The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.     

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.     

Clingy genes: Why were genes for ribosomal proteins retained in many mitochondrial genomes?

Why mitochondria still retain their own genome is a puzzle given the enormous effort to maintain a mitochondrial translation machinery. Most mitochondrially encoded proteins are membrane-embedded subunits of the respiratory chain. Their hydrophobicity presumably impedes their import into mitochondria. However, many mitochondrial genomes also encode protein subunits of the mitochondrial ribosome. These proteins lack transmembrane domains and hydrophobicity cannot explain why their genes remained in mitochondria. In this review, we provide an overview about mitochondrially encoded subunits of mitochondrial ribosomes of fungi, plants and protists. Moreover, we discuss and evaluate different hypotheses which were put forward to explain why (ribosomal) proteins remained mitochondrially encoded. It seems likely that the synthesis of ribosomal proteins in the mitochondrial matrix is used to regulate the assembly of the mitochondrial ribosome within mitochondria and to avoid problems that mitochondrial proteins might pose for cytosolic proteostasis and for the assembly of cytosolic ribosomes.     

Isolation and characterization of the acidic phosphoproteins of 60-S ribosomes from Artemia salina and rat liver.

Eucaryotic L7/L12-type proteins are present in ethanol/salt extracts (P1 protein) of ribosomes from Artemia salina and rat liver. These proteins are partially phosphorylated and occur in two forms of closely related structure: a major form eL12 having methionine at the N-terminal position and a minor form of eL12 (eL12') which seems slightly elongated and contains a blocked N terminus. Purification of the four different forms of this protein, eL12, eL12-P, eL12' and eL12'-P, was performed by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose. Using a radioimmuno assay, 1.8 copies of eL12 and 0.9 of eL12' were found on the 80-S A. salina ribosome. In ribosomes of both rat liver and A. salina, eL12 is present in a larger quantity than eL12'. 40-S and 60-S ribosomal subunits extracted with ethanol/salt were essentially free of eL12 proteins. A large pool of eL12 was found in the cytosol after removal of the ribosomes by centrifugation or molecular sieving. The proteins of rat liver and A. salina are similar with regard to their isoelectric points and molecular weights. Sedimentation equilibrium studies indicated that the isolated protein eL12 occurs as a dimer.     

Ribosome Rescue and Translation Termination at Non-Standard Stop Codons by ICT1 in Mammalian Mitochondria

Release factors (RFs) govern the termination phase of protein synthesis. Human mitochondria harbor four different members of the class 1 RF family: RF1Lmt/mtRF1a, RF1mt, C12orf65 and ICT1. The homolog of the essential ICT1 factor is widely distributed in bacteria and organelles and has the peculiar feature in human mitochondria to be part of the ribosome as a ribosomal protein of the large subunit. The factor has been suggested to rescue stalled ribosomes in a codon-independent manner. The mechanism of action of this factor was obscure and is addressed here. Using a homologous mitochondria system of purified components, we demonstrate that the integrated ICT1 has no rescue activity. Rather, purified ICT1 binds stoichiometrically to mitochondrial ribosomes in addition to the integrated copy and functions as a general rescue factor, i.e. it releases the polypeptide from the peptidyl tRNA from ribosomes stalled at the end or in the middle of an mRNA or even from non-programmed ribosomes. The data suggest that the unusual termination at a sense codon (AGA/G) of the oxidative-phosphorylation enzymes CO1 and ND6 is also performed by ICT1 challenging a previous model, according to which RF1Lmt/mtRF1a is responsible for the translation termination at non-standard stop codons. We also demonstrate by mutational analyses that the unique insertion sequence present in the N-terminal domain of ICT1 is essential for peptide release rather than for ribosome binding. The function of RF1mt, another member of the class1 RFs in mammalian mitochondria, was also examined and is discussed.     

The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present

Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.     

Purification of human mitochondrial ribosomal L7/L12 stalk proteins and reconstitution of functional hybrid ribosomes in Escherichia coli

Bacterial ribosomal L7/L12 stalk is formed by L10, L11, and multiple copies of L7/L12, which plays an essential role in recruiting initiation and elongation factors during translation. The homologs of these proteins, MRPL10, MRPL11, and MRPL12, are present in human mitochondrial ribosomes. To evaluate the role of MRPL10, MRPL11, and MRPL12 in translation, we over-expressed and purified components of the human mitochondrial L7/L12 stalk proteins in Escherichia coli. Here, we designed a construct to co-express MRPL10 and MRPL12 using a duet expression system to form a functional MRPL10-MRPL12 complex. The goal is to demonstrate the homology between the mitochondrial and bacterial L7/L12 stalk proteins and to reconstitute a hybrid ribosome to be used in structural and functional studies of the mitochondrial stalk.     

Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome

Oxidative phosphorylation in mitochondria requires the synthesis of proteins encoded in the mitochondrial DNA. The mitochondrial translation machinery differs significantly from that of the bacterial ancestor of the organelle. This is especially evident from many mitochondria-specific ribosomal proteins. An important site of the ribosome is the polypeptide tunnel exit. Here, nascent chains are exposed to an aqueous environment for the first time. Many biogenesis factors interact with the tunnel exit of pro- and eukaryotic ribosomes to help the newly synthesized proteins to mature. To date, nothing is known about the organization of the tunnel exit of mitochondrial ribosomes. We therefore undertook a comprehensive approach to determine the composition of the yeast mitochondrial ribosomal tunnel exit. Mitochondria contain homologues of the ribosomal proteins located at this site in bacterial ribosomes. Here, we identified proteins located in their proximity by chemical cross-linking and mass spectrometry. Our analysis revealed a complex network of interacting proteins including proteins and protein domains specific to mitochondrial ribosomes. This network includes Mba1, the membrane-bound ribosome receptor of the inner membrane, as well as Mrpl3, Mrpl13, and Mrpl27, which constitute ribosomal proteins exclusively found in mitochondria. This unique architecture of the tunnel exit is presumably an adaptation of the translation system to the specific requirements of the organelle.     

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.     

The stoichiometry of the ribosomal proteins of Escherichia coli

Summary A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.     

Nascent polypeptide-associated complex stimulates protein import into yeast mitochondria

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide-associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.     

Processing of mitochondrial presequences

Mitochondrial proteins are synthesized as precursor proteins on either cytosolic or mitochondrial ribosomes. The synthesized precursors from both translation origins possess targeting signals that guide the protein to its final destination in one of the four subcompartments of the organelle. The majority of nuclear-encoded mitochondrial precursors and also mitochondrial-encoded preproteins have an N-terminal presequence that serves as a targeting sequence. Specific presequence peptidases that are found in the matrix, inner membrane and intermembrane space of mitochondria proteolytically remove the signal sequence upon import or sorting. Besides the classical presequence peptidases MPP, IMP and Oct1, several novel proteases have recently been described to possess precursor processing activity, and analysis of their functional relevance revealed a tight connection between precursor processing, mitochondrial dynamics and protein quality control. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.     

Mitochondrial preprotein translocases as dynamic molecular machines

Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments--outer membrane, intermembrane space (IMS), inner membrane and matrix--requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria.     

Assembly of Saccharomyces cerevisiae ribosomal stalk: binding of P1 proteins is required for the interaction of P2 proteins

The yeast ribosomal stalk is formed by a protein pentamer made of the 38 kDa P0 and four 12 kDa acidic P1/P2. The interaction of recombinant acidic proteins P1 alpha and P2 beta with ribosomes from Saccharomyces cerevisiae D4567, lacking all the 12 kDa stalk components, has been used to study the in vitro assembly of this important ribosomal structure. Stimulation of the ribosome activity was obtained by incubating simultaneously the particles with both proteins, which were nonphosphorylated initially and remained unmodified afterward. The N-terminus state, free or blocked, did not affect either the binding or reactivating activity of both proteins. Independent incubation with each protein did not affect the activity of the particles, however, protein P2 beta alone was unable to bind the ribosome whereas P1 alpha could. The binding of P1 alpha alone is a saturable process in acidic-protein-deficient ribosomes and does not take place in complete wild-type particles. Binding of P1 proteins in the absence of P2 proteins takes also place in vivo, when protein P1 beta is overexpressed in S. cerevisiae. In contrast, protein P2 beta is not detected in the ribosome in the P1-deficient D67 strain despite being accumulated in the cytoplasm. The results confirm that neither phosphorylation nor N-terminal blocking of the 12 kDa acidic proteins is required for the assembly and function of the yeast stalk. More importantly, and regardless of the involvement of other elements, they indicate that stalk assembling is a coordinated process, in which P1 proteins would provide a ribosomal anchorage to P2 proteins, and P2 components would confer functionality to the complex.     

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.     

Studies of the interaction of Escherichia coli YjeQ with the ribosome in vitro

Escherichia coli YjeQ represents a conserved group of bacteria-specific nucleotide-binding proteins of unknown physiological function that have been shown to be essential to the growth of E. coli and Bacillus subtilis. The protein has previously been characterized as possessing a slow steady-state GTP hydrolysis activity (8 h(-1)) (D. M. Daigle, L. Rossi, A. M. Berghuis, L. Aravind, E. V. Koonin, and E. D. Brown, Biochemistry 41: 11109-11117, 2002). In the work reported here, YjeQ from E. coli was found to copurify with ribosomes from cell extracts. The copy number of the protein per cell was nevertheless low relative to the number of ribosomes (ratio of YjeQ copies to ribosomes, 1:200). In vitro, recombinant YjeQ protein interacted strongly with the 30S ribosomal subunit, and the stringency of that interaction, revealed with salt washes, was highest in the presence of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate (GMP-PNP). Likewise, association with the 30S subunit resulted in a 160-fold stimulation of YjeQ GTPase activity, which reached a maximum with stoichiometric amounts of ribosomes. N-terminal truncation variants of YjeQ revealed that the predicted OB-fold region was essential for ribosome binding and GTPase stimulation, and they showed that an N-terminal peptide (amino acids 1 to 20 in YjeQ) was necessary for the GMP-PNP-dependent interaction of YjeQ with the 30S subunit. Taken together, these data indicate that the YjeQ protein participates in a guanine nucleotide-dependent interaction with the ribosome and implicate this conserved, essential GTPase as a novel factor in ribosome function.     

Properties of human mitochondrial ribosomes

Mammalian mitochondrial ribosomes (55S) differ unexpectedly from bacterial (70S) and cytoplasmic ribosomes (80S), as well as other kinds of mitochondrial ribosomes. Typical of mammalian mitochondrial ribosomes, the bovine mitochondrial ribosome has been developed as a model system for the study of human mitochondrial ribosomes, to address several questions related to the structure, function, biosynthesis and evolution of these interesting ribosomes. Bovine mitochondrial ribosomal proteins (MRPs) from each subunit have been identified and characterized with respect to individuality and electrophoretic properties, amino acid sequence, topographic disposition, RNA binding properties, evolutionary relationships and reaction with affinity probes of ribosomal functional domains. Several distinctive properties of these ribosomes are being elucidated, including their antibiotic susceptibility and composition. Human mitochondrial ribosomes lack several of the major RNA stem structures of bacterial ribosomes but they contain a correspondingly higher protein content (as many as 80 proteins), suggesting a model where proteins have replaced RNA structural elements during the evolution of these ribosomes. Despite their lower RNA content they are physically larger than bacterial ribosomes, because of the 'extra' proteins they contain. The extra proteins in mitochondrial ribosomes are 'new' in the sense that they are not homologous to proteins in bacterial or cytoplasmic ribosomes. Some of the new proteins appear to be bifunctional. All of the mammalian MRPs are encoded in nuclear genes (a separate set from those encoding cytoplasmic ribosomal proteins) which are evolving more rapidly than those encoding cytoplasmic ribosomal proteins. The MRPs are imported into mitochondria where they assemble coordinately with mitochondrially transcribed rRNAs into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.     

Functional characterization of yeast mitochondrial release factor 1

The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.     

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.