, which methylates proteins, nucleic acids, and other small molecule metabolites, are involved in the regulation of mRNA stability and translation efficiency [18][19][20] . Several METTLs localize to mitochondria, among which METTL9, METTL12 and METTL20 are responsible for protein methylation, while METTL8 and METTL2A for mt-tRNA methylation, METTL15 for mt-rRNA methylation 10,[21][22][23] . ...
Mitochondrial rRNA modifications are essential for mitoribosome assembly and its proper function. The m ⁴ C methyltransferase METTL15 maintains mitochondrial homeostasis by catalyzing m ⁴ C839 located in 12 S rRNA helix 44 (h44). This modification is essential to fine-tuning the ribosomal decoding center and increasing decoding fidelity according to studies of a conserved site in Escherichia coli . Here, we reported a series of crystal structures of human METTL15–hsRBFA–h44–SAM analog, METTL15–hsRBFA–SAM, METTL15–SAM and apo METTL15. The structures presented specific interactions of METTL15 with different substrates and revealed that hsRBFA recruits METTL15 to mitochondrial small subunit for further modification instead of 12 S rRNA. Finally, we found that METTL15 deficiency caused increased reactive oxygen species, decreased membrane potential and altered cellular metabolic state. Knocking down METTL15 caused an elevated lactate secretion and increased levels of histone H4K12-lactylation and H3K9-lactylation. METTL15 might be a suitable model to study the regulation between mitochondrial metabolism and histone lactylation.
... In nucleus-encoded mRNAs, poly(A) tails are crucial for their stability and control the translation initiation step; differently, the impact of mitochondrial poly(A) tails on mt-RNA stability varies depending on the transcripts examined. Importantly, the polyadenylation process completes the UAA stop codon that is absent in most human mitochondrial mtRNAs [34]. This chain of molecular events leads to the formation of mature mt-mRNA, ready to be translated by mitochondrial ribosomes [35]. ...
Until a few decades ago, most of our knowledge of RNA transcription products was focused on protein-coding sequences, which were later determined to make up the smallest portion of the mammalian genome. Since 2002, we have learnt a great deal about the intriguing world of non-coding RNAs (ncRNAs), mainly due to the rapid development of bioinformatic tools and next-generation sequencing (NGS) platforms. Moreover, interest in non-human ncRNAs and their functions has increased as a result of these technologies and the accessibility of complete genome sequences of species ranging from Archaea to primates. Despite not producing proteins, ncRNAs constitute a vast family of RNA molecules that serve a number of regulatory roles and are essential for cellular physiology and pathology. This review focuses on a subgroup of human ncRNAs, namely mtDNA-encoded long non-coding RNAs (mt-lncRNAs), which are transcribed from the mitochondrial genome and whose disparate localisations and functions are linked as much to mitochondrial metabolism as to cellular physiology and pathology.
... Mt-RNAs are first synthesised as extended polycistronic transcripts. Subsequently, in order to be functional for protein synthesis, these long precursors undergo maturation by coordinated processes of cleavage and post-transcriptional modification, both taking place within the mitochondrial RNA granules [2,3]. The enzymatic cleavage at the 5 and 3 ends of mt-tRNAs is mediated by RNase P and ELAC2, respectively, and is crucial for releasing mature mt-RNAs co-transcriptionally or shortly after RNA synthesis [4]. ...
In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial trans- lation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.
... In the resulting CP dataset, we selected a list of mitochondrial gene expression factors based on r ecent r e vie ws (48)(49)(50)(51)(52) and arranged this list of 164 proteins according to their molecular functions and migration profiles (Supplementary Data S3). Proteins involved in mt-mRNA processing (e.g. ...
Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimized this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA– and RNA–protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE). In combination with enzymatic digestion of DNA, our CP approach improved the identification of a wide range of protein interactors of the mitochondrial gene expression system without compromising the detection of other multiprotein complexes. The utility of this approach was particularly demonstrated by analysing the complexome changes in human mitochondria with impaired gene expression after transient, chemically induced mitochondrial DNA depletion. Effects of RNase on mitochondrial protein complexes were also evaluated and discussed. Overall, our adaptations significantly improved the identification of mitochondrial DNA– and RNA–protein interactions by CP, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome in a single experiment.
... The mitochondrial ATP8 gene is located upstream of the ATP6 gene in the mtDNA and its 3'-end overlaps by 46 nucleotides with the 5'-end of ATP6, while the start codon of the COX3 gene located downstream overlaps with the stop codon of ATP6. ATP8, ATP6 and COX3 are initially expressed in one polycistronic transcript that is subsequently processed into separate ATP8/ATP6 and COX3 polyadenylated mature mRNAs (Dautant et al., 2018;Jedynak-Slyvka et al., 2021). The ATP8/ATP6 bicistronic transcript is translated through a single ribosome/mRNA engagement, while COX3 is translated separately (Cruz-Zaragoza et al., 2021). ...
Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to contribute to rescue such mutations. A number of these were based on the capacity of human mitochondria to import RNAs from the cytosol and were designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of the Brome mosaic virus or the Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy opens a powerful approach to eliminate mutant organellar transcripts and to analyze the control and communication of the human organellar genetic system.
