The human mitochondrial mRNA structurome reveals mechanisms of gene expression.

The human mitochondrial genome encodes crucial oxidative phosphorylation system proteins, pivotal for aerobic energy transduction. They are translated from nine monocistronic and two bicistronic transcripts whose native structures remain unexplored, posing a gap in understanding mitochondrial gene expression. In this work, we devised the mitochondrial dimethyl sulfate mutational profiling with sequencing (mitoDMS-MaPseq) method and applied detection of RNA folding ensembles using expectation-maximization (DREEM) clustering to unravel the native mitochondrial messenger RNA (mt-mRNA) structurome in wild-type (WT) and leucine-rich pentatricopeptide repeat-containing protein (LRPPRC)-deficient cells. Our findings elucidate LRPPRC's role as a holdase contributing to maintaining mt-mRNA folding and efficient translation. mt-mRNA structural insights in WT mitochondria, coupled with metabolic labeling, unveil potential mRNA-programmed translational pausing and a distinct programmed ribosomal frameshifting mechanism. Our data define a critical layer of mitochondrial gene expression regulation. These mt-mRNA folding maps provide a reference for studying mt-mRNA structures in diverse physiological and pathological contexts.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

Suborganellar Localization of Mitochondrial Proteins and Transcripts in Human Cells.

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.

mTRIP, an Imaging Tool to Investigate Mitochondrial DNA Dynamics in Physiology and Disease at the Single-Cell Resolution.

Mitochondrial physiology and metabolism are closely linked to replication and transcription of mitochondrial DNA (mtDNA). However, the characterization of mtDNA processing is poorly defined at the single-cell level. We developed mTRIP (mitochondrial Transcription and Replication Imaging Protocol), an imaging approach based on modified fluorescence in situ hybridization (FISH), which simultaneously reveals mitochondrial structures committed to mtDNA initiation of replication as well as the mitochondrial RNA (mtRNA) content at the single-cell level in human cells. Also specific RNA regions, rather than global RNA, can be tracked with mTRIP. In addition, mTRIP can be coupled to immunofluorescence for in situ protein tracking, or to MitoTracker, thereby allowing for simultaneous labeling of mtDNA, mtRNA, and proteins or mitochondria, respectively. Altogether, qualitative and quantitative alterations of the dynamics of mtDNA processing are detected by mTRIP in human cells undergoing physiological changes, as well as stress and dysfunction. mTRIP helped elucidating mtDNA processing alterations in cancer cells, and has a potential for diagnostic of mitochondrial diseases.

Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

A novel, mitochondrial, internal tRNA-derived RNA fragment possesses clinical utility as a molecular prognostic biomarker in chronic lymphocytic leukemia.

OBJECTIVES: Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults. The prognosis of CLL patients varies considerably. Transfer RNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNA fragments excised from mature tRNAs and pre-tRNAs located in nuclei as well as in mitochondria. In this study, the clinical utility of i-tRF-PheGAA, a novel mitochondrial tRF, was investigated in CLL. DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CLL patients and 43 non-leukemic controls. Total RNA was isolated from each sample, polyadenylated at the 3' end and reversely transcribed. An in-house developed real-time quantitative PCR assay was developed and applied, and the results were biostatistically analyzed. For the normalization of the i-tRF-PheGAA expression levels, the expression of a small nucleolar RNA (RNU48) was used as reference. RESULTS: Mann-Whitney U test showed that i-tRF-PheGAA can distinguish between CLL samples and normal controls (p < 0.001). As determined by Kaplan-Meier survival analysis, overexpression of i-tRF-PheGAA was related to poor overall survival of the CLL patients (p < 0.001). Univariate bootstrap Cox regression analysis exhibited a higher hazard ratio of 7.95 (95% CI = 2.37-26.72, p < 0.001) for patients with positive i-tRF-PheGAA expression status. Multivariate bootstrap Cox regression analysis showed that the prognostic value of this tRF is independent of clinical stage, mutational status of the immunoglobulin heavy chain variable (IGHV) genetic locus, and CD38 expression status (p = 0.010). CONCLUSIONS: Our results show that i-tRF-PheGAA can serve as a molecular biomarker of poor prognosis in CLL, alongside with the existing factors for CLL prognosis.

Tristetraprolin/ZFP36 Regulates the Turnover of Autoimmune-Associated HLA-DQ mRNAs.

HLA class II genes encode highly polymorphic heterodimeric proteins functioning to present antigens to T cells and stimulate a specific immune response. Many HLA genes are strongly associated with autoimmune diseases as they stimulate self-antigen specific CD4+ T cells driving pathogenic responses against host tissues or organs. High expression of HLA class II risk genes is associated with autoimmune diseases, influencing the strength of the CD4+ T-mediated autoimmune response. The expression of HLA class II genes is regulated at both transcriptional and post-transcriptional levels. Protein components of the RNP complex binding the 3'UTR and affecting mRNA processing have previously been identified. Following on from this, the regulation of HLA-DQ2.5 risk genes, the main susceptibility genetic factor for celiac disease (CD), was investigated. The DQ2.5 molecule, encoded by HLA-DQA1*05 and HLA-DQB1*02 alleles, presents the antigenic gluten peptides to CD4+ T lymphocytes, activating the autoimmune response. The zinc-finger protein Tristetraprolin (TTP) or ZFP36 was identified to be a component of the RNP complex and has been described as a factor modulating mRNA stability. The 3'UTR of CD-associated HLA-DQA1*05 and HLA-DQB1*02 mRNAs do not contain canonical TTP binding consensus sequences, therefore an in silico approach focusing on mRNA secondary structure accessibility and stability was undertaken. Key structural differences specific to the CD-associated mRNAs were uncovered, allowing them to strongly interact with TTP through their 3'UTR, conferring a rapid turnover, in contrast to lower affinity binding to HLA non-CD associated mRNA.

Exploration of CCA-added RNAs revealed the expression of mitochondrial non-coding RNAs regulated by CCA-adding enzyme.

Post-transcriptional non-template additions of nucleotides to 3'-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3'-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3'-ends of the sequence 5'-NCCA-3', which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen 'CCA-RNAs' that only contain CCA as non-genomic sequences, and eleven 'NCCA-RNAs' that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Structural significance of hypermodified nucleoside 5-carboxymethylaminomethyluridine (cmnm5U) from 'wobble' (34th) position of mitochondrial tRNAs: Molecular modeling and Markov state model studies.

A quantum chemical semi-empirical RM1 approach was used to deduce the structural role of hypermodified nucleoside 5-carboxymethylaminomethyluridine 5'-monophosphate (pcmnm5U) from 'wobble' (34th) position of mitochondrial tRNAs. The energetically preferred pcmnm5U(34) adopted a 'skew' conformation for C5-substituted side chain (-CH2-NH2+-CH2-COO-) moiety that orient towards the 5'-ribose-phosphate backbone, which support 'anti' orientation of glycosyl (χ34) torsion angle. Preferred conformation of pcmnm5U(34) was stabilized by O(4) … HC(10), O1P⋯HN(11), O(15) … HN(11), O(15) … HC(10), O4' … HC(6) and O(2) … HC2' hydrogen bonding interactions. The high flexibility of side chain moiety displayed different structural properties for pcmnm5U(34). Three different conformations of pcmnm5U(34) were observed in molecular dynamics simulations and Markov state model studies. The unmodified uracil revealed 'syn' and 'anti' orientations for glycosyl (χ34) torsion angle that substantiate the role of "-CH2-NH2+-CH2-COO-" moiety in maintaining the 'anti' orientation of pcmnm5U(34). The preferred conformation of pcmnm5U(34) helps to recognize Guanosine more proficiently than Adenosine from the third position of codons. The role of pcmnm5U(34) in tRNA biogenesis paves the way to understand its structural significance in usual mitochondrial metabolism and respiration.

N1-methyladenosine methylome in messenger RNA and non-coding RNA.

Chemical modifications to rRNA, tRNA and mRNA provide a new regulatory layer of gene expression, which is termed as the `epitranscriptome'. N1-methyladenosine (m1A), first characterized more than 50 years ago, is a well-known modification in rRNA and tRNA. m1A in these abundant non-coding RNAs plays important roles in maintaining their biological functions. Recent studies also reveal that m1A is present in both nuclear-encoded and mitochondrial-encoded mRNA and is dynamically regulated by environmental and developmental conditions; m1A is found in a subset of nuclear-encoded long non-coding RNAs as well. Finally, we also discuss the potential challenges of identifying m1A modification in the human transcriptome.

Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes.

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.