Mammalian Mitochondrial Ribosomes Research Articles

Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome

Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome

Architecture of the large subunit of the mammalian mitochondrial ribosome

Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.

MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation

Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no invivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease ofde novo transcription manifested as reduced 7SRNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

Defects in mitochondrial ribosomal proteins (MRPs) cause various diseases in humans. Because of the essential role of MRPs in synthesizing the essential subunits of oxidative phosphorylation (OXPHOS) complexes, identifying all of the protein components involved in the mitochondrial translational machinery is critical. Initially, we identified 79 MRPs; however, identifying MRPs with no clear homologs in bacteria and yeast mitochondria was challenging, due to limited availability of expressed sequence tags (ESTs) in the databases available at that time. With the improvement in genome sequencing and increased sensitivity of mass spectrometry (MS)-based technologies, we have established four previously known proteins as MRPs and have confirmed the identification of ICT1 (MRP58) as a ribosomal protein. The newly identified MRPs are MRPS37 (Coiled-coil-helix-coiled-coil-helix domain containing protein 1-CHCHD1), MRPS38 (Aurora kinase A interacting protein1, AURKAIP1), MRPS39 (Pentatricopeptide repeat-containing protein 3, PTCD3), in the small subunit and MRPL59 (CR-6 interacting factor 1, CRIF1) in the large subunit. Furthermore, we have demonstrated the essential roles of CHCHD1, AURKAIP1, and CRIF1in mitochondrial protein synthesis by siRNA knock-down studies, which had significant effects on the expression of mitochondrially encoded proteins.

Structural aspects of mitochondrial translational apparatus

During the last decade groundbreaking progress has been made towards the understanding of structure and function of cell's translational machinery. Cryo-electron microscopic (cryo-EM) and X-ray crystallographic structures of cytoplasmic ribosomes from several bacterial and eukaryotic species are now available in various ligand-bound states. Significant advances have also been made in structural studies on ribosomes of the cellular organelles, such as those present in the chloroplasts and mitochondria, using cryo-EM techniques. Here we review the progress made in structure determination of the mitochondrial ribosomes, with an emphasis on the mammalian mitochondrial ribosome and one of its translation initiation factors, and discuss challenges that lie ahead in obtaining their high-resolution structures.

A Newly Identified Protein Regulates Translation in Mammalian Mitochondria

Mitochondria are responsible for providing 90% of the energy in eukaryotic organisms by oxidative phosphorylation (OXPHOS). Mitochondrially encoded components of OXPHOS complexes are all essential and synthesized by a specialized translation system containing 55S ribosomes in mitochondria. In the reevaluation of mitochondrial ribosomal proteins (MRPs) and ribosome‐interacting proteins, we identified Gadd45GIP1 as a bona fide component in purified 55S ribosomes. Here, we report Gadd45GIP1 association with the large subunit of mammalian mitochondrial ribosomes and its role in the regulation of mitochondrial protein synthesis. In siRNA knock‐down studies, reduced expression of Gadd45GIP1 decreased de novo synthesis of the 13 mitochondrially encoded proteins of OXPHOS complexes in vivo. This indicates that the Gadd45GIP1 is an essential member of the mitochondrial ribosome, and its specific role in protein synthesis is currently under investigation.Supported by NIH grant R01GM071034 (E.C.K.)

Three-dimensional localization of the GTP-binding protein within the mammalian mitochondrial ribosome

Three-dimensional localization of the GTP-binding protein within the mammalian mitochondrial ribosome

MTERF4 Regulates Translation by Targeting the Methyltransferase NSUN4 to the Mammalian Mitochondrial Ribosome

Precise control of mitochondrial DNA gene expression is critical for regulation of oxidative phosphorylation capacity in mammals. The MTERF protein family plays a key role in this process, and its members have been implicated in regulation of transcription initiation and site-specific transcription termination. We now demonstrate that a member of this family, MTERF4, directly controls mitochondrial ribosomal biogenesis and translation. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4 and is necessary for recruitment of this factor to the large ribosomal subunit. Loss of MTERF4 leads to defective ribosomal assembly and a drastic reduction in translation. Our results thus show that MTERF4 is an important regulator of translation in mammalian mitochondria.

Identification of Protein-Protein and Protein-Ribosome Interacting Regions of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.

Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes

In humans the mitochondrial inner membrane protein Oxa1L is involved in the biogenesis of membrane proteins and facilitates the insertion of both mitochondrial- and nuclear-encoded proteins from the mitochondrial matrix into the inner membrane. The C-terminal approximately 100-amino acid tail of Oxa1L (Oxa1L-CTT) binds to mitochondrial ribosomes and plays a role in the co-translational insertion of mitochondria-synthesized proteins into the inner membrane. Contrary to suggestions made for yeast Oxa1p, our results indicate that the C-terminal tail of human Oxa1L does not form a coiled-coil helical structure in solution. The Oxa1L-CTT exists primarily as a monomer in solution but forms dimers and tetramers at high salt concentrations. The binding of Oxa1L-CTT to mitochondrial ribosomes is an enthalpy-driven process with a K(d) of 0.3-0.8 microM and a stoichiometry of 2. Oxa1L-CTT cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins L13, L20, and L28 and to mammalian mitochondrial specific ribosomal proteins MRPL48, MRPL49, and MRPL51. Oxa1L-CTT does not cross-link to proteins decorating the conventional exit tunnel of the bacterial large ribosomal subunit (L22, L23, L24, and L29).

Preferential Selection of the 5′-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes

Mammalian mitochondrial mRNAs are basically leaderless, having few or no untranslated nucleotides prior to the 5'-start codon. We demonstrate here that mammalian mitochondrial 55 S ribosomes preferentially form initiation complexes at a 5'-terminal AUG codon over an internal AUG. The preferential use of the 5'-start codon is also seen on mitochondrial 28 S small subunits, which suggests that mitochondrial translation initiation on leaderless mRNAs does not require the large ribosomal subunit. The selection of the 5'-AUG is dependent on the presence of fMet-tRNA and is enhanced by the presence of the mitochondrial initiation factor IF2(mt). In prokaryotes, IF3 is believed to antagonize initiation on leaderless mRNAs. However, IF3(mt) stimulates initiation complex formation on leaderless mRNAs when tested with 55 S ribosomes. The addition of even a few nucleotides 5' to the AUG codon significantly reduces the efficiency of initiation, highlighting the importance of the leaderless or nearly leaderless nature of mitochondrial mRNAs. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of tRNA(Met) attached at the 5'-end of a mitochondrial protein-coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.

Current Views of the Structure of the Mammalian Mitochondrial Ribosome

AbstractMammalian mitochondria synthesize polypeptides crucial for energy generation using ribosomes with a number of unique features. These ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian mitochondrial ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these ribosomes. Among these is a gate‐like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the mitochondrial ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the mitochondrial ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control.

ICT1 comes to the rescue of mitochondrial ribosomes

In the current issue, [Richter et al (2010)][1] show that mammalian mitochondrial ribosomes contain a ribosomal protein (ICT1) that acts as a ribosome‐dependent, codon‐independent peptidyl‐tRNA hydrolase. This ribosomal protein can rescue ribosomes stalled on mRNAs lacking a termination codon. [1]: #ref-3

Preferential Selection of the 5′-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes

Preferential Selection of the 5′-Terminal Start Codon on Leaderless mRNAs by Mammalian Mitochondrial Ribosomes

Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes

Properties of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L and Its Interactions with Mammalian Mitochondrial Ribosomes

The effect of spermine on the initiation of mitochondrial protein synthesis

Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine is a quaternary aliphatic amine that is cationic under physiological conditions. In this paper, we demonstrate that spermine stimulates fMet-tRNA binding to mammalian mitochondrial 55S ribosomes. The stimulatory effect of spermine is independent of the identity of the mRNA. The degree of stimulation of spermine is the same at all concentrations of mitochondrial initiation factor 2 (IF2mt) and mitochondrial initiation factor 3 (IF3mt). This observation indicates that IF2mt and IF3mt, while essential for initiation, are not the primary components of the translation initiation system affected by spermine. IF3mt dissociates 55S ribosomes detectably in the absence of spermine, but this effect is strongly inhibited in the presence of spermine. This observation indicates that the positive effect of spermine on initiation is not due to an increase in the availability of the small subunits for initiation. Spermine also promotes fMet-tRNA binding to small subunits of the mitochondrial ribosome in the presence of IF2mt. The major effect of spermine in promoting initiation complex formation thus appears to be on the interaction of fMet-tRNA with the ribosome.

Phosphorylated Proteins of the Mammalian Mitochondrial Ribosome: Implications in Protein Synthesis

Mitochondria, the powerhouse of eukaryotic cells, have their own translation machinery that is solely responsible for synthesis of 13 mitochondrially encoded protein subunits of oxidative phosphorylation complexes. Phosphorylation is a well-known post-translational modification in regulation of many processes in mammalian mitochondria including oxidative phosphorylation. However, there is still very limited knowledge on phosphorylation of mitochondrial ribosomal proteins and their role(s) in ribosome function. In this study, we have identified the mitochondrial ribosomal proteins that are phosphorylated at serine, threonine or tyrosine residues. Twenty-four phosphorylated proteins were visualized by phosphorylation-specific techniques including in vitro radiolabeling, residue specific antibodies for phosphorylated residues, or ProQ phospho dye and identified by tandem mass spectrometry. Translation assays with isolated ribosomes that were phosphorylated in vitro by kinases PKA, PKCdelta, or Abl Tyr showed up to 30% inhibition due to phosphorylation. Findings from this study should serve as the framework for future studies addressing the regulation mechanisms of mitochondrial translation machinery by phosphorylation and other post-translational modifications.

Structure of a mitochondrial ribosome with minimal RNA

The Leishmania tarentolae mitochondrial ribosome (Lmr) is a minimal ribosomal RNA (rRNA)-containing ribosome. We have obtained a cryo-EM map of the Lmr. The map reveals several features that have not been seen in previously-determined structures of eubacterial or eukaryotic (cytoplasmic or organellar) ribosomes to our knowledge. Comparisons of the Lmr map with X-ray crystallographic and cryo-EM maps of the eubacterial ribosomes and a cryo-EM map of the mammalian mitochondrial ribosome show that (i) the overall structure of the Lmr is considerably more porous, (ii) the topology of the intersubunit space is significantly different, with fewer intersubunit bridges, but more tunnels, and (iii) several of the functionally-important rRNA regions, including the alpha-sarcin-ricin loop, have different relative positions within the structure. Furthermore, the major portions of the mRNA channel, the tRNA passage, and the nascent polypeptide exit tunnel contain Lmr-specific proteins, suggesting that the mechanisms for mRNA recruitment, tRNA interaction, and exiting of the nascent polypeptide in Lmr must differ markedly from the mechanisms deduced for ribosomes in other organisms. Our study identifies certain structural features that are characteristic solely of mitochondrial ribosomes and other features that are characteristic of both mitochondrial and chloroplast ribosomes (i.e., organellar ribosomes).

Methylation of 12S rRNA Is Necessary for In Vivo Stability of the Small Subunit of the Mammalian Mitochondrial Ribosome

The 3' end of the rRNA of the small ribosomal subunit contains two extremely highly conserved dimethylated adenines. This modification and the responsible methyltransferases are present in all three domains of life, but its function has remained elusive. We have disrupted the mouse Tfb1m gene encoding a mitochondrial protein homologous to bacterial dimethyltransferases and demonstrate here that loss of TFB1M is embryonic lethal. Disruption of Tfb1m in heart leads to complete loss of adenine dimethylation of the rRNA of the small mitochondrial ribosomal subunit, impaired assembly of the mitochondrial ribosome, and abolished mitochondrial translation. In addition, we present biochemical evidence that TFB1M does not activate or repress transcription in the presence of TFB2M. Our results thus show that TFB1M is a nonredundant dimethyltransferase in mammalian mitochondria. In addition, we provide a possible explanation for the universal conservation of adenine dimethylation of rRNA by showing a critical role in ribosome maintenance.