scholarly journals Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Victor Tobiasson ◽  
Alexey Amunts
Keyword(s):  
Mitochondrial Genome ◽  
Ribosomal Proteins ◽  
Protein Targeting ◽  
Large Subunit ◽  
Model Organism ◽  
Small Subunit ◽  
Specific Protein ◽  
Mitochondrial Translation ◽  
Functional Protein ◽  
Mitochondrial Ribosome

To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

scholarly journals Structural basis of mitochondrial translation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shintaro Aibara ◽  
Vivek Singh ◽  
Angelika Modelska ◽  
Alexey Amunts
Keyword(s):  
Conformational Changes ◽  
Messenger Rna ◽  
Large Subunit ◽  
Small Subunit ◽  
Specific Protein ◽  
Structural Basis ◽  
Mitochondrial Translation ◽  
Dynamic Interactions ◽  
Sequential Mechanism ◽  
Specific Proteins

Translation of mitochondrial messenger RNA (mt-mRNA) is performed by distinct mitoribosomes comprising at least 36 mitochondria-specific proteins. How these mitoribosomal proteins assist in the binding of mt-mRNA and to what extent they are involved in the translocation of transfer RNA (mt-tRNA) is unclear. To visualize the process of translation in human mitochondria, we report ~3.0 Å resolution structure of the human mitoribosome, including the L7/L12 stalk, and eight structures of its functional complexes with mt-mRNA, mt-tRNAs, recycling factor and additional trans factors. The study reveals a transacting protein module LRPPRC-SLIRP that delivers mt-mRNA to the mitoribosomal small subunit through a dedicated platform formed by the mitochondria-specific protein mS39. Mitoribosomal proteins of the large subunit mL40, mL48, and mL64 coordinate translocation of mt-tRNA. The comparison between those structures shows dynamic interactions between the mitoribosome and its ligands, suggesting a sequential mechanism of conformational changes.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

scholarly journals The proteins of Xenopus ovary ribosomes

1971 ◽  
Vol 125 (4) ◽  
pp. 1091-1107 ◽  
Author(s):  
P J Ford
Keyword(s):  
Potassium Chloride ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Buoyant Density ◽  
Small Subunit ◽  
Chemical Methods ◽  
Ribosomal Subunits ◽  
Molecular Weights ◽  
Caesium Chloride ◽  
Chloride Treatment

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.

scholarly journals Mrpl36 Is Important for Generation of Assembly Competent Proteins during Mitochondrial Translation

2009 ◽  
Vol 20 (10) ◽  
pp. 2615-2625 ◽  
Author(s):  
Martin Prestele ◽  
Frank Vogel ◽  
Andreas S. Reichert ◽  
Johannes M. Herrmann ◽  
Martin Ott
Keyword(s):  
Protein Synthesis ◽  
Respiratory Chain ◽  
Large Subunit ◽  
Critical Role ◽  
Mitochondrial Translation ◽  
Respiratory Chain Complexes ◽  
Ribosomal Subunits ◽  
Terminal Domain ◽  
Chain Complexes ◽  
Mitochondrial Ribosome

The complexes of the respiratory chain represent mosaics of nuclear and mitochondrially encoded components. The processes by which synthesis and assembly of the various subunits are coordinated remain largely elusive. During evolution, many proteins of the mitochondrial ribosome acquired additional domains pointing at specific properties or functions of the translation machinery in mitochondria. Here, we analyzed the function of Mrpl36, a protein associated with the large subunit of the mitochondrial ribosome. This protein, homologous to the ribosomal protein L31 from bacteria, contains a mitochondria-specific C-terminal domain that is not required for protein synthesis per se; however, its absence decreases stability of Mrpl36. Cells lacking this C-terminal domain can still synthesize proteins, but these translation products fail to be properly assembled into respiratory chain complexes and are rapidly degraded. Surprisingly, overexpression of Mrpl36 seems to even increase the efficiency of mitochondrial translation. Our data suggest that Mrpl36 plays a critical role during translation that determines the rate of respiratory chain assembly. This important function seems to be carried out by a stabilizing activity of Mrpl36 on the interaction between large and small ribosomal subunits, which could influence accuracy of protein synthesis.

Subcellular distribution of ribosomal proteins in Dictyostelium discoideum

1990 ◽  
Vol 68 (5) ◽  
pp. 839-845
Author(s):  
S. Ramagopal
Keyword(s):  
Ribosomal Proteins ◽  
Large Subunit ◽  
Small Subunit ◽  
Cellular Slime Mold ◽  
Developmentally Regulated ◽  
Two Dimensional Gel Electrophoresis ◽  
Coomassie Blue ◽  
Cellular Slime ◽  
State Growth

The distribution of ribosomal proteins in monosomes, polysomes, the postribosomal cytosol, and the nucleus was determined during steady-state growth in vegetative amoebae. A partitioning of previously reported cell-specific ribosomal proteins between monosomes and polysomes was observed. L18, one of the two unique proteins in amoeba ribosomes, was distributed equally among monosomes and polysomes. However S5, the other unique protein, was abundant in monosomes but barely visible in polysomes. Of the developmentally regulated proteins, D and S6 were detectable only in polysomes and S14 was more abundant in monosomes. The cystosol revealed no ribosomal proteins. On staining of the nuclear proteins with Coomassie blue, about 18, 7 from 40S subunit and 11 from 60S subunit, were identified as ribosomal proteins. By in vivo labeling of the proteins with [35S]methionine, 24 of the 34 small subunit proteins and 33 of the 42 large subunit proteins were localized in the nucleus. For the majority of the ribosomal proteins, the apparent relative stoichiometry was similar in nuclear preribosomal particles and in cytoplasmic ribosomes. However, in preribosomal particles the relative amount of four proteins (S11, S30, L7, and L10) was two- to four-fold higher and of eight proteins (S14, S15, S20, S34, L12, L27, L34, and L42) was two- to four-fold lower than that of cytoplasmic ribosomes.Key words: cellular slime mold, cell-specific ribosomal proteins, nucleus, cytoplasm, two-dimensional gel electrophoresis.

scholarly journals A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

1990 ◽  
Vol 10 (9) ◽  
pp. 4590-4595 ◽  
Author(s):  
T W McMullin ◽  
P Haffter ◽  
T D Fox
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Nuclear Gene ◽  
Small Subunit ◽  
Rrna Gene ◽  
Mitochondrial Translation ◽  
Activator Proteins ◽  
Translational Activator

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.

scholarly journals MRM2 and MRM3 are involved in biogenesis of the large subunit of the mitochondrial ribosome

2014 ◽  
Vol 25 (17) ◽  
pp. 2542-2555 ◽  
Author(s):  
Joanna Rorbach ◽  
Pierre Boesch ◽  
Payam A. Gammage ◽  
Thomas J. J. Nicholls ◽  
Sarah F. Pearce ◽  
...  
Keyword(s):  
16S Rrna ◽  
Rna Processing ◽  
Large Subunit ◽  
Peptidyl Transferase ◽  
Mitochondrial Translation ◽  
Peptidyl Transferase Center ◽  
Mitochondrial Ribosome ◽  
Translation Apparatus ◽  
Rrna Maturation ◽  
And Function

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Ribosome production in all organisms studied thus far entails a complex, multistep pathway involving a number of auxiliary factors. This includes several RNA processing and modification steps required for correct rRNA maturation. Little is known about the maturation of human mitochondrial 16S rRNA and its role in biogenesis of the mitoribosome. Here we investigate two methyltransferases, MRM2 (also known as RRMJ2, encoded by FTSJ2) and MRM3 (also known as RMTL1, encoded by RNMTL1), that are responsible for modification of nucleotides of the 16S rRNA A-loop, an essential component of the peptidyl transferase center. Our studies show that inactivation of MRM2 or MRM3 in human cells by RNA interference results in respiratory incompetence as a consequence of diminished mitochondrial translation. Ineffective translation in MRM2- and MRM3-depleted cells results from aberrant assembly of the large subunit of the mitochondrial ribosome (mt-LSU). Our findings show that MRM2 and MRM3 are human mitochondrial methyltransferases involved in the modification of 16S rRNA and are important factors for the biogenesis and function of the large subunit of the mitochondrial ribosome.

scholarly journals Decoding regulatory specificity of human ribosomal proteins on global gene expression

2021 ◽  
Author(s):  
Yizhao Luan ◽  
Nan Tang ◽  
Jiaqi Yang ◽  
Shuting Liu ◽  
Chichi Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Ribosomal Proteins ◽  
Translational Regulation ◽  
Large Subunit ◽  
Global Gene Expression ◽  
Small Subunit ◽  
Sequencing Analysis ◽  
Retina Development ◽  
P53 Signaling

Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with few regulatory functions. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is unclear. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We assessed deficiency of 75 RP, including 44 from the large subunit (60S) and 31 from the small subunit (40S), on gene translation and transcription via ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S or the 40S RPs had distinguished effects on the two subunits. The gene ontology analysis revealed that RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by affecting P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis but RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity and provided an important resource which offered novel insights into ribosome regulation in human diseases and cancer.

scholarly journals YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria

2019 ◽  
Author(s):  
Aaron R. D’Souza ◽  
Lindsey Van Haute ◽  
Christopher A. Powell ◽  
Pedro Rebelo-Guiomar ◽  
Joanna Rorbach ◽  
...  
Keyword(s):  
Small Subunit ◽  
Dual Function ◽  
Mitochondrial Translation ◽  
Trna Processing ◽  
Mitochondrial Ribosome ◽  
Severe Reduction ◽  
Translation Apparatus ◽  
Additional Protein ◽  
Processing Steps ◽  
Issue Section

AbstractMitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterised a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, caused by less accurate mitochondrial mt-tRNASer(AGY) processing from the primary transcript and an accumulation of immature mitochondrial small subunit. Our results suggest that YbeY performs a dual function in mitochondria coupling tRNA processing to mitoribosome biogenesis.Issue SectionNucleic Acid EnzymesAbstract Figure

scholarly journals Genetic ablation of the mitochondrial ribosome in Plasmodium falciparum sensitizes the human malaria parasite to antimalarial drugs targeting mitochondrial functions

2020 ◽  
Author(s):  
Liqin Ling ◽  
Maruthi Mulaka ◽  
Justin Munro ◽  
Swati Dass ◽  
Michael W. Mather ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Ribosomal Proteins ◽  
Drug Targets ◽  
Small Subunit ◽  
Rrna Genes ◽  
Pyrimidine Nucleotides ◽  
Genetic Ablation ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Functions ◽  
Mitochondrial Ribosome

ABSTRACTThe mitochondrion of malaria parasites contains clinically validated drug targets. Within Plasmodium spp., the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ∼ 27 small, fragmented rRNA genes in length of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum has been shown to be essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of mitoribosomes in sustaining the metabolic status of the parasite mitochondrion remains unknown. Here, among the 14 annotated mitoribosomal proteins of the small subunit of P. falciparum, we verified the localization and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, PfmtRPS18), employing a CRISPR/Cas9 mediated conditional knockdown tool. Using immuno-electron microscopy, we provided evidence that the mitoribosome is closely associated with the mitochondrial inner membrane in the parasite. Upon knockdown of the mitoribosome, the parasites became hypersensitive to inhibitors targeting the bc1 complex, dihydroorotate dehydrogenase and F1Fo ATP synthase complex. Furthermore, knockdown of the mitoribosome blocked the pyrimidine biosynthesis pathway and reduced the pool of pyrimidine nucleotides. Together, our data suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capability of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors targeting mitochondrial functions.

Sign in / Sign up

Export Citation Format

Share Document