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Sample records for ribosomal protein mrnas

  1. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed Central

    Mangiarotti, Giorgio

    2003-01-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs. PMID:12392449

  2. Two Dictyostelium ribosomal proteins act as RNases for specific classes of mRNAs.

    PubMed

    Mangiarotti, Giorgio

    2003-03-01

    Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.

  3. Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

    PubMed Central

    Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

    2013-01-01

    Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

  4. Appraisal of the Missing Proteins Based on the mRNAs Bound to Ribosomes.

    PubMed

    Xu, Shaohang; Zhou, Ruo; Ren, Zhe; Zhou, Baojin; Lin, Zhilong; Hou, Guixue; Deng, Yamei; Zi, Jin; Lin, Liang; Wang, Quanhui; Liu, Xin; Xu, Xun; Wen, Bo; Liu, Siqi

    2015-12-04

    Considering the technical limitations of mass spectrometry in protein identification, the mRNAs bound to ribosomes (RNC-mRNA) are assumed to reflect the mRNAs participating in the translational process. The RNC-mRNA data are reasoned to be useful for appraising the missing proteins. A set of the multiomics data including free-mRNAs, RNC-mRNAs, and proteomes was acquired from three liver cancer cell lines. On the basis of the missing proteins in neXtProt (release 2014-09-19), the bioinformatics analysis was carried out in three phases: (1) finding how many neXtProt missing proteins have or do not have RNA-seq and/or MS/MS evidence, (2) analyzing specific physicochemical and biological properties of the missing proteins that lack both RNA-seq and MS/MS evidence, and (3) analyzing the combined properties of these missing proteins. Total of 1501 missing proteins were found by neither RNC-mRNA nor MS/MS in the three liver cancer cell lines. For these missing proteins, some are expected higher hydrophobicity, unsuitable detection, or sensory functions as properties at the protein level, while some are predicted to have nonexpressing chromatin structures on the corresponding gene level. With further integrated analysis, we could attribute 93% of them (1391/1501) to these causal factors, which result in the expression products scarcely detected by RNA-seq or MS/MS.

  5. The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs

    PubMed Central

    Thompson, Mary K; Rojas-Duran, Maria F; Gangaramani, Paritosh; Gilbert, Wendy V

    2016-01-01

    Translation is a core cellular process carried out by a highly conserved macromolecular machine, the ribosome. There has been remarkable evolutionary adaptation of this machine through the addition of eukaryote-specific ribosomal proteins whose individual effects on ribosome function are largely unknown. Here we show that eukaryote-specific Asc1/RACK1 is required for efficient translation of mRNAs with short open reading frames that show greater than average translational efficiency in diverse eukaryotes. ASC1 mutants in S. cerevisiae display compromised translation of specific functional groups, including cytoplasmic and mitochondrial ribosomal proteins, and display cellular phenotypes consistent with their gene-specific translation defects. Asc1-sensitive mRNAs are preferentially associated with the translational ‘closed loop’ complex comprised of eIF4E, eIF4G, and Pab1, and depletion of eIF4G mimics the translational defects of ASC1 mutants. Together our results reveal a role for Asc1/RACK1 in a length-dependent initiation mechanism optimized for efficient translation of genes with important housekeeping functions. DOI: http://dx.doi.org/10.7554/eLife.11154.001 PMID:27117520

  6. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum

    PubMed Central

    Jagannathan, Sujatha; Reid, David W.; Cox, Amanda H.

    2014-01-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER. PMID:25142066

  7. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum.

    PubMed

    Jagannathan, Sujatha; Reid, David W; Cox, Amanda H; Nicchitta, Christopher V

    2014-10-01

    The specialized protein synthesis functions of the cytosol and endoplasmic reticulum compartments are conferred by the signal recognition particle (SRP) pathway, which directs the cotranslational trafficking of signal sequence-encoding mRNAs from the cytosol to the endoplasmic reticulum (ER). Although subcellular mRNA distributions largely mirror the binary pattern predicted by the SRP pathway model, studies in mammalian cells, yeast, and Drosophila have also demonstrated that cytosolic protein-encoding mRNAs are broadly represented on ER-bound ribosomes. A mechanism for such noncanonical mRNA localization remains, however, to be identified. Here, we examine the hypothesis that de novo translation initiation on ER-bound ribosomes serves as a mechanism for localizing cytosolic protein-encoding mRNAs to the ER. As a test of this hypothesis, we performed single molecule RNA fluorescence in situ hybridization studies of subcellular mRNA distributions and report that a substantial fraction of mRNAs encoding the cytosolic protein GAPDH resides in close proximity to the ER. Consistent with these data, analyses of subcellular mRNA and ribosome distributions in multiple cell lines demonstrated that cytosolic protein mRNA-ribosome distributions were strongly correlated, whereas signal sequence-encoding mRNA-ribosome distributions were divergent. Ribosome footprinting studies of ER-bound polysomes revealed a substantial initiation codon read density enrichment for cytosolic protein-encoding mRNAs. We also demonstrate that eukaryotic initiation factor 2α is bound to the ER via a salt-sensitive, ribosome-independent mechanism. Combined, these data support ER-localized translation initiation as a mechanism for mRNA recruitment to the ER. © 2014 Jagannathan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

    PubMed

    Root-Bernstein, Robert; Root-Bernstein, Meredith

    2016-05-21

    We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their

  9. The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs

    PubMed Central

    Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

    2017-01-01

    Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNALys(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC. PMID:28685749

  10. The E3 ubiquitin ligase and RNA-binding protein ZNF598 orchestrates ribosome quality control of premature polyadenylated mRNAs.

    PubMed

    Garzia, Aitor; Jafarnejad, Seyed Mehdi; Meyer, Cindy; Chapat, Clément; Gogakos, Tasos; Morozov, Pavel; Amiri, Mehdi; Shapiro, Maayan; Molina, Henrik; Tuschl, Thomas; Sonenberg, Nahum

    2017-07-07

    Cryptic polyadenylation within coding sequences (CDS) triggers ribosome-associated quality control (RQC), followed by degradation of the aberrant mRNA and polypeptide, ribosome disassembly and recycling. Although ribosomal subunit dissociation and nascent peptide degradation are well-understood, the molecular sensors of aberrant mRNAs and their mechanism of action remain unknown. We studied the Zinc Finger Protein 598 (ZNF598) using PAR-CLIP and revealed that it cross-links to tRNAs, mRNAs and rRNAs, thereby placing the protein on translating ribosomes. Cross-linked reads originating from AAA-decoding tRNA(Lys)(UUU) were 10-fold enriched over its cellular abundance, and poly-lysine encoded by poly(AAA) induced RQC in a ZNF598-dependent manner. Encounter with translated polyA segments by ZNF598 triggered ubiquitination of several ribosomal proteins, requiring the E2 ubiquitin ligase UBE2D3 to initiate RQC. Considering that human CDS are devoid of >4 consecutive AAA codons, sensing of prematurely placed polyA tails by a specialized RNA-binding protein is a novel nucleic-acid-based surveillance mechanism of RQC.

  11. Glucose triggers different global responses in yeast, depending on the strength of the signal, and transiently stabilizes ribosomal protein mRNAs.

    PubMed

    Yin, Zhikang; Wilson, Séan; Hauser, Nicole C; Tournu, Helene; Hoheisel, Jörg D; Brown, Alistair J P

    2003-05-01

    Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of> 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

  12. Alteration of a novel dispensable mitochondrial ribosomal small-subunit protein, Rsm28p, allows translation of defective COX2 mRNAs.

    PubMed

    Williams, Elizabeth H; Bsat, Nada; Bonnefoy, Nathalie; Butler, Christine A; Fox, Thomas D

    2005-02-01

    Mutations affecting the RNA sequence of the first 10 codons of the Saccharomyces cerevisiae mitochondrial gene COX2 strongly reduce translation of the mRNA, which encodes the precursor of cytochrome c oxidase subunit II. A dominant chromosomal mutation that suppresses these defects is an internal in-frame deletion of 67 codons from the gene YDR494w. Wild-type YDR494w encodes a 361-residue polypeptide with no similarity to proteins of known function. The epitope-tagged product of this gene, now named RSM28, is both peripherally associated with the inner surface of the inner mitochondrial membrane and soluble in the matrix. Epitope-tagged Rsm28p from Triton X-100-solubilized mitochondria sedimented with the small subunit of mitochondrial ribosomes in a sucrose gradient containing 500 mM NH4Cl. Complete deletion of RSM28 caused only a modest decrease in growth on nonfermentable carbon sources in otherwise wild-type strains and enhanced the respiratory defect of the suppressible cox2 mutations. The rsm28 null mutation also reduced translation of an ARG8m reporter sequence inserted at the COX1, COX2, and COX3 mitochondrial loci. We tested the ability of RSM28-1 to suppress a variety of cox2 and cox3 mutations and found that initiation codon mutations in both genes were suppressed. We conclude that Rsm28p is a dispensable small-subunit mitochondrial ribosomal protein previously undetected in systematic investigations of these ribosomes, with a positive role in translation of several mitochondrial mRNAs.

  13. Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.

    PubMed

    Shi, Zhen; Fujii, Kotaro; Kovary, Kyle M; Genuth, Naomi R; Röst, Hannes L; Teruel, Mary N; Barna, Maria

    2017-07-06

    Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

    PubMed Central

    Masuda, Tomoaki; Petrov, Alexey N.; Iizuka, Ryo; Funatsu, Takashi; Puglisi, Joseph D.; Uemura, Sotaro

    2012-01-01

    Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation. PMID:22411833

  15. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  16. Ribosome Traffic on mRNAs Maps to Gene Ontology: Genome-wide Quantification of Translation Initiation Rates and Polysome Size Regulation

    PubMed Central

    Ciandrini, Luca

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function. PMID:23382661

  17. Ribosome traffic on mRNAs maps to gene ontology: genome-wide quantification of translation initiation rates and polysome size regulation.

    PubMed

    Ciandrini, Luca; Stansfield, Ian; Romano, M Carmen

    2013-01-01

    To understand the complex relationship governing transcript abundance and the level of the encoded protein, we integrate genome-wide experimental data of ribosomal density on mRNAs with a novel stochastic model describing ribosome traffic dynamics during translation elongation. This analysis reveals that codon arrangement, rather than simply codon bias, has a key role in determining translational efficiency. It also reveals that translation output is governed both by initiation efficiency and elongation dynamics. By integrating genome-wide experimental data sets with simulation of ribosome traffic on all Saccharomyces cerevisiae ORFs, mRNA-specific translation initiation rates are for the first time estimated across the entire transcriptome. Our analysis identifies different classes of mRNAs characterised by their initiation rates, their ribosome traffic dynamics, and by their response to ribosome availability. Strikingly, this classification based on translational dynamics maps onto key gene ontological classifications, revealing evolutionary optimisation of translation responses to be strongly influenced by gene function.

  18. Novel factor rescues ribosomes trapped on non-stop mRNAs.

    PubMed

    Himeno, Hyouta

    2010-11-01

    Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.

  19. Ribosomal stress activates eEF2K-eEF2 pathway causing translation elongation inhibition and recruitment of terminal oligopyrimidine (TOP) mRNAs on polysomes.

    PubMed

    Gismondi, Angelo; Caldarola, Sara; Lisi, Gaia; Juli, Giada; Chellini, Lidia; Iadevaia, Valentina; Proud, Christopher G; Loreni, Fabrizio

    2014-11-10

    The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. BALANCED PRODUCTION OF RIBOSOMAL PROTEINS

    PubMed Central

    Perry, Robert P.

    2017-01-01

    Eukaryotic ribosomes contain one molecule each of 79 different proteins. The genes encoding these proteins are usually at widely scattered loci and have distinctive promoters with certain common features. This minireview discusses the means by which cells manage to balance the production of ribosomal proteins so as to end up with equimolar quantities in the ribosome. Regulation at all levels of gene expression, from transcription to protein turnover, is considered. PMID:17689889

  1. Mitochondrial ribosomal proteins (MRPs) of yeast.

    PubMed Central

    Graack, H R; Wittmann-Liebold, B

    1998-01-01

    Mitochondrial ribosomal proteins (MRPs) are the counterparts in that organelle of the cytoplasmic ribosomal proteins in the host. Although the MRPs fulfil similar functions in protein biosynthesis, they are distinct in number, features and primary structures from the latter. Most progress in the eludication of the properties of individual MRPs, and in the characterization of the corresponding genes, has been made in baker's yeast (Saccharomyces cerevisiae). To date, 50 different MRPs have been determined, although biochemical data and mutational analysis propose a total number which is substantially higher. Surprisingly, only a minority of the MRPs that have been characterized show significant sequence similarities to known ribosomal proteins from other sources, thus limiting the deduction of their functions by simple comparison of amino acid sequences. Further, individual MRPs have been characterized functionally by mutational studies, and the regulation of expression of MRP genes has been described. The interaction of the mitochondrial ribosomes with transcription factors specific for individual mitochondrial mRNAs, and the communication between mitochondria and the nucleus for the co-ordinated expression of ribosomal constituents, are other aspects of current MRP research. Although the mitochondrial translational system is still far from being described completely, the yeast MRP system serves as a model for other organisms, including that of humans. PMID:9445368

  2. Supernumerary proteins of mitochondrial ribosomes.

    PubMed

    Rackham, Oliver; Filipovska, Aleksandra

    2014-04-01

    Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes. Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions. The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems. Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research. © 2013.

  3. Paradigms of ribosome synthesis: Lessons learned from ribosomal proteins

    PubMed Central

    Gamalinda, Michael; Woolford, John L

    2015-01-01

    The proteome in all cells is manufactured via the intricate process of translation by multimolecular factories called ribosomes. Nevertheless, these ribonucleoprotein particles, the largest of their kind, also have an elaborate assembly line of their own. Groundbreaking discoveries that bacterial ribosomal subunits can be self-assembled in vitro jumpstarted studies on how ribosomes are constructed. Until recently, ribosome assembly has been investigated almost entirely in vitro with bacterial small subunits under equilibrium conditions. In light of high-resolution ribosome structures and a more sophisticated toolkit, the past decade has been defined by a burst of kinetic studies in vitro and, importantly, also a shift to examining ribosome maturation in living cells, especially in eukaryotes. In this review, we summarize the principles governing ribosome assembly that emerged from studies focusing on ribosomal proteins and their interactions with rRNA. Understanding these paradigms has taken center stage, given the linkage between anomalous ribosome biogenesis and proliferative disorders. PMID:26779413

  4. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  5. Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana.

    PubMed

    Juntawong, Piyada; Sorenson, Reed; Bailey-Serres, Julia

    2013-06-01

    RNA binding proteins (RBPs) function post-transcriptionally to fine-tune gene regulation. Arabidopsis thaliana has four Gly-rich, zinc finger-containing RBPs called cold shock proteins 1-4 (CSP1-CSP4), that possess an evolutionary conserved cold shock domain. Here, we determined that CSP1 associates with polyribosomes (polysomes) via an RNA-mediated interaction. Both the abundance and polysomal co-fractionation of CSP1 was enhanced in the cold (4°C), but did not influence global levels of polysomes, which were minimally perturbed by above freezing cold temperatures. Using a polyclonal antiserum, CSP1 was co-immunopurified with several hundred transcripts from rosettes of plants cultivated at 23°C or transferred to 4°C for 12 h. CSP1-associated mRNAs were characterized by G+C-rich 5' untranslated regions and gene ontologies related to cellular respiration, mRNA binding and translation. The majority of the CSP1-associated mRNAs were constitutively expressed and stable in the cold. CSP1 abundance was correlated with improved translation of ribosomal protein mRNAs during cold stress and improved maintenance of homeostasis and translation of mRNAs under water-deficit stress. In summary, CSP1 selectively chaperones mRNAs, providing translational enhancement during stress.

  6. Ribosome-messenger recognition: mRNA target sites for ribosomal protein S1.

    PubMed Central

    Boni, I V; Isaeva, D M; Musychenko, M L; Tzareva, N V

    1991-01-01

    Ribosomal protein S1 is known to play an important role in translational initiation, being directly involved in recognition and binding of mRNAs by 30S ribosomal particles. Using a specially developed procedure based on efficient crosslinking of S1 to mRNA induced by UV irradiation, we have identified S1 binding sites on several phage RNAs in preinitiation complexes. Targets for S1 on Q beta and fr RNAs are localized upstream from the coat protein gene and contain oligo(U)-sequences. In the case of Q beta RNA, this S1 binding site overlaps the S-site for Q beta replicase and the site for S1 binding within a binary complex. It is reasonable that similar U-rich sequences represent S1 binding sites on bacterial mRNAs. To test this idea we have used E. coli ssb mRNA prepared in vitro with the T7 promoter/RNA polymerase system. By the methods of toeprinting, enzymatic footprinting, and UV crosslinking we have shown that binding of the ssb mRNA to 30S ribosomes is S1-dependent. The oligo(U)-sequence preceding the SD domain was found to be the target for S1. We propose that S1 binding sites, represented by pyrimidine-rich sequences upstream from the SD region, serve as determinants involved in recognition of mRNA by the ribosome. Images PMID:2011495

  7. An extra-ribosomal function of ribosomal protein L13a in macrophage resolves inflammation

    PubMed Central

    Poddar, Darshana; Basu, Abhijit; Baldwin, William; Kondratov, Roman V; Barik, Sailen; Mazumder, Barsanjit

    2013-01-01

    Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of pro-inflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout (KO) mice here we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these KO animals show unregulated expression of several chemokines e.g. CXCL13, CCL22, CCL8 and CCR3. These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, our studies provide the first evidence of an essential extra-ribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host. PMID:23460747

  8. YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes.

    PubMed

    Handa, Yoshihiro; Inaho, Noriyuki; Nameki, Nobukazu

    2011-03-01

    In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl-tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.

  9. Neuron-Like Networks Between Ribosomal Proteins Within the Ribosome

    NASA Astrophysics Data System (ADS)

    Poirot, Olivier; Timsit, Youri

    2016-05-01

    From brain to the World Wide Web, information-processing networks share common scale invariant properties. Here, we reveal the existence of neural-like networks at a molecular scale within the ribosome. We show that with their extensions, ribosomal proteins form complex assortative interaction networks through which they communicate through tiny interfaces. The analysis of the crystal structures of 50S eubacterial particles reveals that most of these interfaces involve key phylogenetically conserved residues. The systematic observation of interactions between basic and aromatic amino acids at the interfaces and along the extension provides new structural insights that may contribute to decipher the molecular mechanisms of signal transmission within or between the ribosomal proteins. Similar to neurons interacting through “molecular synapses”, ribosomal proteins form a network that suggest an analogy with a simple molecular brain in which the “sensory-proteins” innervate the functional ribosomal sites, while the “inter-proteins” interconnect them into circuits suitable to process the information flow that circulates during protein synthesis. It is likely that these circuits have evolved to coordinate both the complex macromolecular motions and the binding of the multiple factors during translation. This opens new perspectives on nanoscale information transfer and processing.

  10. Import of ribosomal proteins into yeast mitochondria.

    PubMed

    Woellhaf, Michael W; Hansen, Katja G; Garth, Christoph; Herrmann, Johannes M

    2014-12-01

    Mitochondrial ribosomes of baker's yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences, suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and indeed showed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix, and thereby specifically focus on proteins of the mitochondrial ribosome.

  11. The global translation profile in a ribosomal protein mutant resembles that of an eIF3 mutant.

    PubMed

    Tiruneh, Bayu Sisay; Kim, Byung-Hoon; Gallie, Daniel R; Roy, Bijoyita; von Arnim, Albrecht G

    2013-12-30

    Genome-wide assays performed in Arabidopsis and other organisms have revealed that the translation status of mRNAs responds dramatically to different environmental stresses and genetic lesions in the translation apparatus. To identify additional features of the global landscape of translational control, we used microarray analysis of polysomal as well as non-polysomal mRNAs to examine the defects in translation in a poly(A) binding protein mutant, pab2 pab8, as well as in a mutant of a large ribosomal subunit protein, rpl24b/shortvalve1. The mutation of RPL24B stimulated the ribosome occupancy of mRNAs for nuclear encoded ribosomal proteins. Detailed analysis yielded new insights into the translational regulon containing the ribosomal protein mRNAs. First, the ribosome occupancy defects in the rpl24b mutant partially overlapped with those in a previously analyzed initiation factor mutant, eif3h. Second, a group of mRNAs with incomplete coding sequences appeared to be uncoupled from the regulon, since their dependence on RPL24B differed from regular mRNAs. Third, different sister paralogs of the ribosomal proteins differed in their translation state in the wild-type. Some sister paralogs also differed in their response to the rpl24b mutation. In contrast to rpl24b, the pab2 pab8 mutant revealed few gene specific translational defects, but a group of seed storage protein mRNAs were stimulated in their ribosome occupancy. In the course of this work, while optimizing the statistical analysis of ribosome occupancy data, we collected 12 biological replicates of translation states from wild-type seedlings. We defined 20% of mRNAs as having a high variance in their translation state. Many of these mRNAs were functionally associated with responses to the environment, suggesting that subtle variation in the environmental conditions is sensed by plants and transduced to affect the translational efficiency of hundreds of mRNAs. These data represent the first genome

  12. Ribosome Inactivating Proteins from Rosaceae.

    PubMed

    Shang, Chenjing; Rougé, Pierre; Van Damme, Els J M

    2016-08-22

    Ribosome-inactivating proteins (RIPs) are widespread among higher plants of different taxonomic orders. In this study, we report on the RIP sequences found in the genome/transcriptome of several important Rosaceae species, including many economically important edible fruits such as apple, pear, peach, apricot, and strawberry. All RIP domains from Rosaceae share high sequence similarity with conserved residues in the catalytic site and the carbohydrate binding sites. The genomes of Malus domestica and Pyrus communis contain both type 1 and type 2 RIP sequences, whereas for Prunus mume, Prunus persica, Pyrus bretschneideri, and Pyrus communis a complex set of type 1 RIP sequences was retrieved. Heterologous expression and purification of the type 1 as well as the type 2 RIP from apple allowed to characterize the biological activity of the proteins. Both RIPs from Malus domestica can inhibit protein synthesis. Furthermore, molecular modelling suggests that RIPs from Rosaceae possess three-dimensional structures that are highly similar to the model proteins and can bind to RIP substrates. Screening of the recombinant type 2 RIP from apple on a glycan array revealed that this type 2 RIP interacts with terminal sialic acid residues. Our data suggest that the RIPs from Rosaceae are biologically active proteins.

  13. Synthesis of ribosomal proteins in developing Dictyostelium discoideum cells is controlled by the methylation of proteins S24 and S31.

    PubMed

    Mangiarotti, Giorgio

    2002-01-01

    Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.

  14. The Ribosome Modulates Nascent Protein Folding

    PubMed Central

    Kaiser, Christian M.; Goldman, Daniel H.; Chodera, John D.; Tinoco, Ignacio; Bustamante, Carlos

    2014-01-01

    Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state. PMID:22194581

  15. Ribosome-associated protein quality control

    PubMed Central

    Brandman, Onn; Hegde, Ramanujan S

    2016-01-01

    Protein synthesis by the ribosome can fail for numerous reasons including faulty mRNA, insufficient availability of charged tRNAs and genetic errors. All organisms have evolved mechanisms to recognize stalled ribosomes and initiate pathways for recycling, quality control and stress signaling. Here we review the discovery and molecular dissection of the eukaryotic ribosome-associated quality-control pathway for degradation of nascent polypeptides arising from interrupted translation. PMID:26733220

  16. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes

    PubMed Central

    Aphasizheva, Inna; Maslov, Dmitri A.; Qian, Yu; Huang, Lan; Wang, Qi; Costello, Catherine E.; Aphasizhev, Ruslan

    2016-01-01

    Summary Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3′ adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation. PMID:26713541

  17. Differential Stoichiometry among Core Ribosomal Proteins

    PubMed Central

    Slavov, Nikolai; Semrau, Stefan; Airoldi, Edoardo; Budnik, Bogdan; van Oudenaarden, Alexander

    2015-01-01

    Summary Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC) and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function. PMID:26565899

  18. The other lives of ribosomal proteins

    PubMed Central

    2010-01-01

    Despite the fact that ribosomal proteins are the constituents of an organelle that is present in every cell, they show a surprising level of regulation, and several of them have also been shown to have other extra-ribosomal functions, such in replication, transcription, splicing or even ageing. This review provides a comprehensive summary of these important aspects. PMID:20650820

  19. A new system for naming ribosomal proteins.

    PubMed

    Ban, Nenad; Beckmann, Roland; Cate, Jamie H D; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis L J; Lindahl, Lasse; Liljas, Anders; Lipton, Jeffrey M; McAlear, Michael A; Moore, Peter B; Noller, Harry F; Ortega, Joaquin; Panse, Vikram Govind; Ramakrishnan, V; Spahn, Christian M T; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

    2014-02-01

    A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.

  20. Protein synthesis by ribosomes with tethered subunits.

    PubMed

    Orelle, Cédric; Carlson, Erik D; Szal, Teresa; Florin, Tanja; Jewett, Michael C; Mankin, Alexander S

    2015-08-06

    The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  1. Illuminating parasite protein production by ribosome profiling

    PubMed Central

    Parsons, Marilyn; Myler, Peter J.

    2016-01-01

    While technologies for global enumeration of transcript abundance are well-developed, those that assess protein abundance require tailoring to penetrate to low abundance proteins. Ribosome profiling circumvents this challenge by measuring global protein production via sequencing small mRNA fragments protected by the assembled ribosome. This powerful approach is now being applied to protozoan parasites, including trypanosomes and Plasmodium. It has been used to identify new protein coding sequences (CDSs) and clarify the boundaries of previously annotated CDSs in Trypanosoma brucei. Ribosome profiling has demonstrated that translation efficiencies vary widely between genes and, for trypanosomes at least, for the same gene across stages. The ribosomal proteins are themselves subjected to translational control, suggesting a means of reinforcing global translational regulation. PMID:27061497

  2. Model Uracil-Rich RNAs and Membrane Protein mRNAs Interact Specifically with Cold Shock Proteins in Escherichia coli.

    PubMed

    Benhalevy, Daniel; Bochkareva, Elena S; Biran, Ido; Bibi, Eitan

    2015-01-01

    Are integral membrane protein-encoding mRNAs (MPRs) different from other mRNAs such as those encoding cytosolic mRNAs (CPRs)? This is implied from the emerging concept that MPRs are specifically recognized and delivered to membrane-bound ribosomes in a translation-independent manner. MPRs might be recognized through uracil-rich segments that encode hydrophobic transmembrane helices. To investigate this hypothesis, we designed DNA sequences encoding model untranslatable transcripts that mimic MPRs or CPRs. By utilizing in vitro-synthesized biotinylated RNAs mixed with Escherichia coli extracts, we identified a highly specific interaction that takes place between transcripts that mimic MPRs and the cold shock proteins CspE and CspC, which are normally expressed under physiological conditions. Co-purification studies with E. coli expressing 6His-tagged CspE or CspC confirmed that the specific interaction occurs in vivo not only with the model uracil-rich untranslatable transcripts but also with endogenous MPRs. Our results suggest that the evolutionarily conserved cold shock proteins may have a role, possibly as promiscuous chaperons, in the biogenesis of MPRs.

  3. Comparative anatomy of a regulatory ribosomal protein.

    PubMed

    Worbs, Michael; Wahl, Markus C; Lindahl, Lasse; Zengel, Janice M

    2002-08-01

    Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.

  4. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  5. Toward the mechanism of eIF4F-mediated ribosomal attachment to mammalian capped mRNAs

    PubMed Central

    Kumar, Parimal; Hellen, Christopher U.T.; Pestova, Tatyana V.

    2016-01-01

    Ribosomal attachment to mammalian capped mRNAs is achieved through the cap–eukaryotic initiation factor 4E (eIF4E)–eIF4G–eIF3–40S chain of interactions, but the mechanism by which mRNA enters the mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5′-terminal AUGs was stimulated by the eIF4E–cap interaction and followed “the first AUG” rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5′ end of mRNA, implying that Met-tRNAiMet inspects mRNA from the first nucleotide and that initiation does not have a “blind spot.” In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E–eIF4G–eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E–eIF4G–eIF3–40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAiMet can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m7G cap specifically interacts with eIF3l. PMID:27401559

  6. Reconstitution of functional eukaryotic ribosomes from Dictyostelium discoideum ribosomal proteins and RNA.

    PubMed

    Mangiarotti, G; Chiaberge, S

    1997-08-08

    40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).

  7. Functional Importance of Mobile Ribosomal Proteins.

    PubMed

    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  8. Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones

    NASA Astrophysics Data System (ADS)

    Pausch, Patrick; Singh, Ujjwala; Ahmed, Yasar Luqman; Pillet, Benjamin; Murat, Guillaume; Altegoer, Florian; Stier, Gunter; Thoms, Matthias; Hurt, Ed; Sinning, Irmgard; Bange, Gert; Kressler, Dieter

    2015-06-01

    Exponentially growing yeast cells produce every minute >160,000 ribosomal proteins. Owing to their difficult physicochemical properties, the synthesis of assembly-competent ribosomal proteins represents a major challenge. Recent evidence highlights that dedicated chaperone proteins recognize the N-terminal regions of ribosomal proteins and promote their soluble expression and delivery to the assembly site. Here we explore the intuitive possibility that ribosomal proteins are captured by dedicated chaperones in a co-translational manner. Affinity purification of four chaperones (Rrb1, Syo1, Sqt1 and Yar1) selectively enriched the mRNAs encoding their specific ribosomal protein clients (Rpl3, Rpl5, Rpl10 and Rps3). X-ray crystallography reveals how the N-terminal, rRNA-binding residues of Rpl10 are shielded by Sqt1's WD-repeat β-propeller, providing mechanistic insight into the incorporation of Rpl10 into pre-60S subunits. Co-translational capturing of nascent ribosomal proteins by dedicated chaperones constitutes an elegant mechanism to prevent unspecific interactions and aggregation of ribosomal proteins on their road to incorporation.

  9. Direct Interaction of the N-Terminal Domain of Ribosomal Protein S1 with Protein S2 in Escherichia coli

    PubMed Central

    Byrgazov, Konstantin; Manoharadas, Salim; Kaberdina, Anna C.; Vesper, Oliver; Moll, Isabella

    2012-01-01

    Despite of the high resolution structure available for the E. coli ribosome, hitherto the structure and localization of the essential ribosomal protein S1 on the 30 S subunit still remains to be elucidated. It was previously reported that protein S1 binds to the ribosome via protein-protein interaction at the two N-terminal domains. Moreover, protein S2 was shown to be required for binding of protein S1 to the ribosome. Here, we present evidence that the N-terminal domain of S1 (amino acids 1–106; S1106) is necessary and sufficient for the interaction with protein S2 as well as for ribosome binding. We show that over production of protein S1106 affects E. coli growth by displacing native protein S1 from its binding pocket on the ribosome. In addition, our data reveal that the coiled-coil domain of protein S2 (S2α2) is sufficient to allow protein S1 to bind to the ribosome. Taken together, these data uncover the crucial elements required for the S1/S2 interaction, which is pivotal for translation initiation on canonical mRNAs in Gram-negative bacteria. The results are discussed in terms of a model wherein the S1/S2 interaction surface could represent a possible target to modulate the selectivity of the translational machinery and thereby alter the translational program under distinct conditions. PMID:22412910

  10. Listerin-dependent nascent protein ubiquitination relies on ribosome subunit dissociation.

    PubMed

    Shao, Sichen; von der Malsburg, Karina; Hegde, Ramanujan S

    2013-06-06

    Quality control of defective mRNAs relies on their translation to detect the lesion. Aberrant proteins are therefore an obligate byproduct of mRNA surveillance and must be degraded to avoid disrupting protein homeostasis. These defective translation products are thought to be ubiquitinated at the ribosome, but the mechanism of ubiquitin ligase selectivity for these ribosomes is not clear. Here, we in vitro reconstitute ubiquitination of nascent proteins produced from aberrant mRNAs. Stalled 80S ribosome-nascent chain complexes are dissociated by the ribosome recycling factors Hbs1/Pelota/ABCE1 to a unique 60S-nascent chain-tRNA complex. The ubiquitin ligase Listerin preferentially recognizes 60S-nascent chains and triggers efficient nascent chain ubiquitination. Interfering with Hbs1 function stabilizes 80S complexes, precludes efficient Listerin recruitment, and reduces nascent chain ubiquitination. Thus, ribosome recycling factors control Listerin localization, explaining how translation products of mRNA surveillance are efficiently ubiquitinated while sparing translating ribosomes.

  11. Fragile X mental retardation protein regulates translation by binding directly to the ribosome.

    PubMed

    Chen, Eileen; Sharma, Manjuli R; Shi, Xinying; Agrawal, Rajendra K; Joseph, Simpson

    2014-05-08

    Fragile X syndrome (FXS) is the most common form of inherited mental retardation, and it is caused by loss of function of the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is involved in the translational regulation of several neuronal mRNAs. However, the precise mechanism of translational inhibition by FMRP is unknown. Here, we show that FMRP inhibits translation by binding directly to the L5 protein on the 80S ribosome. Furthermore, cryoelectron microscopic reconstruction of the 80S ribosome⋅FMRP complex shows that FMRP binds within the intersubunit space of the ribosome such that it would preclude the binding of tRNA and translation elongation factors on the ribosome. These findings suggest that FMRP inhibits translation by blocking the essential components of the translational machinery from binding to the ribosome.

  12. Analysis of Ribosome-Associated mRNAs in Rice Reveals the Importance of Transcript Size and GC Content in Translation

    PubMed Central

    Zhao, Dongyan; Hamilton, John P.; Hardigan, Michael; Yin, Dongmei; He, Tao; Vaillancourt, Brieanne; Reynoso, Mauricio; Pauluzzi, Germain; Funkhouser, Scott; Cui, Yuehua; Bailey-Serres, Julia; Jiang, Jiming; Buell, C. Robin; Jiang, Ning

    2016-01-01

    Gene expression is controlled at transcriptional and post-transcriptional levels including decoding of messenger RNA (mRNA) into polypeptides via ribosome-mediated translation. Translational regulation has been intensively studied in the model dicot plant Arabidopsis thaliana, and in this study, we assessed the translational status [proportion of steady-state mRNA associated with ribosomes] of mRNAs by Translating Ribosome Affinity Purification followed by mRNA-sequencing (TRAP-seq) in rice (Oryza sativa), a model monocot plant and the most important food crop. A survey of three tissues found that most transcribed rice genes are translated whereas few transposable elements are associated with ribosomes. Genes with short and GC-rich coding regions are overrepresented in ribosome-associated mRNAs, suggesting that the GC-richness characteristic of coding sequences in grasses may be an adaptation that favors efficient translation. Transcripts with retained introns and extended 5′ untranslated regions are underrepresented on ribosomes, and rice genes belonging to different evolutionary lineages exhibited differential enrichment on the ribosomes that was associated with GC content. Genes involved in photosynthesis and stress responses are preferentially associated with ribosomes, whereas genes in epigenetic regulation pathways are the least enriched on ribosomes. Such variation is more dramatic in rice than that in Arabidopsis and is correlated with the wide variation of GC content of transcripts in rice. Taken together, variation in the translation status of individual transcripts reflects important mechanisms of gene regulation, which may have a role in evolution and diversification. PMID:27852012

  13. Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes.

    PubMed

    Jakob, Steffen; Ohmayer, Uli; Neueder, Andreas; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Hochmuth, Eduard; Deutzmann, Rainer; Griesenbeck, Joachim; Tschochner, Herbert; Milkereit, Philipp

    2012-01-01

    Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.

  14. Ribosomal protein methyltransferases in the yeast Saccharomyces cerevisiae: Roles in ribosome biogenesis and translation.

    PubMed

    Al-Hadid, Qais; White, Jonelle; Clarke, Steven

    2016-02-12

    A significant percentage of the methyltransferasome in Saccharomyces cerevisiae and higher eukaryotes is devoted to methylation of the translational machinery. Methylation of the RNA components of the translational machinery has been studied extensively and is important for structure stability, ribosome biogenesis, and translational fidelity. However, the functional effects of ribosomal protein methylation by their cognate methyltransferases are still largely unknown. Previous work has shown that the ribosomal protein Rpl3 methyltransferase, histidine protein methyltransferase 1 (Hpm1), is important for ribosome biogenesis and translation elongation fidelity. In this study, yeast strains deficient in each of the ten ribosomal protein methyltransferases in S. cerevisiae were examined for potential defects in ribosome biogenesis and translation. Like Hpm1-deficient cells, loss of four of the nine other ribosomal protein methyltransferases resulted in defects in ribosomal subunit synthesis. All of the mutant strains exhibited resistance to the ribosome inhibitors anisomycin and/or cycloheximide in plate assays, but not in liquid culture. Translational fidelity assays measuring stop codon readthrough, amino acid misincorporation, and programmed -1 ribosomal frameshifting, revealed that eight of the ten enzymes are important for translation elongation fidelity and the remaining two are necessary for translation termination efficiency. Altogether, these results demonstrate that ribosomal protein methyltransferases in S. cerevisiae play important roles in ribosome biogenesis and translation.

  15. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

    PubMed

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

  16. The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

    PubMed Central

    Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A.; Hanson, Johannes; Meyer, Christian

    2016-01-01

    Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase. PMID:27877176

  17. Hold on to your friends: Dedicated chaperones of ribosomal proteins: Dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation.

    PubMed

    Pillet, Benjamin; Mitterer, Valentin; Kressler, Dieter; Pertschy, Brigitte

    2017-01-01

    Eukaryotic ribosomes are assembled from their components, the ribosomal RNAs and ribosomal proteins, in a tremendously complex, multi-step process, which primarily takes place in the nuclear compartment. Therefore, most ribosomal proteins have to travel from the cytoplasm to their incorporation site on pre-ribosomes within the nucleus. However, due to their particular characteristics, such as a highly basic amino acid composition and the presence of unstructured extensions, ribosomal proteins are especially prone to aggregation and degradation in their unassembled state, hence specific mechanisms must operate to ensure their safe delivery. Recent studies have uncovered a group of proteins, termed dedicated chaperones, specialized in accompanying and guarding individual ribosomal proteins. In this essay, we review how these dedicated chaperones utilize different folds to interact with their ribosomal protein clients and how they ensure their soluble expression and interconnect their intracellular transport with their efficient assembly into pre-ribosomes.

  18. Acidic Ribosomal Proteins from the Extreme ’Halobacterium cutirubrum’,

    DTIC Science & Technology

    the extreme halophilic bacterium, Halobacterium cutirubrum. The identification of the protein moieties involved in these and other interactions in...the halophile ribosome requires a rapid and reproducible screening method for the separation, enumeration and identification of these acidic...polypeptides in the complex ribosomal protein mixtures. In this paper the authors present the results of analyses of the halophile ribosomal proteins using a

  19. Insights into the Mechanism of Ribosomal Incorporation of Mammalian L13a Protein during Ribosome Biogenesis

    PubMed Central

    Das, Priyanka; Basu, Abhijit; Biswas, Aditi; Poddar, Darshana; Andrews, Joel; Barik, Sailen; Komar, Anton A.

    2013-01-01

    In contrast to prokaryotes, the precise mechanism of incorporation of ribosomal proteins into ribosomes in eukaryotes is not well understood. For the majority of eukaryotic ribosomal proteins, residues critical for rRNA binding, a key step in the hierarchical assembly of ribosomes, have not been well defined. In this study, we used the mammalian ribosomal protein L13a as a model to investigate the mechanism(s) underlying eukaryotic ribosomal protein incorporation into ribosomes. This work identified the arginine residue at position 68 of L13a as being essential for L13a binding to rRNA and incorporation into ribosomes. We also demonstrated that incorporation of L13a takes place during maturation of the 90S preribosome in the nucleolus, but that translocation of L13a into the nucleolus is not sufficient for its incorporation into ribosomes. Incorporation of L13a into the 90S preribosome was required for rRNA methylation within the 90S complex. However, mutations abolishing ribosomal incorporation of L13a did not affect its ability to be phosphorylated or its extraribosomal function in GAIT element-mediated translational silencing. These results provide new insights into the mechanism of ribosomal incorporation of L13a and will be useful in guiding future studies aimed at fully deciphering mammalian ribosome biogenesis. PMID:23689135

  20. Translation initiation of viral mRNAs.

    PubMed

    López-Lastra, Marcelo; Ramdohr, Pablo; Letelier, Alejandro; Vallejos, Maricarmen; Vera-Otarola, Jorge; Valiente-Echeverría, Fernando

    2010-05-01

    Viruses depend on cells for their replication but have evolved mechanisms to achieve this in an efficient and, in some instances, a cell-type-specific manner. The expression of viral proteins is frequently subject to translational control. The dominant target of such control is the initiation step of protein synthesis. Indeed, during the early stages of infection, viral mRNAs must compete with their host counterparts for the protein synthetic machinery, especially for the limited pool of eukaryotic translation initiation factors (eIFs) that mediate the recruitment of ribosomes to both viral and cellular mRNAs. To circumvent this competition viruses use diverse strategies so that ribosomes can be recruited selectively to viral mRNAs. In this review we focus on the initiation of protein synthesis and outline some of the strategies used by viruses to ensure efficient translation initiation of their mRNAs.

  1. Translating mRNAs strongly correlate to proteins in a multivariate manner and their translation ratios are phenotype specific.

    PubMed

    Wang, Tong; Cui, Yizhi; Jin, Jingjie; Guo, Jiahui; Wang, Guibin; Yin, Xingfeng; He, Qing-Yu; Zhang, Gong

    2013-05-01

    As a well-known phenomenon, total mRNAs poorly correlate to proteins in their abundances as reported. Recent findings calculated with bivariate models suggested even poorer such correlation, whereas focusing on the translating mRNAs (ribosome nascent-chain complex-bound mRNAs, RNC-mRNAs) subset. In this study, we analysed the relative abundances of mRNAs, RNC-mRNAs and proteins on genome-wide scale, comparing human lung cancer A549 and H1299 cells with normal human bronchial epithelial (HBE) cells, respectively. As discovered, a strong correlation between RNC-mRNAs and proteins in their relative abundances could be established through a multivariate linear model by integrating the mRNA length as a key factor. The R(2) reached 0.94 and 0.97 in A549 versus HBE and H1299 versus HBE comparisons, respectively. This correlation highlighted that the mRNA length significantly contributes to the translational modulation, especially to the translational initiation, favoured by its correlation with the mRNA translation ratio (TR) as observed. We found TR is highly phenotype specific, which was substantiated by both pathway analysis and biased TRs of the splice variants of BDP1 gene, which is a key transcription factor of transfer RNAs. These findings revealed, for the first time, the intrinsic and genome-wide translation modulations at translatomic level in human cells at steady-state, which are tightly correlated to the protein abundance and functionally relevant to cellular phenotypes.

  2. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  3. TSC1 Sets the Rate of Ribosome Export and Protein Synthesis through Nucleophosmin Translation

    PubMed Central

    Pelletier, Corey L.; Maggi, Leonard B.; Brady, Suzanne N.; Scheidenhelm, Danielle K.; Gutmann, David H.; Weber, Jason D.

    2008-01-01

    Nucleophosmin (B23) is a nucleolar phosphoprotein that has been implicated in numerous cellular processes. In particular, nucleophosmin interacts with nucleolar components of newly synthesized ribosomes to promote ribosome nuclear export. Nucleophosmin is a classic mitogen-induced protein, with changes in its expression correlating with growth factor stimulation. In this study, we examined the underlying mechanism of nucleophosmin induction and showed that hyperproliferative signals emanating from oncogenic H-RasV12 cause tremendous increases in nucleophosmin protein expression. Nucleophosmin protein accumulation was dependent on mammalian target of rapamycin (mTOR) activation, as rapamycin completely prevented nucleophosmin induction. Consistent with this finding, genetic ablation of Tsc1, a major upstream inhibitor of mTOR, resulted in nucleophosmin protein induction through increased translation of existing nucleophosmin mRNAs. Increases in nucleophosmin protein accumulation were suppressed by reintroduction of TSC1. Induction of nucleophosmin through Tsc1 loss resulted in a greater pool of actively translating ribosomes in the cytoplasm, higher overall rates of protein synthesis, and increased cell proliferation, all of which were dependent on efficient nucleophosmin nuclear export. Nucleophosmin protein accumulation in the absence of Tsc1 promoted the nuclear export of maturing ribosome subunits, providing a mechanistic link between TSC1/mTOR signaling, nucleophosmin-mediated nuclear export of ribosome subunits, protein synthesis levels, and cell growth. PMID:17308101

  4. Selective translational control and nonspecific posttranscriptional regulation of ribosomal protein gene expression during development and regeneration of rat liver.

    PubMed Central

    Aloni, R; Peleg, D; Meyuhas, O

    1992-01-01

    Mammalian liver development is accompanied by a transition from rapid growth in the fetus to a quiescent state in the adult. However, extensive proliferation can be induced in the adult liver by partial hepatectomy. In this study, we examined the regulation of ribosomal protein (rp) gene expression in the developing and regenerating rat liver. Our results indicate that the translation of rp mRNAs is selectively repressed by about 70% upon development from fetal to adult life, as illustrated by the decrease in ribosomal loading. In addition, the relative abundance of these mRNAs, like that of several other, but not all, housekeeping mRNAs, declines during development through a posttranscriptional mechanism. When liver cells commence growth following partial hepatectomy, translation of rp mRNAs is resumed to near-maximal capacity, as judged by their very efficient recruitment into polysomes. The concomitant increase in the abundance rp mRNAs under these circumstances is achieved by a posttranscriptional mechanism. The apparent fluctuations in the translation efficiency of rp mRNAs are accompanied by parallel changes in the expression of the genes encoding the initiation factors eIF-4E and eIF-4A. Our results indicate that selective translational control of rp mRNAs in mammals is not confined to manipulated cells in culture but constitutes an important regulatory mechanism operating in vivo in the course of liver development and regeneration. Images PMID:1373810

  5. Functional signature for the recognition of specific target mRNAs by human Staufen1 protein

    PubMed Central

    de Lucas, Susana; Oliveros, Juan Carlos; Chagoyen, Mónica; Ortín, Juan

    2014-01-01

    Cellular messenger RNAs (mRNAs) are associated to proteins in the form of ribonucleoprotein particles. The double-stranded RNA-binding (DRB) proteins play important roles in mRNA synthesis, modification, activity and decay. Staufen is a DRB protein involved in the localized translation of specific mRNAs during Drosophila early development. The human Staufen1 (hStau1) forms RNA granules that contain translation regulation proteins as well as cytoskeleton and motor proteins to allow the movement of the granule on microtubules, but the mechanisms of hStau1-RNA recognition are still unclear. Here we used a combination of affinity chromatography, RNAse-protection, deep-sequencing and bioinformatic analyses to identify mRNAs differentially associated to hStau1 or a mutant protein unable to bind RNA and, in this way, defined a collection of mRNAs specifically associated to wt hStau1. A common sequence signature consisting of two opposite-polarity Alu motifs was present in the hStau1-associated mRNAs and was shown to be sufficient for binding to hStau1 and hStau1-dependent stimulation of protein expression. Our results unravel how hStau1 identifies a wide spectrum of cellular target mRNAs to control their localization, expression and fate. PMID:24470147

  6. Potential extra-ribosomal functions of ribosomal proteins in Saccharomyces cerevisiae.

    PubMed

    Lu, Hui; Zhu, Yi-Fei; Xiong, Juan; Wang, Rong; Jia, Zhengping

    2015-08-01

    Ribosomal proteins (RPs), are essential components of the ribosomes, the molecular machines that turn mRNA blueprints into proteins, as they serve to stabilize the structure of the rRNA, thus improving protein biosynthesis. In addition, growing evidence suggests that RPs can function in other cellular roles. In the present review, we summarize several potential extra-ribosomal functions of RPs in ribosomal biogenesis, transcription activity, translation process, DNA repair, replicative life span, adhesive growth, and morphological transformation in Saccharomyces cerevisiae. However, the future in-depth studies are needed to identify these novel secondary functions of RPs in S. cerevisiae.

  7. RPG: the Ribosomal Protein Gene database

    PubMed Central

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes. PMID:14681386

  8. RPG: the Ribosomal Protein Gene database.

    PubMed

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

  9. Isolation of Mitochondrial Ribosomes.

    PubMed

    Carroll, Adam J

    2017-01-01

    Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

  10. Stepwise splitting of ribosomal proteins from yeast ribosomes by LiCl.

    PubMed

    Piir, Kerli; Tamm, Tiina; Kisly, Ivan; Tammsalu, Triin; Remme, Jaanus

    2014-01-01

    Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5-1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.

  11. Antibiotic-induced ribosomal assembly defects result from changes in the synthesis of ribosomal proteins.

    PubMed

    Siibak, Triinu; Peil, Lauri; Dönhöfer, Alexandra; Tats, Age; Remm, Maido; Wilson, Daniel N; Tenson, Tanel; Remme, Jaanus

    2011-04-01

    Inhibitors of protein synthesis cause defects in the assembly of ribosomal subunits. In response to treatment with the antibiotics erythromycin or chloramphenicol, precursors of both large and small ribosomal subunits accumulate. We have used a pulse-labelling approach to demonstrate that the accumulating subribosomal particles maturate into functional 70S ribosomes. The protein content of the precursor particles is heterogeneous and does not correspond with known assembly intermediates. Mass spectrometry indicates that production of ribosomal proteins in the presence of the antibiotics correlates with the amounts of the individual ribosomal proteins within the precursor particles. Thus, treatment of cells with chloramphenicol or erythromycin leads to an unbalanced synthesis of ribosomal proteins, providing the explanation for formation of assembly-defective particles. The operons for ribosomal proteins show a characteristic pattern of antibiotic inhibition where synthesis of the first proteins is inhibited weakly but gradually increases for the subsequent proteins in the operon. This phenomenon most likely reflects translational coupling and allows us to identify other putative coupled non-ribosomal operons in the Escherichia coli chromosome. © 2011 Blackwell Publishing Ltd.

  12. Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.

    PubMed

    Márquez, Viter; Fröhlich, Thomas; Armache, Jean-Paul; Sohmen, Daniel; Dönhöfer, Alexandra; Mikolajka, Aleksandra; Berninghausen, Otto; Thomm, Michael; Beckmann, Roland; Arnold, Georg J; Wilson, Daniel N

    2011-02-04

    Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.

  13. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA

    PubMed Central

    Pathak, Bani K.; Mondal, Surojit; Banerjee, Senjuti; Ghosh, Amar Nath; Barat, Chandana

    2017-01-01

    An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our in vitro studies unexpectedly reveal that the partial unfolding or aggregation of lysozyme under reducing conditions in presence of the ribosome can induce aggregation of ribosomal components. Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity. PMID:28169307

  14. Functional analysis of Saccharomyces cerevisiae ribosomal protein Rpl3p in ribosome synthesis

    PubMed Central

    Rosado, Iván V.; Kressler, Dieter; de la Cruz, Jesús

    2007-01-01

    Ribosome synthesis in eukaryotes requires a multitude of trans-acting factors. These factors act at many steps as the pre-ribosomal particles travel from the nucleolus to the cytoplasm. In contrast to the well-studied trans-acting factors, little is known about the contribution of the ribosomal proteins to ribosome biogenesis. Herein, we have analysed the role of ribosomal protein Rpl3p in 60S ribosomal subunit biogenesis. In vivo depletion of Rpl3p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. This phenotype is likely due to the instability of early and intermediate pre-ribosomal particles, as evidenced by the low steady-state levels of 27SA3, 27SBS and 7SL/S precursors. Furthermore, depletion of Rpl3p impairs the nucleocytoplasmic export of pre-60S ribosomal particles. Interestingly, flow cytometry analysis indicates that Rpl3p-depleted cells arrest in the G1 phase. Altogether, we suggest that upon depletion of Rpl3p, early assembly of 60S ribosomal subunits is aborted and subsequent steps during their maturation and export prevented. PMID:17569673

  15. Convergent evolution led ribosome inactivating proteins to interact with ribosomal stalk.

    PubMed

    Lapadula, Walter J; Sanchez-Puerta, M Virginia; Ayub, Maximiliano Juri

    2012-03-01

    Ribosome-inactivating proteins (RIPs) inhibit protein synthesis by depurinating an adenine on the sarcin-ricin loop (SRL) of the large subunit ribosomal RNA. Several RIPs interact with the C-terminal end of ribosomal stalk P proteins, and this interaction is required for their full activity. In contrast, the activity of Pokeweed Antiviral Protein is not affected by blocking this stalk component. Here, we provide evidence from phylogenetic analyses and sequence alignments suggesting that the interaction with the C-terminal end of P proteins evolved independently in different RIPs by convergent evolution.

  16. Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron.

    PubMed Central

    Christopher, D A; Hallick, R B

    1990-01-01

    We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons. We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit. The primary transcript also contains at least 15 group II and group III introns. Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases. This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns. Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA. A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon. Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized. The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date. The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases. PMID:2136640

  17. Ribosomal History Reveals Origins of Modern Protein Synthesis

    PubMed Central

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world. PMID:22427882

  18. Ribosomal history reveals origins of modern protein synthesis.

    PubMed

    Harish, Ajith; Caetano-Anollés, Gustavo

    2012-01-01

    The origin and evolution of the ribosome is central to our understanding of the cellular world. Most hypotheses posit that the ribosome originated in the peptidyl transferase center of the large ribosomal subunit. However, these proposals do not link protein synthesis to RNA recognition and do not use a phylogenetic comparative framework to study ribosomal evolution. Here we infer evolution of the structural components of the ribosome. Phylogenetic methods widely used in morphometrics are applied directly to RNA structures of thousands of molecules and to a census of protein structures in hundreds of genomes. We find that components of the small subunit involved in ribosomal processivity evolved earlier than the catalytic peptidyl transferase center responsible for protein synthesis. Remarkably, subunit RNA and proteins coevolved, starting with interactions between the oldest proteins (S12 and S17) and the oldest substructure (the ribosomal ratchet) in the small subunit and ending with the rise of a modern multi-subunit ribosome. Ancestral ribonucleoprotein components show similarities to in vitro evolved RNA replicase ribozymes and protein structures in extant replication machinery. Our study therefore provides important clues about the chicken-or-egg dilemma associated with the central dogma of molecular biology by showing that ribosomal history is driven by the gradual structural accretion of protein and RNA structures. Most importantly, results suggest that functionally important and conserved regions of the ribosome were recruited and could be relics of an ancient ribonucleoprotein world.

  19. La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

    PubMed

    Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

    2017-04-07

    The 5'terminal oligopyrimidine (5'TOP) motif is a cis-regulatory RNA element located immediately downstream of the 7-methylguanosine [m(7)G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m(7)GTP), and a capped cytidine (m(7)GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

  20. Ribosome-Inactivating and Related Proteins

    PubMed Central

    Schrot, Joachim; Weng, Alexander; Melzig, Matthias F.

    2015-01-01

    Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs. PMID:26008228

  1. Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins*

    PubMed Central

    Moore, Graham; Crichton, Robert R.

    1974-01-01

    Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex. PMID:4462744

  2. On the Ribosomal Density that Maximizes Protein Translation Rate

    PubMed Central

    Zarai, Yoram; Margaliot, Michael; Tuller, Tamir

    2016-01-01

    During mRNA translation, several ribosomes attach to the same mRNA molecule simultaneously translating it into a protein. This pipelining increases the protein translation rate. A natural and important question is what ribosomal density maximizes the protein translation rate. Using mathematical models of ribosome flow along both a linear and a circular mRNA molecules we prove that typically the steady-state protein translation rate is maximized when the ribosomal density is one half of the maximal possible density. We discuss the implications of our results to endogenous genes under natural cellular conditions and also to synthetic biology. PMID:27861564

  3. Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs

    PubMed Central

    Boyne, James R; Jackson, Brian R; Taylor, Adam; Macnab, Stuart A; Whitehouse, Adrian

    2010-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts. PMID:20436455

  4. Ribosome Inactivating Proteins from an evolutionary perspective.

    PubMed

    Lapadula, Walter Jesús; Ayub, Maximiliano Juri

    2017-09-15

    Ribosome Inactivating Proteins (RIPs) are rRNA N-glycosidases that inhibit protein synthesis through the elimination of a single adenine residue from 28S rRNA. Many of these toxins have been characterized in depth from a biochemical and molecular point of view. In addition, their potential use in medicine as highly selective toxins is being explored. In contrast, the evolutionary history of RIP encoding genes has remained traditionally underexplored. In recent years, accumulation of large genomic data has fueled research on this issue and revealed unexpected information about the origin and evolution of RIP toxins. In this review we summarize the current evidence available on the occurrence of different evolutionary mechanisms (gene duplication and losses, horizontal gene transfer, synthesis de novo and domain combination) involved in the evolution of the RIP gene family. Finally, we propose a revised nomenclature for RIP genes based on their evolutionary history. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. The effect of ribosomal protein S1 from Escherichia coli and Micrococcus luteus on protein synthesis in vitro by E. coli and Bacillus subtilis.

    PubMed

    Farwell, M A; Roberts, M W; Rabinowitz, J C

    1992-11-01

    We have designed a set of nine plasmids containing the Bacillus pumilis cat gene with one of three Shine-Dalgarno (SD) sequences (weak, strong or stronger) and one of three initiation codons (AUG, GUG or UUG). These constructions have been used to determine the effect of ribosomal protein S1, SD and initiation codon sequences and Escherichia coli ribosomal protein S1 on translation in vitro by E. coli and B. subtilis ribosomes. Translation of these nine constructions was determined with three types of ribosomes: E. coli containing ribosomal protein S1, E. coli depleted of S1, and B. subtilis which is naturally free of S1. E. coli ribosomes were able to translate all nine transcripts with variable efficiencies. B. subtilis and S1-depleted E. coli ribosomes were similar to each other and differed from non-depleted E. coli ribosomes in that they required strong or stronger SD sequences and were unable to translate any of the weak transcripts. Addition of S1 from either E. coli or Micrococcus luteus, a Gram-positive bacterium, enabled S1-depleted E. coli ribosomes to translate mRNAs with weak SD sequences but had no effect on B. subtilis ribosomes. AUG was the preferred initiation codon for all ribosome types; however, B. subtilis ribosomes showed greater tolerance for the non-AUG codons than either type of E. coli ribosome. The presence of a strong or stronger SD sequence increased the efficiency by which E. coli ribosomes could utilize non-AUG codons.(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function.

    PubMed

    Babiano, Reyes; Gamalinda, Michael; Woolford, John L; de la Cruz, Jesús

    2012-08-01

    Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.

  7. In vitro read-through polysome/ribosome display of full-length protein ORF and it's applications.

    PubMed

    Ogawa, Atsushi; Sando, Shinsuke; Aoyama, Yasuhiro

    2005-01-01

    Combination of nonsense suppression and protein-ribosome-mRNA (PRM) complexation techniques leads to a new strategy "read-through polysome/ribosome display", which is designed to display full-length open reading frame (ORF) domain of the protein on the natural mRNA templates. The optimised conditions are to use the anticodon-adjusted tRNA for Leu as a nonsense suppressor in a reconstituted translation system containing diminished amounts of release factors (RFs). When applied to pseudo-natural mRNAs of Escherichia coli dihydrofolate reductase (E. coli DHFR), the input mRNA was recovered as a polysome complex displaying full-length DHFR.

  8. Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

    PubMed Central

    2016-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79–80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type–specific disorders that often transition from hypoproliferative to hyperproliferative growth. PMID:25706898

  9. Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

    PubMed

    de la Cruz, Jesús; Karbstein, Katrin; Woolford, John L

    2015-01-01

    The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

  10. Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes

    PubMed Central

    1985-01-01

    The stability and movement of several polyadenylated (poly A+) and nonpolyadenylated (poly A-) mRNAs in Xenopus oocytes have been examined. At least 50% of the poly A+ mRNA molecules (9S rabbit globin mRNA, chicken ovalbumin, and lysozyme) were stable in oocytes over a 48- h period, irrespective of the amount injected. About 50% of injected poly A- reovirus mRNAs was degraded within the first 24 h of injection, irrespective of the amount injected, although no further degradation was observed over an additional 24 h. The movement of all poly A+ mRNAs injected at either the animal or vegetal pole of the oocyte was very slow. Little movement of RNA from the animal half to the vegetal half was observed even 48 h after injection. In contrast, similar amounts of mRNA were present in both halves 48 h after vegetal pole injection. Similar results were obtained after injection of poly A- reovirus mRNAs. The movement of the proteins encoded by the poly A+ mRNAs was studied in the 6-h period after injection when little mRNA movement had occurred. 85% of the globin synthesized accumulated in the animal half irrespective of injection site. The movement of the sequestered secretory proteins ovalbumin and lysozyme in the same oocytes as globin was much slower; very little lysozyme appeared in the half of the oocyte opposite the site of injection. PMID:2858488

  11. Role of ribosomal protein mutations in tumor development (Review)

    PubMed Central

    GOUDARZI, KAVEH M.; LINDSTRÖM, MIKAEL S.

    2016-01-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  12. S1 ribosomal protein and the interplay between translation and mRNA decay

    PubMed Central

    Delvillani, Francesco; Papiani, Giulia; Dehò, Gianni; Briani, Federica

    2011-01-01

    S1 is an ‘atypical’ ribosomal protein weakly associated with the 30S subunit that has been implicated in translation, transcription and control of RNA stability. S1 is thought to participate in translation initiation complex formation by assisting 30S positioning in the translation initiation region, but little is known about its role in other RNA transactions. In this work, we have analysed in vivo the effects of different intracellular S1 concentrations, from depletion to overexpression, on translation, decay and intracellular distribution of leadered and leaderless messenger RNAs (mRNAs). We show that the cspE mRNA, like the rpsO transcript, may be cleaved by RNase E at multiple sites, whereas the leaderless cspE transcript may also be degraded via an alternative pathway by an unknown endonuclease. Upon S1 overexpression, RNase E-dependent decay of both cspE and rpsO mRNAs is suppressed and these transcripts are stabilized, whereas cleavage of leaderless cspE mRNA by the unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and that part of the Escherichia coli ribosomes may actually lack S1. PMID:21685451

  13. Post-translational Modification of Ribosomal Proteins

    PubMed Central

    Arragain, Simon; Garcia-Serres, Ricardo; Blondin, Geneviève; Douki, Thierry; Clemancey, Martin; Latour, Jean-Marc; Forouhar, Farhad; Neely, Helen; Montelione, Gaetano T.; Hunt, John F.; Mulliez, Etienne; Fontecave, Marc; Atta, Mohamed

    2010-01-01

    Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family. PMID:20007320

  14. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

    PubMed Central

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-01-01

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. DOI: http://dx.doi.org/10.7554/eLife.03473.001 PMID:25144938

  15. A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

    PubMed

    Schütz, Sabina; Fischer, Ute; Altvater, Martin; Nerurkar, Purnima; Peña, Cohue; Gerber, Michaela; Chang, Yiming; Caesar, Stefanie; Schubert, Olga T; Schlenstedt, Gabriel; Panse, Vikram G

    2014-08-21

    Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers-termed here escortins-to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles. Copyright © 2014, Schütz et al.

  16. Ribosome recycling: An essential process of protein synthesis.

    PubMed

    Kiel, Michael C; Kaji, Hideko; Kaji, Akira

    2007-01-01

    A preponderance of textbooks outlines cellular protein synthesis (translation) in three basic steps: initiation, elongation, and termination. However, researchers in the field of translation accept that a vital fourth step exists; this fourth step is called ribosome recycling. Ribosome recycling occurs after the nascent polypeptide has been released during the termination step. Despite the release of the polypeptide, ribosomes remain bound to the mRNA and tRNA. It is only during the fourth step of translation that ribosomes are ultimately released from the mRNA, split into subunits, and are free to bind new mRNA, thus the term "ribosome recycling." This step is essential to the viability of cells. In bacteria, it is catalyzed by two proteins, elongation factor G and ribosome recycling factor, a near perfect structural mimic of tRNA. Eukaryotic organelles such as mitochondria and chloroplasts possess ribosome recycling factor and elongation factor G homologues, but the nature of ribosome recycling in eukaryotic cytoplasm is still under investigation. In this review, the discovery of ribosome recycling and the basic mechanisms involved are discussed so that textbook writers and teachers can include this vital step, which is just as important as the three conventional steps, in sections dealing with protein synthesis.

  17. Ribosomal protein S1 unwinds double-stranded RNA in multiple steps

    PubMed Central

    Qu, Xiaohui; Lancaster, Laura; Noller, Harry F.; Bustamante, Carlos; Tinoco, Ignacio

    2012-01-01

    The sequence and secondary structure of the 5′-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5′-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure (“stand-by site”) before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode. PMID:22908248

  18. Molecular mechanisms of ribosomal protein gene coregulation

    PubMed Central

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B. Franklin

    2015-01-01

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20–50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1–TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. PMID:26385964

  19. Molecular mechanisms of ribosomal protein gene coregulation.

    PubMed

    Reja, Rohit; Vinayachandran, Vinesh; Ghosh, Sujana; Pugh, B Franklin

    2015-09-15

    The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair resolution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20-50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1-TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. © 2015 Reja et al.; Published by Cold Spring Harbor Laboratory Press.

  20. Prefabrication of a ribosomal protein subcomplex essential for eukaryotic ribosome formation

    PubMed Central

    Peña, Cohue; Schütz, Sabina; Fischer, Ute; Chang, Yiming; Panse, Vikram G

    2016-01-01

    Spatial clustering of ribosomal proteins (r-proteins) through tertiary interactions is a striking structural feature of the eukaryotic ribosome. However, the functional importance of these intricate inter-connections, and how they are established is currently unclear. Here, we reveal that a conserved ATPase, Fap7, organizes interactions between neighboring r-proteins uS11 and eS26 prior to their delivery to the earliest ribosome precursor, the 90S. In vitro, uS11 only when bound to Fap7 becomes competent to recruit eS26 through tertiary contacts found between these r-proteins on the mature ribosome. Subsequently, Fap7 ATPase activity unloads the uS11:eS26 subcomplex onto its rRNA binding site, and therefore ensures stoichiometric integration of these r-proteins into the 90S. Fap7-depletion in vivo renders uS11 susceptible to proteolysis, and precludes eS26 incorporation into the 90S. Thus, prefabrication of a native-like r-protein subcomplex drives efficient and accurate construction of the eukaryotic ribosome. DOI: http://dx.doi.org/10.7554/eLife.21755.001 PMID:27929371

  1. Single Molecule Force Measurement for Protein Synthesis on the Ribosome

    NASA Astrophysics Data System (ADS)

    Uemura, Sotaro

    2008-04-01

    The ribosome is a molecular machine that translates the genetic code described on the messenger RNA (mRNA) into an amino acid sequence through repetitive cycles of transfer RNA (tRNA) selection, peptide bond formation and translocation. Although the detailed interactions between the translation components have been revealed by extensive structural and biochemical studies, it is not known how the precise regulation of macromolecular movements required at each stage of translation is achieved. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence. The removal of the SD sequence significantly reduced the rupture force, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA in a pre-peptidyl transfer state. In contrast, the post-peptidyl transfer state weakened the rupture force as compared to the complex in a pre-peptidyl transfer state and it was the same for both the SD-containing and SD-deficient mRNAs. The results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step. In this article, we discuss about the above new results including the introduction of the ribosome translation mechanism and the optical tweezer method.

  2. The RNA recognition motif protein CP33A is a global ligand of chloroplast mRNAs and is essential for plastid biogenesis and plant development.

    PubMed

    Teubner, Marlene; Fuß, Janina; Kühn, Kristina; Krause, Kirsten; Schmitz-Linneweber, Christian

    2017-02-01

    Chloroplast RNA metabolism depends on a multitude of nuclear-encoded RNA-binding proteins (RBPs). Most known chloroplast RBPs address specific RNA targets and RNA-processing functions. However, members of the small chloroplast ribonucleoprotein family (cpRNPs) play a global role in processing and stabilizing chloroplast RNAs. Here, we show that the cpRNP CP33A localizes to a distinct sub-chloroplastic domain and is essential for chloroplast development. The loss of CP33A yields albino seedlings that exhibit aberrant leaf development and can only survive in the presence of an external carbon source. Genome-wide RNA association studies demonstrate that CP33A associates with all chloroplast mRNAs. For a given transcript, quantification of CP33A-bound versus free RNAs demonstrates that CP33A associates with the majority of most mRNAs analyzed. Our results further show that CP33A is required for the accumulation of a number of tested mRNAs, and is particularly relevant for unspliced and unprocessed precursor mRNAs. Finally, CP33A fails to associate with polysomes or to strongly co-precipitate with ribosomal RNA, suggesting that it defines a ribodomain that is separate from the chloroplast translation machinery. Collectively, these findings suggest that CP33A contributes to globally essential RNA processes in the chloroplasts of higher plants.

  3. Cotranslational Protein Folding inside the Ribosome Exit Tunnel.

    PubMed

    Nilsson, Ola B; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D; O'Brien, Edward P; Beckmann, Roland; von Heijne, Gunnar

    2015-09-08

    At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Cotranslational Protein Folding inside the Ribosome Exit Tunnel

    PubMed Central

    Nilsson, Ola B.; Hedman, Rickard; Marino, Jacopo; Wickles, Stephan; Bischoff, Lukas; Johansson, Magnus; Müller-Lucks, Annika; Trovato, Fabio; Puglisi, Joseph D.; O’Brien, Edward P.; Beckmann, Roland; von Heijne, Gunnar

    2015-01-01

    Summary At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins. PMID:26321634

  5. Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits.

    PubMed

    O'Donohue, Marie-Françoise; Choesmel, Valérie; Faubladier, Marlène; Fichant, Gwennaële; Gleizes, Pierre-Emmanuel

    2010-09-06

    Our knowledge of the functions of metazoan ribosomal proteins in ribosome synthesis remains fragmentary. Using siRNAs, we show that knockdown of 31 of the 32 ribosomal proteins of the human 40S subunit (ribosomal protein of the small subunit [RPS]) strongly affects pre-ribosomal RNA (rRNA) processing, which often correlates with nucleolar chromatin disorganization. 16 RPSs are strictly required for initiating processing of the sequences flanking the 18S rRNA in the pre-rRNA except at the metazoan-specific early cleavage site. The remaining 16 proteins are necessary for progression of the nuclear and cytoplasmic maturation steps and for nuclear export. Distribution of these two subsets of RPSs in the 40S subunit structure argues for a tight dependence of pre-rRNA processing initiation on the folding of both the body and the head of the forming subunit. Interestingly, the functional dichotomy of RPS proteins reported in this study is correlated with the mutation frequency of RPS genes in Diamond-Blackfan anemia.

  6. Ribosomal Protein S14 Unties the MDM2-p53 Loop Upon Ribosomal Stress

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Zhang, Qi; Lu, Hua

    2013-01-01

    The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26, or RPS7, have been shown to play a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, like RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 play distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress. PMID:22391559

  7. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing

    PubMed Central

    Hang, Runlai; Liu, Chunyan; Ahmad, Ayaz; Zhang, Yong; Lu, Falong; Cao, Xiaofeng

    2014-01-01

    Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis. PMID:25352672

  8. Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

    PubMed

    Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

    2016-01-22

    It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

  9. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  10. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomes.

    PubMed

    Soung, George Y; Miller, Jennifer L; Koc, Hasan; Koc, Emine C

    2009-07-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in 24 Escherichia coli ribosomal proteins by tandem mass spectrometry. Detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining, and antibodies for phospho-Ser, Thr, and Tyr; or by mass spectrometry equipped with a capability to detect addition and loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, and L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given phosphorylation sites in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins.

  11. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli

    PubMed Central

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-01-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover. PMID:18824515

  12. Polynucleotide phosphorylase hinders mRNA degradation upon ribosomal protein S1 overexpression in Escherichia coli.

    PubMed

    Briani, Federica; Curti, Serena; Rossi, Francesca; Carzaniga, Thomas; Mauri, Pierluigi; Dehò, Gianni

    2008-11-01

    The exoribonuclease polynucleotide phosphorylase (PNPase, encoded by pnp) is a major player in bacterial RNA decay. In Escherichia coli, PNPase expression is post-transcriptionally regulated at the level of mRNA stability. The primary transcript is very efficiently processed by the endonuclease RNase III at a specific site and the processed pnp mRNA is rapidly degraded in a PNPase-dependent manner. While investigating the PNPase autoregulation mechanism we found, by UV-cross-linking experiments, that the ribosomal protein S1 in crude extracts binds to the pnp-mRNA leader region. We assayed the potential role of S1 protein in pnp gene regulation by modulating S1 expression from depletion to overexpression. We found that S1 depletion led to a sharp decrease of the amount of pnp and other tested mRNAs, as detected by Northern blotting, whereas S1 overexpression caused a strong stabilization of pnp and the other transcripts. Surprisingly, mRNA stabilization depended on PNPase, as it was not observed in a pnp deletion strain. PNPase-dependent stabilization, however, was not detected by chemical decay assay of bulk mRNA. Overall, our data suggest that PNPase exonucleolytic activity may be modulated by the translation potential of the target mRNAs and that, upon ribosomal protein S1 overexpression, PNPase protects from degradation a set of full-length mRNAs. It thus appears that a single mRNA species may be differentially targeted to either decay or PNPase-dependent stabilization, thus preventing its depletion in conditions of fast turnover.

  13. Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

    PubMed Central

    Davis, Joseph H.; Williamson, James R.; Britton, Robert A.

    2014-01-01

    RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins. PMID:25330043

  14. Ribosome biogenesis; the KsgA protein throws a methyl-mediated switch in ribosome assembly.

    PubMed

    Mangat, Chand S; Brown, Eric D

    2008-12-01

    Many trans-acting factors that aid in ribosome biogenesis have been identified in higher organisms but relatively few such factors are known in prokaryotes. In bacteria, the list of such factors includes ATP-energized helicases and chaperones as well as an emerging cadre of switch GTPases. The KsgA protein is a universally conserved methyltransferase that dimethylates both A1518 and A1519 of the 16S rRNA of the small ribosomal subunit. Methylation has long been thought to be solely for fine-tuning of protein translation. In this issue of Molecular Microbiology, Connolly et al. present data suggesting KsgA might function in the assembly of the small subunit of the ribosome. Indeed, the work indicates that KsgA might have a checkpoint role in ribosome biogenesis where methylation by this protein marks the completion of its assembly role. These findings open our thinking to new candidate assembly factors and provide a new direction for understanding ribosome assembly.

  15. Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly.

    PubMed

    Gulati, Megha; Jain, Nikhil; Davis, Joseph H; Williamson, James R; Britton, Robert A

    2014-10-01

    RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.

  16. Fragile X mental retardation protein FMRP binds mRNAs in the nucleus.

    PubMed

    Kim, Miri; Bellini, Michel; Ceman, Stephanie

    2009-01-01

    The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

  17. Functional Specialization of Ribosomes?

    PubMed Central

    Gilbert, Wendy V.

    2011-01-01

    Ribosomes are highly conserved macromolecular machines responsible for protein synthesis in all living organisms. Work published in the past year shows that changes to the ribosome core can affect the mechanism of translation initiation that is favored in the cell, potentially leading to specific changes in the relative efficiencies with which different proteins are made. Here I examine recent data from expression and proteomic studies suggesting that cells make slightly different ribosomes under different growth conditions and discuss genetic evidence that such differences are functional. In particular, I will argue that eukaryotic cells likely produce ribosomes that lack one or more ‘core’ ribosomal proteins (RPs) under some conditions, and that ‘core’ RPs contribute differentially to translation of distinct subpopulations of mRNAs. PMID:21242088

  18. Bypassing of stems versus linear base-by-base inspection of mammalian mRNAs during ribosomal scanning.

    PubMed

    Abaeva, Irina S; Marintchev, Assen; Pisareva, Vera P; Hellen, Christopher U T; Pestova, Tatyana V

    2011-01-05

    Initiation codon selection in eukaryotes involves base-by-base inspection of the 5'-untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA-binding cleft in a single-stranded form and therefore undergoes base-by-base inspection during scanning.

  19. Ribosome-inactivating proteins from plant cells in culture.

    PubMed Central

    Barbieri, L; Bolognesi, A; Cenini, P; Falasca, A I; Minghetti, A; Garofano, L; Guicciardi, A; Lappi, D; Miller, S P; Stirpe, F

    1989-01-01

    1. Ribosome-inactivating proteins were found in high amounts in one line of cells of Phytolacca americana (pokeweed) cultured in vitro and, in less quantity, in lines of Saponaria officinalis (soapwort) and of Zea mays (corn) cells. 2. The main ribosome-inactivating protein from pokeweed cells was purified to homogeneity. It is a protein with Mr 29,000 and basic pI, similar to the 'pokeweed antiviral protein' (PAP), a ribosome-inactivating protein from pokeweed leaves. We propose to call the pokeweed antiviral protein isolated from pokeweed cells PAP-C. 3. PAP-C inactivates ribosomes in a less-than-equimolar ratio, thus inhibiting protein synthesis by a rabbit reticulocyte lysate with an IC50 (concentration causing 50% inhibition) of 0.067 nM (2 ng/ml), and modifies rRNA in a manner apparently identical to that of ricin and other ribosome-inactivating proteins. It inhibits protein synthesis by intact cells with an IC50 of 0.7-3.4 microM, and is toxic to mice with an LD50 of 0.95 mg/kg. Images Fig. 1. Fig. 3. Fig. 4. PMID:2930487

  20. Structures of Eukaryotic Ribosomal Stalk Proteins and Its Complex with Trichosanthin, and Their Implications in Recruiting Ribosome-Inactivating Proteins to the Ribosomes

    PubMed Central

    Choi, Andrew K. H.; Wong, Eddie C. K.; Lee, Ka-Ming; Wong, Kam-Bo

    2015-01-01

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA. PMID:25723321

  1. Structures of eukaryotic ribosomal stalk proteins and its complex with trichosanthin, and their implications in recruiting ribosome-inactivating proteins to the ribosomes.

    PubMed

    Choi, Andrew K H; Wong, Eddie C K; Lee, Ka-Ming; Wong, Kam-Bo

    2015-02-25

    Ribosome-inactivating proteins (RIP) are RNA N-glycosidases that inactivate ribosomes by specifically depurinating a conserved adenine residue at the α-sarcin/ricin loop of 28S rRNA. Recent studies have pointed to the involvement of the C-terminal domain of the eukaryotic stalk proteins in facilitating the toxic action of RIPs. This review highlights how structural studies of eukaryotic stalk proteins provide insights into the recruitment of RIPs to the ribosomes. Since the C-terminal domain of eukaryotic stalk proteins is involved in specific recognition of elongation factors and some eukaryote-specific RIPs (e.g., trichosanthin and ricin), we postulate that these RIPs may have evolved to hijack the translation-factor-recruiting function of ribosomal stalk in reaching their target site of rRNA.

  2. Shwachman–Bodian–Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs

    PubMed Central

    In, Kyungmin; Zaini, Mohamad A.; Müller, Christine; Warren, Alan J.; von Lindern, Marieke; Calkhoven, Cornelis F.

    2016-01-01

    Mutations in the Shwachman–Bodian–Diamond Syndrome (SBDS) gene cause Shwachman–Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5′ untranslated regions (5′ UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. PMID:26762974

  3. Shwachman-Bodian-Diamond syndrome (SBDS) protein deficiency impairs translation re-initiation from C/EBPα and C/EBPβ mRNAs.

    PubMed

    In, Kyungmin; Zaini, Mohamad A; Müller, Christine; Warren, Alan J; von Lindern, Marieke; Calkhoven, Cornelis F

    2016-05-19

    Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -β mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPβ-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -β deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Translational control of ribosomal protein synthesis during early Dictyostelium discoideum development.

    PubMed Central

    Steel, L F; Jacobson, A

    1987-01-01

    Throughout the developmental program of Dictyostelium discoideum there are substantial changes in the rates of both ribosome utilization and rRNA transcription and processing. We examined the regulation of ribosomal protein (r-protein) gene expression and found that, at the start of development, expression of these genes was drastically and specifically reduced by a block to translational initiation. An apparently separate event signals a sudden decrease in the relative amount of r-protein mRNA at about 10 h of development, a time when aggregated amoebae are forming tight cell-cell contacts. For the first 9 h of development, the relative amount of r-protein mRNA remained essentially unchanged and comparable to levels detected in growing cells. While the r-protein mRNAs were almost fully loaded on polysomes during vegetative growth, they were specifically excluded from polysomes at the start of development. The translational block was not the result of irreversible structural changes which inactivate the r-protein mRNAs since they remained translatable both in vitro, in wheat germ extracts, and in vivo, where they were recruited onto polysomes in the presence of the elongation inhibitor cycloheximide. In addition, precise measurements of poly(A) tail lengths on individual hybrid-selected mRNA species showed that there is no difference in the poly(A) tail length of r-protein mRNA isolated from growing cells and 1-h developing cells. Therefore, changes in translational efficiency cannot be attributed to cleavage of poly(A) tails. Images PMID:2882416

  5. Cryo-EM structure of the spinach chloroplast ribosome reveals the location of plastid-specific ribosomal proteins and extensions.

    PubMed

    Graf, Michael; Arenz, Stefan; Huter, Paul; Dönhöfer, Alexandra; Nováček, Jiří; Wilson, Daniel N

    2016-12-15

    Ribosomes are the protein synthesizing machines of the cell. Recent advances in cryo-EM have led to the determination of structures from a variety of species, including bacterial 70S and eukaryotic 80S ribosomes as well as mitoribosomes from eukaryotic mitochondria, however, to date high resolution structures of plastid 70S ribosomes have been lacking. Here we present a cryo-EM structure of the spinach chloroplast 70S ribosome, with an average resolution of 5.4 Å for the small 30S subunit and 3.6 Å for the large 50S ribosomal subunit. The structure reveals the location of the plastid-specific ribosomal proteins (RPs) PSRP1, PSRP4, PSRP5 and PSRP6 as well as the numerous plastid-specific extensions of the RPs. We discover many features by which the plastid-specific extensions stabilize the ribosome via establishing additional interactions with surrounding ribosomal RNA and RPs. Moreover, we identify a large conglomerate of plastid-specific protein mass adjacent to the tunnel exit site that could facilitate interaction of the chloroplast ribosome with the thylakoid membrane and the protein-targeting machinery. Comparing the Escherichia coli 70S ribosome with that of the spinach chloroplast ribosome provides detailed insight into the co-evolution of RP and rRNA.

  6. Overexpressed ribosomal proteins suppress defective chaperonins in Saccharomyces cerevisiae.

    PubMed

    Kabir, M Anaul; Sherman, Fred

    2008-12-01

    The chaperonin Cct complex of the yeast Saccharomyces cerevisiae is composed of eight different subunits encoded by eight essential genes, CCT1-CCT8. This Cct complex is responsible for the folding of a number of proteins including actin and tubulin. We have isolated and characterized 22 multicopy suppressors of the temperature-sensitive allele, cct4-1, which encodes an altered protein with a G345D replacement that diminishes ATP hydrolysis. Fourteen of the suppressors encode ribosomal proteins, four have roles in ribosome biogenesis, two have phosphatase activities, one is involved in protein synthesis and one of the suppressors corresponded to Cct4p. Some of the suppressors also acted on certain cct1, cct2, cct3 and cct6 mutations. We suggest that certain overexpressed ribosomal and other proteins can act as weak chaperones, phenotypically alleviating the partial defects of mutationally altered Cct subunits.

  7. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap.

    PubMed

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J E; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 mul of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

  8. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  9. Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

    PubMed

    Giraud, Pierre; Créchet, Jean-Bernard; Uzan, Marc; Bontems, François; Sizun, Christina

    2015-04-01

    Ribosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E. coli S1 is a modular protein that contains six repeats of an S1 motif, which have distinct functions despite structural homology. Whereas the three central repeats have been shown to be involved in mRNA recognition, the two first repeats that constitute the N-terminal domain of S1 are responsible for binding to the 30S subunit. Here we report the almost complete (1)H, (13)C and (15)N resonance assignment of two fragments of the 30S binding region of S1. The first fragment comprises only the first repeat. The second corresponds to the entire ribosome binding domain. Since S1 is absent from all high resolution X-ray structures of prokaryotic ribosomes, these data provide a first step towards atomic level structural characterization of this domain by NMR. Chemical shift analysis of the first repeat provides evidence for structural divergence from the canonical OB-fold of an S1 motif. In contrast the second domain displays the expected topology for an S1 motif, which rationalizes the functional specialization of the two subdomains.

  10. Ribosomal frameshifting in decoding antizyme mRNAs from yeast and protists to humans: close to 300 cases reveal remarkable diversity despite underlying conservation

    PubMed Central

    Ivanov, Ivaylo P.; Atkins, John F.

    2007-01-01

    The protein antizyme is a negative regulator of intracellular polyamine levels. Ribosomes synthesizing antizyme start in one ORF and at the codon 5′ adjacent to its stop codon, shift +1 to a second and partially overlapping ORF which encodes most of the protein. The ribosomal frameshifting is a sensor and effector of an autoregulatory circuit which is conserved in animals, fungi and protists. Stimulatory signals encoded 5′ and 3′ of the shift site act to program the frameshifting. Despite overall conservation, many individual branches have evolved specific features surrounding the frameshift site. Among these are RNA pseudoknots, RNA stem-loops, conserved primary RNA sequences, nascent peptide sequences and branch-specific ‘shifty’ codons. PMID:17332016

  11. The Ribosomal Protein Rpl22 Controls Ribosome Composition by Directly Repressing Expression of Its Own Paralog, Rpl22l1

    PubMed Central

    O'Leary, Monique N.; Schreiber, Katherine H.; Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Hale, J. Scott; Academia, Emmeline C.; Shah, Shreya R.; Morton, John F.; Holstein, Carly A.; Martin, Dan B.; Kaeberlein, Matt; Ladiges, Warren C.; Fink, Pamela J.; MacKay, Vivian L.; Wiest, David L.; Kennedy, Brian K.

    2013-01-01

    Most yeast ribosomal protein genes are duplicated and their characterization has led to hypotheses regarding the existence of specialized ribosomes with different subunit composition or specifically-tailored functions. In yeast, ribosomal protein genes are generally duplicated and evidence has emerged that paralogs might have specific roles. Unlike yeast, most mammalian ribosomal proteins are thought to be encoded by a single gene copy, raising the possibility that heterogenous populations of ribosomes are unique to yeast. Here, we examine the roles of the mammalian Rpl22, finding that Rpl22−/− mice have only subtle phenotypes with no significant translation defects. We find that in the Rpl22−/− mouse there is a compensatory increase in Rpl22-like1 (Rpl22l1) expression and incorporation into ribosomes. Consistent with the hypothesis that either ribosomal protein can support translation, knockdown of Rpl22l1 impairs growth of cells lacking Rpl22. Mechanistically, Rpl22 regulates Rpl22l1 directly by binding to an internal hairpin structure and repressing its expression. We propose that ribosome specificity may exist in mammals, providing evidence that one ribosomal protein can influence composition of the ribosome by regulating its own paralog. PMID:23990801

  12. Ribosome Dwell Times and the Protein Copy Number Distribution

    NASA Astrophysics Data System (ADS)

    Gorissen, Mieke; Vanderzande, Carlo

    2012-09-01

    Translation is the cellular process in which ribosomes make proteins from information encoded on messenger RNA (mRNA). We model translation with an exclusion process taking into account the experimentally determined, non-exponential, waiting time between steps of a ribosome. From numerical simulations using realistic parameter values, we determine the distribution P( E) of the number of proteins E produced by one mRNA. We find that for small E this distribution is not geometric. We present a simplified and analytically solvable model that relates P( E) to the distributions of the times to produce the first E proteins.

  13. Ribosome recycling induces optimal translation rate at low ribosomal availability.

    PubMed

    Marshall, E; Stansfield, I; Romano, M C

    2014-09-06

    During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3' end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called 'closed-loop' model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.

  14. Ribosomal Proteins Control or Bypass p53 during Nucleolar Stress

    PubMed Central

    Russo, Annapina; Russo, Giulia

    2017-01-01

    The nucleolus is the site of ribosome biogenesis, a complex process that requires the coordinate activity of all three RNA polymerases and hundreds of non-ribosomal factors that participate in the maturation of ribosomal RNA (rRNA) and assembly of small and large subunits. Nevertheless, emerging studies have highlighted the fundamental role of the nucleolus in sensing a variety of cellular stress stimuli that target ribosome biogenesis. This condition is known as nucleolar stress and triggers several response pathways to maintain cell homeostasis, either p53-dependent or p53-independent. The mouse double minute (MDM2)-p53 stress signaling pathways are activated by multiple signals and are among the most important regulators of cellular homeostasis. In this review, we will focus on the role of ribosomal proteins in p53-dependent and p53-independent response to nucleolar stress considering novel identified regulators of these pathways. We describe, in particular, the role of ribosomal protein uL3 (rpL3) in p53-independent nucleolar stress signaling pathways. PMID:28085118

  15. Post-transcriptional regulation of ribosomal protein genes during serum starvation in Entamoeba histolytica.

    PubMed

    Ahamad, Jamaluddin; Ojha, Sandeep; Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha

    2015-06-01

    Ribosome synthesis involves all three RNA polymerases which are co-ordinately regulated to produce equimolar amounts of rRNAs and ribosomal proteins (RPs). Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in E. histolytica rDNA transcription continues but pre-rRNA processing slows down and unprocessed pre-rRNA accumulates during serum starvation. To investigate the regulation of RP genes under stress we measured transcription of six selected RP genes from the small- and large-ribosomal subunits (RPS6, RPS3, RPS19, RPL5, RPL26, RPL30) representing the early-, mid-, and late-stages of ribosomal assembly. Transcripts of these genes persisted in growth-stressed cells. Expression of luciferase reporter under the control of two RP genes (RPS19 and RPL30) was studied during serum starvation and upon serum replenishment. Although luciferase transcript levels remained unchanged during starvation, luciferase activity steadily declined to 7.8% and 15% of control cells, respectively. After serum replenishment the activity increased to normal levels, suggesting post-transcriptional regulation of these genes. Mutations in the sequence -2 to -9 upstream of AUG in the RPL30 gene resulted in the phenotype expected of post-transcriptional regulation. Transcription of luciferase reporter was unaffected in this mutant, and luciferase activity did not decline during serum starvation, showing that this sequence is required to repress translation of RPL30 mRNA, and mutations in this region relieve repression. Our data show that during serum starvation E. histolytica blocks ribosome biogenesis post-transcriptionally by inhibiting pre-rRNA processing on the one hand, and the translation of RP mRNAs on the other.

  16. Mechanisms of the initiation of protein synthesis: in reading frame binding of ribosomes to mRNA.

    PubMed

    Nakamoto, Tokumasa

    2011-02-01

    The various mechanisms proposed to describe the initiation of protein synthesis are reviewed with a focus on their initiation signals. A characteristic feature of the various mechanisms is that each one of them postulates a distinct initiation signal. The signals of the Shine-Dalgarno (SD), the scanning and the internal ribosome entry site (IRES) mechanisms are all located exclusively in the 5' leader sequence, whereas, the signal of the cumulative specificity (CS) mechanism includes the entire initiation site (IS). Computer analysis of known E. coli IS sequences showed signal characteristics in the entire model IS consisting of 47 bases, in segments of the 5' leader and of the protein-coding regions. The proposal that eukaryotic translation actually occurs in two steps is scrutinized. In a first step, initiation factors (eIF4F) interact with the cap of the mRNA, thereby enhancing the accessibility of the IS. In the second step, initiation is by the conserved prokaryotic mechanism in which the ribosomes bind directly to the mRNA without ribosomal scanning. This binding occurs by the proposed process of in reading frame binding of ribosomes to mRNA, which is consistent with the CS mechanism. The basic CS mechanism is able to account for the initiation of translation of leaderless mRNAs, as well as for that of canonical mRNAs. The SD, the scanning and the IRES mechanisms, on the other hand, are inconsistent with the initiation of translation of leaderless mRNAs. Based on these and other observations, it is deemed that the CS mechanism is the universal initiation mechanism.

  17. RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

    PubMed Central

    PELLETIER, MICHEL; READ, LAURIE K.

    2003-01-01

    RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by ∼98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%–80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs. PMID:12649497

  18. Protein-guided RNA dynamics during early ribosome assembly.

    PubMed

    Kim, Hajin; Abeysirigunawarden, Sanjaya C; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2014-02-20

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  19. Protein-guided RNA dynamics during early ribosome assembly

    NASA Astrophysics Data System (ADS)

    Kim, Hajin; Abeysirigunawarden, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-02-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the ribosomal RNA structure so that later proteins may join the complex is poorly understood. Here we use single-molecule fluorescence resonance energy transfer (FRET) to observe real-time encounters between Escherichia coli ribosomal protein S4 and the 16S 5' domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free-energy path to protein-RNA recognition. Three-colour FRET and molecular dynamics simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. These protein-guided dynamics offer an alternative explanation for induced fit in RNA-protein complexes.

  20. Crosslinking of Ribosomal Proteins to RNA in Maize Ribosomes by UV-B and Its Effects on Translation1[w

    PubMed Central

    Casati, Paula; Walbot, Virginia

    2004-01-01

    Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers. PMID:15466230

  1. Polyribosome targeting to microtubules: enrichment of specific mRNAs in a reconstituted microtubule preparation from sea urchin embryos

    PubMed Central

    1994-01-01

    A subset of mRNAs, polyribosomes, and poly(A)-binding proteins copurify with microtubules from sea urchin embryos. Several lines of evidence indicate that the interaction of microtubules with ribosomes is specific: a distinct stalk-like structure appears to mediate their association; ribosomes bind to microtubules with a constant stoichiometry through several purification cycles; and the presence of ribosomes in these preparations depends on the presence of intact microtubules. Five specific mRNAs are enriched with the microtubule- bound ribosomes, indicating that translation of specific proteins may occur on the microtubule scaffolding in vivo. PMID:7962079

  2. Regulation of ribosomal protein synthesis in Vibrio cholerae.

    PubMed

    Allen, Todd D; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M

    2004-09-01

    We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria.

  3. Regulation of Ribosomal Protein Synthesis in Vibrio cholerae

    PubMed Central

    Allen, Todd D.; Watkins, Tonya; Lindahl, Lasse; Zengel, Janice M.

    2004-01-01

    We have investigated the regulation of the S10 and spc ribosomal protein (r-protein) operons in Vibrio cholerae. Both operons are under autogenous control; they are mediated by r-proteins L4 and S8, respectively. Our results suggest that Escherichia coli-like strategies for regulating r-protein synthesis extend beyond the enteric members of the gamma subdivision of proteobacteria. PMID:15317799

  4. Ribosome-inactivating proteins: from toxins to useful proteins.

    PubMed

    Stirpe, Fiorenzo

    2013-06-01

    Ribosome-inactivating proteins (RIPs) either single-chain (type 1) or two-chain (type 2) are frequent in plants, often in multiple forms. They are RNA N-glycosidases, have antiviral, antifungal and insecticidal activity. Their expression in plants is increased under stressful conditions. They are investigated for practical applications in medicine and in agriculture. In medicine, RIPs have been linked to, or fused with, appropriate antibodies or other carriers to form "immunotoxins" or other conjugates specifically toxic to the cells target of the carrier, with the aim of eliminating malignant or other undesired cells. In agriculture, it has been observed that an enhanced expression of RIPs confers to plants an increased resistance to viruses, fungi, insects, and also to drought and salinity.

  5. Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

    SciTech Connect

    Zhdanov, V. P.

    2010-10-15

    Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

  6. Inactivation of Ribosomal Protein Genes in Bacillus subtilis Reveals Importance of Each Ribosomal Protein for Cell Proliferation and Cell Differentiation

    PubMed Central

    Akanuma, Genki; Nanamiya, Hideaki; Natori, Yousuke; Yano, Koichi; Suzuki, Shota; Omata, Shuya; Ishizuka, Morio; Sekine, Yasuhiko

    2012-01-01

    Among the 57 genes that encode ribosomal proteins in the genome of Bacillus subtilis, a Gram-positive bacterium, 50 genes were targeted by systematic inactivation. Individual deletion mutants of 16 ribosomal proteins (L1, L9, L15, L22, L23, L28, L29, L32, L33.1, L33.2, L34, L35, L36, S6, S20, and S21) were obtained successfully. In conjunction with previous reports, 22 ribosomal proteins have been shown to be nonessential in B. subtilis, at least for cell proliferation. Although several mutants that harbored a deletion of a ribosomal protein gene did not show any significant differences in any of the phenotypes that were tested, various mutants showed a reduced growth rate and reduced levels of 70S ribosomes compared with the wild type. In addition, severe defects in the sporulation frequency of the ΔrplA (L1) mutant and the motility of the ΔrpsU (S21) mutant were observed. These data provide the first evidence in B. subtilis that L1 and S21 are required for the progression of cellular differentiation. PMID:23002217

  7. A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

    PubMed

    Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

    2016-11-17

    Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    PubMed Central

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-01-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds. PMID:27669417

  9. Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

    PubMed

    Li, Kunhua; Condurso, Heather L; Li, Gengnan; Ding, Yousong; Bruner, Steven D

    2016-11-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  10. Eukaryote-specific motif of ribosomal protein S15 neighbors A site codon during elongation and termination of translation.

    PubMed

    Khairulina, Julia; Graifer, Dmitri; Bulygin, Konstantin; Ven'yaminova, Aliya; Frolova, Ludmila; Karpova, Galina

    2010-07-01

    The eukaryotic ribosomal protein S15 is a key component of the decoding site in contrast to its prokaryotic counterpart, S19p, which is located away from the mRNA binding track on the ribosome. Here, we determined the oligopeptide of S15 neighboring the A site mRNA codon on the human 80S ribosome with the use of mRNA analogues bearing perfluorophenyl azide-modified nucleotides in the sense or stop codon targeted to the 80S ribosomal A site. The protein was cross-linked to mRNA analogues in specific ribosomal complexes that were obtained in the presence of eRF1 in the experiments with mRNAs bearing stop codon. Digestion of modified S15 with various specific proteolytic agents followed by identification of the resulting modified oligopeptides showed that cross-link was in C-terminal fragment in positions 131-145, most probably, in decapeptide 131-PGIGATHSSR-140. The position of cross-linking site on the S15 protein did not depend on the nature of the A site-bound codon (sense or stop codon) and on the presence of polypeptide chain release factor eRF1 in the ribosomal complexes with mRNA analogues bearing a stop codon. The results indicate an involvement of the mentioned decapeptide in the formation of the ribosomal decoding site during elongation and termination of translation. Alignment of amino acid sequences of eukaryotic S15 and its prokaryotic counterpart, S19p from eubacteria and archaea, revealed that decapeptide PGIGATHSSR in positions 131-140 is strongly conserved in eukaryotes and has minor variations in archaea but has no homology with any sequence in C-terminal part of eubacterial S19p, which suggests involvement of the decapeptide in the translation process in a eukaryote-specific manner.

  11. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

    PubMed Central

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J. E.; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies. PMID:18632729

  12. Epigenetic engineering of ribosomal RNA genes enhances protein production.

    PubMed

    Santoro, Raffaella; Lienemann, Philipp; Fussenegger, Martin

    2009-08-14

    Selection of mammalian high-producer cell lines remains a major challenge for the biopharmaceutical manufacturing industry. Ribosomal RNA (rRNA) genes encode the major component of the ribosome but many rRNA gene copies are not transcribed due to epigenetic silencing by the nucleolar remodelling complex (NoRC) [6], which may limit the cell's full production capacity. Here we show that the knockdown of TIP5, a subunit of NoRC, decreases the number of silent rRNA genes, upregulates rRNA transcription, enhances ribosome synthesis and increases production of recombinant proteins. However, general enhancement of rRNA transcription rate did not stimulate protein synthesis. Our data demonstrates that the number of transcriptionally competent rRNA genes limits efficient ribosome synthesis. Epigenetic engineering of ribosomal RNA genes offers new possibilities for improving biopharmaceutical manufacturing and provides novel insights into the complex regulatory network which governs the translation machinery in normal cellular processes as well as in pathological conditions like cancer.

  13. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

  14. Charge segregation and low hydrophobicity are key features of ribosomal proteins from different organisms.

    PubMed

    Fedyukina, Daria V; Jennaro, Theodore S; Cavagnero, Silvia

    2014-03-07

    Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.

  15. Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms*

    PubMed Central

    Fedyukina, Daria V.; Jennaro, Theodore S.; Cavagnero, Silvia

    2014-01-01

    Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge. PMID:24398678

  16. Ribosome-inactivating proteins: potent poisons and molecular tools.

    PubMed

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-11-15

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

  17. N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis.

    PubMed

    Kamita, Masahiro; Kimura, Yayoi; Ino, Yoko; Kamp, Roza M; Polevoda, Bogdan; Sherman, Fred; Hirano, Hisashi

    2011-04-01

    N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

    PubMed Central

    Singh, Dharam; Chang, Ssu-Jean; Lin, Pei-Hsun; Averina, Olga V.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2009-01-01

    Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments. PMID:19144914

  19. Protein-guided RNA dynamics during early ribosome assembly

    PubMed Central

    Kim, Hajin; Abeysirigunawardena, Sanjaya C.; Chen, Ke; Mayerle, Megan; Ragunathan, Kaushik; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A.

    2014-01-01

    The assembly of 30S ribosomes requires the precise addition of 20 proteins to the 16S ribosomal RNA. How early binding proteins change the rRNA structure so that later proteins may join the complex is poorly understood. Here we use single molecule fluorescence resonance energy transfer (smFRET) to observe real-time encounters between ribosomal protein S4 and the 16S 5′ domain RNA at an early stage of 30S assembly. Dynamic initial S4-RNA complexes pass through a stable non-native intermediate before converting to the native complex, showing that non-native structures can offer a low free energy path to protein-RNA recognition. Three-color FRET and molecular dynamics (MD) simulations reveal how S4 changes the frequency and direction of RNA helix motions, guiding a conformational switch that enforces the hierarchy of protein addition. This protein-guided dynamics offers an alternative explanation for induced fit in RNA-protein complexes. PMID:24522531

  20. Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

    PubMed Central

    Dang, H; Ellis, S R

    1990-01-01

    We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome. Images PMID:2263452

  1. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  2. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    SciTech Connect

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-11-11

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein–protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  3. Mutational analysis of the ribosomal protein Rpl10 from yeast.

    PubMed

    Hofer, Anne; Bussiere, Cyril; Johnson, Arlen W

    2007-11-09

    Yeast Rpl10 belongs to the L10e family of ribosomal proteins. In the large (60 S) subunit, Rpl10 is positioned in a cleft between the central protuberance and the GTPase-activating center. It is loaded into the 60 S subunit at a late step in maturation. We have shown previously that Rpl10 is required for the release of the Crm1-dependent nuclear export adapter Nmd3, an event that also requires the cytoplasmic GTPase Lsg1. Here we have carried out an extensive mutational analysis of Rpl10 to identify mutations that would allow us to map activities to distinct domains of the protein to begin to understand the molecular interaction between Rpl10 and Nmd3. We found that mutations in a central loop (amino acids 102-112) had a significant impact on the release of Nmd3. This loop is unstructured in the crystal and solution structures of prokaryotic Rpl10 orthologs. Thus, the loop is not necessary for stable interaction of Rpl10 with the ribosome, suggesting that it plays a dynamic role in ribosome function or regulating the association of other factors. We also found that mutant Rpl10 proteins were engineered to be unable to bind to the ribosome accumulated in the nucleus. This was unexpected and may suggest a nuclear role for Rpl10.

  4. Protein folding on the ribosome studied using NMR spectroscopy

    PubMed Central

    Waudby, Christopher A.; Launay, Hélène; Cabrita, Lisa D.; Christodoulou, John

    2013-01-01

    NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome–nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity. PMID:24083462

  5. FRET-Based Identification of mRNAs Undergoing Translation

    PubMed Central

    Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L.; Smilansky, Zeev; Cooperman, Barry S.; Goldman, Yale E.

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed. PMID:22693619

  6. FRET-based identification of mRNAs undergoing translation.

    PubMed

    Stevens, Benjamin; Chen, Chunlai; Farrell, Ian; Zhang, Haibo; Kaur, Jaskiran; Broitman, Steven L; Smilansky, Zeev; Cooperman, Barry S; Goldman, Yale E

    2012-01-01

    We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

  7. Ribosomal protein S6 is highly expressed in non-Hodgkin lymphoma and associates with mRNA containing a 5' terminal oligopyrimidine tract.

    PubMed

    Hagner, P R; Mazan-Mamczarz, K; Dai, B; Balzer, E M; Corl, S; Martin, S S; Zhao, X F; Gartenhaus, R B

    2011-03-31

    The molecular mechanism(s) linking tumorigenesis and morphological alterations in the nucleolus are presently coming into focus. The nucleolus is the cellular organelle in which the formation of ribosomal subunits occurs. Ribosomal biogenesis occurs through the transcription of ribosomal RNA (rRNA), rRNA processing and production of ribosomal proteins. An error in any of these processes may lead to deregulated cellular translation, evident in multiple cancers and 'ribosomopathies'. Deregulated protein synthesis may be achieved through the overexpression of ribosomal proteins as seen in primary leukemic blasts with elevated levels of ribosomal proteins S11 and S14. In this study, we demonstrate that ribosomal protein S6 (RPS6) is highly expressed in primary diffuse large B-cell lymphoma (DLBCL) samples. Genetic modulation of RPS6 protein levels with specifically targeted short hairpin RNA (shRNA) lentiviruses led to a decrease in the actively proliferating population of cells compared with control shRNA. Low-dose rapamycin treatments have been shown to affect the translation of 5' terminal oligopyrimidine (5' TOP) tract mRNA, which encodes the translational machinery, implicating RPS6 in 5' TOP translation. Recently, it was shown that disruption of 40S ribosomal biogenesis through specific small inhibitory RNA knockdown of RPS6 defined RPS6 as a critical regulator of 5' TOP translation. For the first time, we show that RPS6 associates with multiple mRNAs containing a 5' TOP tract. These findings expand our understanding of the mechanism(s) involved in ribosomal biogenesis and deregulated protein synthesis in DLBCL.

  8. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency.

    PubMed

    Mailliot, Justine; Garreau de Loubresse, Nicolas; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D; Yusupov, Marat

    2016-05-22

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a "WT-like" configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome.

  9. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency

    PubMed Central

    Mailliot, Justine; de Loubresse, Nicolas Garreau; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D.; Yusupov, Marat

    2017-01-01

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a “WT-like” configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome. PMID:26906928

  10. A Plasma Protein Indistinguishable from Ribosomal Protein S19

    PubMed Central

    Semba, Umeko; Chen, Jun; Ota, Yoshihiko; Jia, Nan; Arima, Hidetoshi; Nishiura, Hiroshi; Yamamoto, Tetsuro

    2010-01-01

    A monocyte-chemoattracting factor is generated during blood coagulation and during clotting of platelet-rich plasma. This chemotactic factor attracts monocytes as a ligand of the C5a receptor; however, it inhibits C5a-induced neutrophil chemotaxis as an apparent receptor antagonist. The curious dual function of the serum monocyte chemotactic factor resembles that of the cross-linked homodimer of ribosomal protein S19 (RP S19). Indeed, the inactive precursor of the monocyte chemotactic factor was present in plasma, and the precursor molecule and RP S19, as well as the active form and the RP S19 dimer, were indistinguishable in terms of immunological reactivity and molecular size. Coagulation factor XIIIa, plasma transglutaminase, and membrane phosphatidylserine on the activated platelets were required for conversion of the precursor to the active form. In addition, the precursor molecule in plasma could be replaced by wild-type recombinant RP S19 but not by mutant forms of it. These results indicate that a molecule indistinguishable from RP S19 was present in plasma, and that the RP S19-like molecule was converted to the active form by a transglutaminase-catalyzed reaction on a scaffold that included the phosphatidylserine-exposed platelet membrane. PMID:20093496

  11. Protein-directed ribosomal frameshifting temporally regulates gene expression

    PubMed Central

    Napthine, Sawsan; Ling, Roger; Finch, Leanne K.; Jones, Joshua D.; Bell, Susanne; Brierley, Ian; Firth, Andrew E.

    2017-01-01

    Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins. PMID:28593994

  12. Genome-wide mRNA processing in methanogenic archaea reveals post-transcriptional regulation of ribosomal protein synthesis

    PubMed Central

    Qi, Lei; Yue, Lei; Feng, Deqin; Qi, Fengxia

    2017-01-01

    Abstract Unlike stable RNAs that require processing for maturation, prokaryotic cellular mRNAs generally follow an ‘all-or-none’ pattern. Herein, we used a 5΄ monophosphate transcript sequencing (5΄P-seq) that specifically captured the 5΄-end of processed transcripts and mapped the genome-wide RNA processing sites (PSSs) in a methanogenic archaeon. Following statistical analysis and stringent filtration, we identified 1429 PSSs, among which 23.5% and 5.4% were located in 5΄ untranslated region (uPSS) and intergenic region (iPSS), respectively. A predominant uridine downstream PSSs served as a processing signature. Remarkably, 5΄P-seq detected overrepresented uPSS and iPSS in the polycistronic operons encoding ribosomal proteins, and the majority upstream and proximal ribosome binding sites, suggesting a regulatory role of processing on translation initiation. The processed transcripts showed increased stability and translation efficiency. Particularly, processing within the tricistronic transcript of rplA-rplJ-rplL enhanced the translation of rplL, which can provide a driving force for the 1:4 stoichiometry of L10 to L12 in the ribosome. Growth-associated mRNA processing intensities were also correlated with the cellular ribosomal protein levels, thereby suggesting that mRNA processing is involved in tuning growth-dependent ribosome synthesis. In conclusion, our findings suggest that mRNA processing-mediated post-transcriptional regulation is a potential mechanism of ribosomal protein synthesis and stoichiometry. PMID:28520982

  13. Translation factors and ribosomal proteins control tumor onset and progression: how?

    PubMed

    Loreni, F; Mancino, M; Biffo, S

    2014-04-24

    Gene expression is shaped by translational control. The modalities and the extent by which translation factors modify gene expression have revealed therapeutic scenarios. For instance, eukaryotic initiation factor (eIF)4E activity is controlled by the signaling cascade of growth factors, and drives tumorigenesis by favoring the translation of specific mRNAs. Highly specific drugs target the activity of eIF4E. Indeed, the antitumor action of mTOR complex 1 (mTORc1) blockers like rapamycin relies on their capability to inhibit eIF4E assembly into functional eIF4F complexes. eIF4E biology, from its inception to recent pharmacological targeting, is proof-of-principle that translational control is druggable. The case for eIF4E is not isolated. The translational machinery is involved in the biology of cancer through many other mechanisms. First, untranslated sequences on mRNAs as well as noncoding RNAs regulate the translational efficiency of mRNAs that are central for tumor progression. Second, other initiation factors like eIF6 show a tumorigenic potential by acting downstream of oncogenic pathways. Third, genetic alterations in components of the translational apparatus underlie an entire class of inherited syndromes known as 'ribosomopathies' that are associated with increased cancer risk. Taken together, data suggest that in spite of their evolutionary conservation and ubiquitous nature, variations in the activity and levels of ribosomal proteins and translation factors generate highly specific effects. Beside, as the structures and biochemical activities of several noncoding RNAs and initiation factors are known, these factors may be amenable to rational pharmacological targeting. The future is to design highly specific drugs targeting the translational apparatus.

  14. Roles of eukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA and ribosome function.

    PubMed

    Ferreira-Cerca, Sébastien; Pöll, Gisela; Gleizes, Pierre-Emmanuel; Tschochner, Herbert; Milkereit, Philipp

    2005-10-28

    Despite the rising knowledge about ribosome function and structure and how ribosomal subunits assemble in vitro in bacteria, the in vivo role of many ribosomal proteins remains obscure both in pro- and eukaryotes. Our systematic analysis of yeast ribosomal proteins (r-proteins) of the small subunit revealed that most eukaryotic r-proteins fulfill different roles in ribosome biogenesis, making them indispensable for growth. Different r-proteins control distinct steps of nuclear and cytoplasmic pre-18S rRNA processing and, thus, ensure that only properly assembled ribosomes become engaged in translation. Comparative analysis of dynamic and steady-state maturation assays revealed that several r-proteins are required for efficient nuclear export of pre-18S rRNA, suggesting that they form an interaction platform with the export machinery. In contrast, the presence of other r-proteins is mainly required before nuclear export is initiated. Our studies draw a correlation between the in vitro assembly, structural localization, and in vivo function of r-proteins.

  15. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes

    PubMed Central

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md.; Gregory, Brian; Samsel, Leigh; Zengel, Janice M.; Lindahl, Lasse

    2013-01-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle. PMID:24109599

  16. Repressed synthesis of ribosomal proteins generates protein-specific cell cycle and morphological phenotypes.

    PubMed

    Thapa, Mamata; Bommakanti, Ananth; Shamsuzzaman, Md; Gregory, Brian; Samsel, Leigh; Zengel, Janice M; Lindahl, Lasse

    2013-12-01

    The biogenesis of ribosomes is coordinated with cell growth and proliferation. Distortion of the coordinated synthesis of ribosomal components affects not only ribosome formation, but also cell fate. However, the connection between ribosome biogenesis and cell fate is not well understood. To establish a model system for inquiries into these processes, we systematically analyzed cell cycle progression, cell morphology, and bud site selection after repression of 54 individual ribosomal protein (r-protein) genes in Saccharomyces cerevisiae. We found that repression of nine 60S r-protein genes results in arrest in the G2/M phase, whereas repression of nine other 60S and 22 40S r-protein genes causes arrest in the G1 phase. Furthermore, bud morphology changes after repression of some r-protein genes. For example, very elongated buds form after repression of seven 60S r-protein genes. These genes overlap with, but are not identical to, those causing the G2/M cell cycle phenotype. Finally, repression of most r-protein genes results in changed sites of bud formation. Strikingly, the r-proteins whose repression generates similar effects on cell cycle progression cluster in the ribosome physical structure, suggesting that different topological areas of the precursor and/or mature ribosome are mechanistically connected to separate aspects of the cell cycle.

  17. UVB and gamma-radiation induce the expression of mRNAs encoding the ribosomal subunit L13A in rat keratinocytes.

    PubMed

    Shahmolky, N; Lefebvre, D L; Poon, R; Bai, Y; Sharma, M; Rosen, C F

    1999-09-01

    Ultraviolet B radiation produces an array of cellular perturbations in the skin. We isolated a keratinocyte cDNA encoding the rat 60S ribosomal subunit protein L13a following differential cDNA library screening with UVB-enriched probes. In contrast to the reported structure of liver L13a, the keratinocyte L13a cDNA contains a longer 3'-untranslated region. Northern blot analysis detected two L13a mRNA transcripts, approximately 800 bp and approximately 1.2 kb, in keratinocytes and a variety of rat tissues. Both L13a mRNA transcripts were induced by UVB irradiation, forskolin and gamma-irradiation. In contrast, no induction of L13a mRNA transcript levels was observed following exposure of keratinocytes to 12-O-tetradecanoylphorbol-13-acetate, serum and the DNA damage-inducing agents methyl methanesulfonate or 4-nitroquinoline-N-oxide. These observations suggest that increased expression of ribosomal subunit genes may be a molecular component of the keratinocyte response to UVB in particular and not part of a nonspecific response to DNA damage.

  18. Ubiquitination of newly synthesized proteins at the ribosome.

    PubMed

    Wang, Feng; Canadeo, Larissa A; Huibregtse, Jon M

    2015-07-01

    Newly synthesized proteins can be misfolded or damaged because of errors during synthesis or environmental insults (e.g., heat shock), placing a significant burden on protein quality control systems. In addition, numerous human diseases are associated with a deficiency in eliminating aberrant proteins or accumulation of aggregated proteins. Understanding the mechanisms of protein quality control and disposal pathways for misfolded proteins is therefore crucial for therapeutic intervention in these diseases. Quality control processes function at many points in the life cycle of proteins, and a subset act at the actual site of protein synthesis, the ribosome. Here we summarize recent advances in the role of the ubiquitin proteasome system in protein quality control during the process of translation.

  19. Ubiquitination of Newly Synthesized Proteins at the Ribosome

    PubMed Central

    Wang, Feng; Canadeo, Larissa A.; Huibregtse, Jon M.

    2015-01-01

    Newly synthesized proteins can be misfolded or damaged because of errors during synthesis or environmental insults (e.g., heat shock), placing a significant burden on protein quality control systems. In addition, numerous human diseases are associated with a deficiency in eliminating aberrant proteins or accumulation of aggregated proteins. Understanding the mechanisms of protein quality control and disposal pathways for misfolded proteins is therefore crucial for therapeutic intervention in these diseases. Quality control processes function at many points in the life cycle of proteins, and a subset act at the actual site of protein synthesis, the ribosome. Here we summarize recent advances in the role of the ubiquitin proteasome system in protein quality control during the process of translation. PMID:25701549

  20. Protein folding: When ribosomes pick the structure

    NASA Astrophysics Data System (ADS)

    Sivertsson, Elin M.; Itzhaki, Laura S.

    2014-05-01

    Anfinsen's principle tells us that the folded structure of a protein is determined solely by its sequence. Now, it has been shown that the rate at which a polypeptide chain is synthesized in the cell can affect which of two alternative folded structures it adopts.

  1. RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

    PubMed

    Yu, DongMei; Zhang, Chao; Qin, PeiWu; Cornish, Peter V; Xu, Dong

    2014-11-01

    Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

  2. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    PubMed Central

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production. PMID:28091612

  3. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    NASA Astrophysics Data System (ADS)

    Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

  4. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  5. Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

    SciTech Connect

    Kartasova, T.; Ponec, M.; van de Putte, P.

    1988-02-01

    uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with (/sup 35/S)methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

  6. Interplay of viral miRNAs and host mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-10-01

    Recent experiments indicate that several viruses may encode microRNAs (miRNAs) in cells. Such RNAs may interfere with the host mRNAs and proteins. We present a kinetic analysis of this interplay. In our treatment, the viral miRNA is considered to be able to associate with the host mRNA with subsequent degradation. This process may result in a decline of the mRNA population and also in a decline of the population of the protein encoded by this mRNA. With these ingredients, we first show the types of the corresponding steady-state kinetics in the cases of positive and negative regulation of the miRNA synthesis by the protein. In addition, we scrutinize the situation when the protein regulates the virion replication or, in other words, provides a feedback for the replication. For the negative feedback, the replication rate is found to increase with increasing the intracellular virion population. For the positive feedback, the replication rate first increases and then drops. These features may determine the stability of steady states.

  7. Protein-tyrosine-kinase-dependent expression of cyclo-oxygenase-1 and -2 mRNAs in human endothelial cells.

    PubMed Central

    Hirai, K; Takayama, H; Tomo, K; Okuma, M

    1997-01-01

    Endothelial cells possess constitutive or inducible cyclo-oxygenase (COX) isoenzymes for prostacyclin production, but the mechanisms for their expression are largely unknown. We found that vanadate, an inhibitor of protein-tyrosine phosphatases, induced the expression of two COX isoenzyme mRNAs in human umbilical vein endothelial cells (HUVEC) in a time- and dose-dependent manner. Vanadate also stimulated an increase in COX-2 protein levels, but did not affect significantly the levels of constitutively expressed COX-1 protein. Synergistic enhancement of expression of the two COX isoenzyme mRNAs was observed on stimulation of HUVEC with vanadate plus interleukin-1alpha. Tyrphostin-47, which as an inhibitor of protein-tyrosine kinases abolished vanadate-induced protein-tyrosine phosphorylation, inhibited expression of the two COX isoenzyme mRNAs in HUVEC stimulated with vanadate or interleukin-1alpha. These data provide conclusive evidence that activation of protein-tyrosine kinases is causally linked to expression of the mRNAs for the two COX isoenzymes in HUVEC. PMID:9065752

  8. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    SciTech Connect

    Feng, Yingang; Song, Xiaxia; Lin, Jinzhong; Xuan, Jinsong; Cui, Qiu; Wang, Jinfeng

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  9. Posttranscriptional regulation of ribosomal protein S20 and stability of the S20 mRNA species.

    PubMed Central

    Mackie, G A

    1987-01-01

    I have tested whether selective degradation of mRNA for ribosomal protein S20 of Escherichia coli occurs under conditions for which the expression of S20 is regulated posttranscriptionally. Blot hybridization of total RNA extracted from cultures at different times after addition of rifampin has permitted the estimation of relative levels of the two S20 mRNA species and their half-lives. In a strain harboring a plasmid containing the complete gene for S20, including the transcriptional terminator, moderate posttranscriptional repression of S20 synthesis is accompanied by a substantial increase in the half-lives of both S20 mRNAs relative to those in the haploid parental strain. In an otherwise identical strain in which the transcriptional terminator is deleted from the plasmid-borne S20 genes, the half-life of total S20 mRNA declines more than twofold relative to that in the haploid parent. Thus accelerated decay of the mRNAs for ribosomal protein S20 appears to be an artifact of deletion of the transcriptional terminator, rather than a physiologically significant consequence of translational repression. Images PMID:2438268

  10. The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

    PubMed

    Dauber, Bianca; Saffran, Holly A; Smiley, James R

    2014-09-01

    We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during

  11. [C-terminal fragment of ribosomal protein S15 is located at the decoding site of the human ribosome].

    PubMed

    Khaĭrulina, Iu S; Molotkov, M V; Bulygin, K N; Graĭfer, D M; Ven'iaminova, A G; Karpova, G G

    2008-01-01

    Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors mRNA codon at the decoding site and the downstream triplets. In this study we determined S15 protein fragments located close to mRNA positions +4 to +12 with respect to the first nucleotide of the P site codon on the human ribosome. For cross-linking to ribosomal protein S15, a set of mRNA was used that contained triplet UUU/UUC at the 5'-termini and a perfluorophenyl azide-modified uridine in position 3' of this triplet. The locations of mRNA analogues on the ribosome were governed by tRNAPhe cognate to the UUU/UUC triplet targeted to the P site. Cross-linked S15 protein was isolated from the irradiated with mild UV light complexes of 80S ribosomes with tRNAPhe and mRNA analogues with subsequent cleavage with CNBr that splits polypeptide chain after methionines. Analysis of modified oligopeptides resulted from the cleavage revealed that in all cases cross-linking site was located in C-terminal fragment 111-145 of protein S15 indicating that this fragment is involved in formation of decoding site of the eukaryotic ribosome.

  12. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

  13. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs

    PubMed Central

    Soto-Rifo, Ricardo; Rubilar, Paulina S; Limousin, Taran; de Breyne, Sylvain; Décimo, Didier; Ohlmann, Théophile

    2012-01-01

    Here, we have characterized a step in translation initiation of viral and cellular mRNAs that contain RNA secondary structures immediately at the vicinity of their m7GTP cap. This is mediated by the DEAD-box helicase DDX3 which can directly bind to the 5′ of the target mRNA where it clamps the entry of eIF4F through an eIF4G and Poly A-binding protein cytoplasmic 1 (PABP) double interaction. This could induce limited local strand separation of the secondary structure to allow 43S pre-initiation complex attachment to the 5′ free extremity of the mRNA. We further demonstrate that the requirement for DDX3 is highly specific to some selected transcripts, cannot be replaced or substituted by eIF4A and is only needed in the very early steps of ribosome binding and prior to 43S ribosomal scanning. Altogether, these data define an unprecedented role for a DEAD-box RNA helicase in translation initiation. PMID:22872150

  14. Dynamic evolution of mitochondrial ribosomal proteins in Holozoa.

    PubMed

    Scheel, Bettina M; Hausdorf, Bernhard

    2014-07-01

    We studied the highly dynamic evolution of mitochondrial ribosomal proteins (MRPs) in Holozoa. Most major clades within Holozoa are characterized by gains and/or losses of MRPs. The usefulness of gains of MRPs as rare genomic changes in phylogenetics is undermined by the high frequency of secondary losses. However, phylogenetic analyses of the MRP sequences provide evidence for the Acrosomata hypothesis, a sister group relationship between Ctenophora and Bilateria. An extensive restructuring of the mitochondrial genome and, as a consequence, of the mitochondrial ribosomes occurred in the ancestor of metazoans. The last MRP genes encoded in the mitochondrial genome were either moved to the nuclear genome or were lost. The strong decrease in size of the mitochondrial genome was probably caused by selection for rapid replication of mitochondrial DNA during oogenesis in the metazoan ancestor. A phylogenetic analysis of MRPL56 sequences provided evidence for a horizontal gene transfer of the corresponding MRP gene between metazoans and Dictyostelidae (Amoebozoa). The hypothesis that the requisition of additional MRPs compensated for a loss of rRNA segments in the mitochondrial ribosomes is corroborated by a significant negative correlation between the number of MRPs and length of the rRNA. Newly acquired MRPs evolved faster than bacterial MRPs and positions in eukaryote-specific MRPs were more strongly affected by coevolution than positions in prokaryotic MRPs in accordance with the necessity to fit these proteins into the pre-existing structure of the mitoribosome. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Selenocysteine insertion sequence (SECIS)-binding protein 2 alters conformational dynamics of residues involved in tRNA accommodation in 80 S ribosomes.

    PubMed

    Caban, Kelvin; Copeland, Paul R

    2012-03-23

    Sec-tRNA(Sec) is site-specifically delivered at defined UGA codons in selenoprotein mRNAs. This recoding event is specified by the selenocysteine insertion sequence (SECIS) element and requires the selenocysteine (Sec)-specific elongation factor, eEFSec, and the SECIS binding protein, SBP2. Sec-tRNA(Sec) is delivered to the ribosome by eEFSec-GTP, but this ternary complex is not sufficient for Sec incorporation, indicating that its access to the ribosomal A-site is regulated. SBP2 stably associates with ribosomes, and mutagenic analysis indicates that this interaction is essential for Sec incorporation. However, the ribosomal function of SBP2 has not been elucidated. To shed light on the functional relevance of the SBP2-ribosome interaction, we screened the functional centers of the 28 S rRNA in translationally competent 80 S ribosomes using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). We demonstrate that SBP2 specifically alters the reactivity of specific residues in Helix 89 (H89) and expansion segment 31 (ES31). These results are indicative of a conformational change in response to SBP2 binding. Based on the known functions of H89 during translation, we propose that SBP2 allows Sec incorporation by either promoting Sec-tRNA(Sec) accommodation into the peptidyltransferase center and/or by stimulating the ribosome-dependent GTPase activity of eEFSec.

  16. Evolution of Drosophila ribosomal protein gene core promoters

    PubMed Central

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2011-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

  17. Evolution of Drosophila ribosomal protein gene core promoters.

    PubMed

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  18. Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly.

    PubMed

    Gamalinda, Michael; Woolford, John L

    2014-11-01

    Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function. © 2014 Gamalinda and Woolford; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  19. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  20. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale.

    PubMed

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

    PubMed Central

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

  2. Transactivation of programmed ribosomal frameshifting by a viral protein

    PubMed Central

    Li, Yanhua; Treffers, Emmely E.; Napthine, Sawsan; Tas, Ali; Zhu, Longchao; Sun, Zhi; Bell, Susanne; Mark, Brian L.; van Veelen, Peter A.; van Hemert, Martijn J.; Firth, Andrew E.; Brierley, Ian; Snijder, Eric J.; Fang, Ying

    2014-01-01

    Programmed −1 ribosomal frameshifting (−1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes −1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual −2 frameshifting (−2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of −1 PRF, yielding a third, truncated nsp2 variant named “nsp2N.” Remarkably, we now show that both −2 and −1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β’s papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection. PMID:24825891

  3. A general procedure for the production of antibody reagents against eukaryotic ribosomal proteins.

    PubMed

    Dieci, Giorgio; Bottarelli, Lorena; Ottonello, Simone

    2005-08-01

    Despite recent progress in the structural and functional analysis of bacterial and archaeal ribosomes, the structure and biogenesis of eukaryotic ribosomes still awaits a detailed characterization. Ribosomal protein-specific antibodies would be valuable tools for such studies, but their production is commonly hindered by the poor expression and solubility of eukaryotic ribosomal proteins in E. coli. We report here an improved general procedure for the over-production of recombinant eukaryotic ribosomal proteins and for the generation of the corresponding polyclonal antibodies. The specificity and sensitivity of detection of the antibodies produced by this procedure are documented.

  4. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

    PubMed

    Koonin, E V; Bork, P; Sander, C

    1994-06-11

    Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.

  5. Ribosomes containing mutants of L4 ribosomal protein from Thermus thermophilus display multiple defects in ribosomal functions and sensitivity against erythromycin

    PubMed Central

    TSAGKALIA, AIKATERINI; LEONTIADOU, FOTINI; XAPLANTERI, MARIA A.; PAPADOPOULOS, GEORGIOS; KALPAXIS, DIMITRIOS L.; CHOLI-PAPADOPOULOU, THEODORA

    2005-01-01

    Protein L4 from Thermus thermophilus (TthL4) was heterologously overproduced in Escherichia coli cells. To study the implication of the extended loop of TthL4 in the exit-tunnel and peptidyltransferase functions, the highly conserved E56 was replaced by D or Q, while the semiconserved G55 was changed to E or S. Moreover, the sequence -G55E56- was inverted to -E55G56-. When we incorporated these mutants into E. coli ribosomes and investigated their impact on poly(Phe) synthesis, high variations in the synthetic activity and response to erythromycin of the resulting ribosomes were observed. In the absence of erythromycin, ribosomes harboring mutations G55E and E56D in TthL4 protein were characterized by low activity in synthesizing poly(Phe) and decreased capability in binding tRNA at the A site. On the other hand, ribosomes possessing mutations G55E, G55S, G55E-E56G, or E56Q in TthL4 protein were unexpectedly more sensitive to erythromycin. Evidence in support of these findings was drawn by in vivo experiments, assessing the erythromycin sensitivity of E. coli cells expressing wild-type or mutant TthL4 proteins. Our results emphasize the role of the extended loop of L4 ribosomal protein in the exit-tunnel and peptidyltransferase center functions. PMID:16244130

  6. Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli.

    PubMed

    Di Pietro, Fabio; Brandi, Anna; Dzeladini, Nadire; Fabbretti, Attilio; Carzaniga, Thomas; Piersimoni, Lolita; Pon, Cynthia L; Giuliodori, Anna Maria

    2013-04-01

    Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock. © 2013 The Authors. Published by Blackwell Publishing Ltd.

  7. Role of the ribosome-associated protein PY in the cold-shock response of Escherichia coli

    PubMed Central

    Di Pietro, Fabio; Brandi, Anna; Dzeladini, Nadire; Fabbretti, Attilio; Carzaniga, Thomas; Piersimoni, Lolita; Pon, Cynthia L; Giuliodori, Anna Maria

    2013-01-01

    Protein Y (PY) is an Escherichia coli cold-shock protein which has been proposed to be responsible for the repression of bulk protein synthesis during cold adaptation. Here, we present in vivo and in vitro data which clarify the role of PY and its mechanism of action. Deletion of yfiA, the gene encoding protein PY, demonstrates that this protein is dispensable for cold adaptation and is not responsible for the shutdown of bulk protein synthesis at the onset of the stress, although it is able to partially inhibit translation. In vitro assays reveal that the extent of PY inhibition changes with different mRNAs and that this inhibition is related to the capacity of PY of binding 30S subunits with a fairly strong association constant, thus stimulating the formation of 70S monomers. Furthermore, our data provide evidence that PY competes with the other ribosomal ligands for the binding to the 30S subunits. Overall these results suggest an alternative model to explain PY function during cold shock and to reconcile the inhibition caused by PY with the active translation observed for some mRNAs during cold shock. PMID:23420694

  8. Coordinated Ribosomal L4 Protein Assembly into the Pre-Ribosome Is Regulated by Its Eukaryote-Specific Extension.

    PubMed

    Stelter, Philipp; Huber, Ferdinand M; Kunze, Ruth; Flemming, Dirk; Hoelz, André; Hurt, Ed

    2015-06-04

    Eukaryotic ribosome biogenesis requires nuclear import and hierarchical incorporation of ∼80 ribosomal proteins (RPs) into the ribosomal RNA core. In contrast to prokaryotes, many eukaryotic RPs possess long extensions that interdigitate in the mature ribosome. RpL4 is a prime example, with an ∼80-residue-long surface extension of unknown function. Here, we identify assembly chaperone Acl4 that initially binds the universally conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain, thereby restricting RpL4 loop insertion at its cognate nascent rRNA site. RpL4 release from Acl4 is orchestrated with pre-ribosome assembly, during which the eukaryote-specific RpL4 extension makes several distinct interactions with the 60S surface, including a co-evolved site on neighboring RpL18. Consequently, mutational inactivation of this contact site, on either RpL4 or RpL18, impairs RpL4-Acl4 disassembly and RpL4 pre-ribosome incorporation. We propose that hierarchical ribosome assembly can be achieved by eukaryotic RP extensions and dedicated assembly chaperones. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Positive modulation of RNA polymerase III transcription by ribosomal proteins

    SciTech Connect

    Dieci, Giorgio; Carpentieri, Andrea; Amoresano, Angela; Ottonello, Simone

    2009-02-06

    A yeast nuclear fraction of unknown composition, named TFIIIE, was reported previously to enhance transcription of tRNA and 5S rRNA genes in vitro. We show that TFIIIE activity co-purifies with a specific subset of ribosomal proteins (RPs) which, as revealed by chromatin immunoprecipitation analysis, generally interact with tRNA and 5S rRNA genes, but not with a Pol II-specific promoter. Only Rpl6Ap and Rpl6Bp, among the tested RPs, were found associated to a TATA-containing tRNA{sup Ile}(TAT) gene. The RPL6A gene also emerged as a strong multicopy suppressor of a conditional mutation in the basal transcription factor TFIIIC, while RPL26A and RPL14A behaved as weak suppressors. The data delineate a novel extra-ribosomal role for one or a few RPs which, by influencing 5S rRNA and tRNA synthesis, could play a key role in the coordinate regulation of the different sub-pathways required for ribosome biogenesis and functionality.

  10. Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.

    PubMed

    Kaur, Jasvinder; Stuart, Rosemary A

    2011-09-01

    Mitochondrial ribosomal protein 20 (Mrp20) is a component of the yeast mitochondrial large (54S) ribosomal subunit and is homologous to the bacterial L23 protein, located at the ribosomal tunnel exit site. The carboxy-terminal mitochondrial-specific domain of Mrp20 was found to have a crucial role in the assembly of the ribosomes. A new, membrane-bound, ribosomal-assembly subcomplex composed of known tunnel-exit-site proteins, an uncharacterized ribosomal protein, MrpL25, and the mitochondrial peroxiredoxin (Prx), Prx1, accumulates in an mrp20ΔC yeast mutant. Finally, data supporting the idea that the inner mitochondrial membrane acts as a platform for the ribosome assembly process are discussed.

  11. Proteins at the Polypeptide Tunnel Exit of the Yeast Mitochondrial Ribosome*

    PubMed Central

    Gruschke, Steffi; Gröne, Kerstin; Heublein, Manfred; Hölz, Stefanie; Israel, Lars; Imhof, Axel; Herrmann, Johannes M.; Ott, Martin

    2010-01-01

    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. PMID:20404317

  12. Ribosome-binding proteins Mdm38 and Mba1 display overlapping functions for regulation of mitochondrial translation.

    PubMed

    Bauerschmitt, Heike; Mick, David U; Deckers, Markus; Vollmer, Christine; Funes, Soledad; Kehrein, Kirsten; Ott, Martin; Rehling, Peter; Herrmann, Johannes M

    2010-06-15

    Biogenesis of respiratory chain complexes depends on the expression of mitochondrial-encoded subunits. Their synthesis occurs on membrane-associated ribosomes and is probably coupled to their membrane insertion. Defects in expression of mitochondrial translation products are among the major causes of mitochondrial disorders. Mdm38 is related to Letm1, a protein affected in Wolf-Hirschhorn syndrome patients. Like Mba1 and Oxa1, Mdm38 is an inner membrane protein that interacts with ribosomes and is involved in respiratory chain biogenesis. We find that simultaneous loss of Mba1 and Mdm38 causes severe synthetic defects in the biogenesis of cytochrome reductase and cytochrome oxidase. These defects are not due to a compromised membrane binding of ribosomes but the consequence of a mis-regulation in the synthesis of Cox1 and cytochrome b. Cox1 expression is restored by replacing Cox1-specific regulatory regions in the mRNA. We conclude, that Mdm38 and Mba1 exhibit overlapping regulatory functions in translation of selected mitochondrial mRNAs.

  13. Fluorescent protein tagging confirms the presence of ribosomal proteins at Drosophila polytene chromosomes

    PubMed Central

    Ramanathan, Preethi; Matina, Tina; Wen, Jikai

    2013-01-01

    Most ribosomal proteins (RPs) are stoichiometrically incorporated into ribosomal subunits and play essential roles in ribosome biogenesis and function. However, a number of RPs appear to have non-ribosomal functions, which involve direct association with pre-mRNA and transcription factors at transcription sites. The consensus is that the RPs found at these sites are off ribosomal subunits, but observation that different RPs are usually found together suggests that ribosomal or ribosomal-like subunits might be present. Notably, it has previously been reported that antibodies against 20 different RPs stain the same Pol II transcription sites in Drosophila polytene chromosomes. Some concerns, however, were raised about the specificity of the antibodies. To investigate further whether RPs are present at transcription sites in Drosophila, we have generated several transgenic flies expressing RPs (RpS2, RpS5a, RpS9, RpS11, RpS13, RpS18, RpL8, RpL11, RpL32, and RpL36) tagged with either green or red fluorescent protein. Imaging of salivary gland cells showed that these proteins are, as expected, abundant in the cytoplasm as well as in the nucleolus. However, these RPs are also apparent in the nucleus in the region occupied by the chromosomes. Indeed, polytene chromosome immunostaining of a representative subset of tagged RPs confirms the association with transcribed loci. Furthermore, characterization of a strain expressing RpL41 functionally tagged at its native genomic locus with YFP, also showed apparent nuclear accumulation and chromosomal association, suggesting that such a nuclear localization pattern might be a shared feature of RPs and is biologically important. We anticipate that the transgenes described here should provide a useful research tool to visualize ribosomal subunits in Drosophila tissues and to study the non-ribosomal functions of RPs. PMID:23638349

  14. Proteins on ribosome surface: Measurements of protein exposure by hot tritium bombardment technique

    PubMed Central

    Agafonov, Dmitry E.; Kolb, Vyacheslav A.; Spirin, Alexander S.

    1997-01-01

    The hot tritium bombardment technique [Goldanskii, V. I., Kashirin, I. A., Shishkov, A. V., Baratova, L. A. & Grebenshchikov, N. I. (1988) J. Mol. Biol. 201, 567–574] has been applied to measure the exposure of proteins on the ribosomal surface. The technique is based on replacement of hydrogen by high energy tritium atoms in thin surface layer of macromolecules. Quantitation of tritium radioactivity of each protein has revealed that proteins S1, S4, S5, S7, S18, S20, and S21 of the small subunit, and proteins L7/L12, L9, L10, L11, L16, L17, L24, and L27 of the large subunit are well exposed on the surface of the Escherichia coli 70 S ribosome. Proteins S8, S10, S12, S16, S17, L14, L20, L29, L30, L31, L32, L33, and L34 have virtually no groups exposed on the ribosomal surface. The remaining proteins are found to be exposed to lesser degree than the well exposed ones. No additional ribosomal proteins was exposed upon dissociation of ribosomes into subunits, thus indicating the absence of proteins on intersubunit contacting surfaces. PMID:9371771

  15. Mosaic tetracycline resistance genes encoding ribosomal protection proteins

    PubMed Central

    Warburton, Philip J.; Amodeo, Nina; Roberts, Adam P.

    2016-01-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria. PMID:27494928

  16. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    PubMed

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  17. Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences

    PubMed Central

    Green, Lisa; Houck-Loomis, Brian; Yueh, Andrew

    2012-01-01

    Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release. PMID:22718819

  18. Molecular evolution of the mammalian ribosomal protein gene, RPS14.

    PubMed

    Rhoads, D D; Roufa, D J

    1991-07-01

    Ribosomal protein S14 genes (RPS14) in eukaryotic species from protozoa to primates exhibit dramatically different intron-exon structures yet share homologous polypeptide-coding sequences. To recognize common features of RPS14 gene architectures in closely related mammalian species and to evaluate similarities in their noncoding DNA sequences, we isolated the intron-containing S14 locus from Chinese hamster ovary (CHO) cell DNA by using a PCR strategy and compared it with human RPS14. We found that rodent and primate S14 genes are composed of identical protein-coding exons interrupted by introns at four conserved DNA sites. However, the structures of corresponding CHO and human RPS14 introns differ significantly. Nonetheless, individual intron splice donor, splice acceptor, and upstream flanking motifs have been conserved within mammalian S14 homologues as well as within RPS14 gene fragments PCR amplified from other vertebrate genera (birds and bony fish). Our data indicate that noncoding, intronic DNA sequences within highly conserved, single-copy ribosomal protein genes are useful molecular landmarks for phylogenetic analysis of closely related vertebrate species.

  19. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

    PubMed Central

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-01-01

    AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

  20. Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

    PubMed

    Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

    2016-02-15

    To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

  1. Interplay between trigger factor and other protein biogenesis factors on the ribosome

    NASA Astrophysics Data System (ADS)

    Bornemann, Thomas; Holtkamp, Wolf; Wintermeyer, Wolfgang

    2014-06-01

    Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.

  2. Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system

    PubMed Central

    Sung, Min-Kyung; Reitsma, Justin M.; Sweredoski, Michael J.; Hess, Sonja; Deshaies, Raymond J.

    2016-01-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin–proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

  3. Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry*

    PubMed Central

    Lauber, Matthew A.; Rappsilber, Juri; Reilly, James P.

    2012-01-01

    Ribosomal protein S1 has been shown to be a significant effector of prokaryotic translation. The protein is in fact capable of efficiently initiating translation, regardless of the presence of a Shine-Dalgarno sequence in mRNA. Structural insights into this process have remained elusive, as S1 is recalcitrant to traditional techniques of structural analysis, such as x-ray crystallography. Through the application of protein cross-linking and high resolution mass spectrometry, we have detailed the ribosomal binding site of S1 and have observed evidence of its dynamics. Our results support a previous hypothesis that S1 acts as the mRNA catching arm of the prokaryotic ribosome. We also demonstrate that in solution the major domains of the 30S subunit are remarkably flexible, capable of moving 30–50Å with respect to one another. PMID:23033476

  4. A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein.

    PubMed Central

    Hellen, C U; Witherell, G W; Schmid, M; Shin, S H; Pestova, T V; Gil, A; Wimmer, E

    1993-01-01

    Initiation of translation of the RNA genomes of picornaviruses such as poliovirus and encephalomyocarditis virus is cap-independent and results from interaction of ribosomes with a segment of the 5' noncoding region of these mRNAs termed the internal ribosomal entry site. Genetic and biochemical studies have previously shown that a 57-kDa cytoplasmic RNA-binding protein (p57) plays an essential role in this translation mechanism. We have now found that p57 shares physical, biochemical, and antigenic properties with the pyrimidine tract-binding protein (PTB), a nuclear protein that has been implicated in various processes involving pre-mRNA. These data indicate that p57 and PTB are the same protein. Purified recombinant PTB bound specifically to a bulged hairpin within the internal ribosomal entry site of encephalomyocarditis virus and had a much lower affinity for a mutated derivative of this hairpin and for unrelated RNAs. Immunodepletion of p57/PTB from a HeLa cell-free lysate inhibited translation of poliovirus and encephalomyocarditis virus mRNAs but had no effect on translation of beta-globin mRNA, confirming the essential role of p57 in translation by internal ribosomal entry. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8395052

  5. Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition

    PubMed Central

    Hafner, Markus; Max, Klaas E.A.; Bandaru, Pradeep; Morozov, Pavel; Gerstberger, Stefanie; Brown, Miguel; Molina, Henrik; Tuschl, Thomas

    2013-01-01

    Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ∼3000 mRNAs at ∼9500 sites located in the 3′ UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. PMID:23481595

  6. The conformation of a nascent polypeptide inside the ribosome tunnel affects protein targeting and protein folding

    PubMed Central

    Peterson, Janine H.; Woolhead, Cheryl A.; Bernstein, Harris D.

    2010-01-01

    In this report we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP1-25) associated with the signal peptide of the E. coli EspP protein. It was previously shown that EspP1-25 inhibits signal peptide recognition by the signal recognition particle (SRP), and we now show that fusion of EspP1-25 to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP1-25 inside the ribosome tunnel. First, we found that mutations in EspP1-25 that abolished its effects on protein targeting and protein folding altered the crosslinking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP1-25 mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli. PMID:20804452

  7. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation.

    PubMed

    Lawrence, Marlon G; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M; Lindahl, Lasse

    2016-07-08

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the ONLY: sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA(crb) pause peptide. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. The extended loops of ribosomal proteins uL4 and uL22 of Escherichia coli contribute to ribosome assembly and protein translation

    PubMed Central

    Lawrence, Marlon G.; Shamsuzzaman, Md; Kondopaka, Maithri; Pascual, Clarence; Zengel, Janice M.; Lindahl, Lasse

    2016-01-01

    Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlAcrb pause peptide. PMID:27257065

  9. Elucidation of a C-rich signature motif in target mRNAs of RNA-binding protein TIAR.

    PubMed

    Kim, Henry S; Kuwano, Yuki; Zhan, Ming; Pullmann, Rudolf; Mazan-Mamczarz, Krystyna; Li, Huai; Kedersha, Nancy; Anderson, Paul; Wilce, Matthew C J; Gorospe, Myriam; Wilce, Jacqueline A

    2007-10-01

    The RNA-binding protein TIAR (related to TIA-1 [T-cell-restricted intracellular antigen 1]) was shown to associate with subsets of mRNAs bearing U-rich sequences in their 3' untranslated regions. TIAR can function as a translational repressor, particularly in response to cytotoxic agents. Using unstressed colon cancer cells, collections of mRNAs associated with TIAR were isolated by immunoprecipitation (IP) of (TIAR-RNA) ribonucleoprotein (RNP) complexes, identified by microarray analysis, and used to elucidate a common signature motif present among TIAR target transcripts. The predicted TIAR motif was an unexpectedly cytosine-rich, 28- to 32-nucleotide-long element forming a stem and a loop of variable size with an additional side loop. The ability of TIAR to bind an RNA oligonucleotide with a representative C-rich TIAR motif sequence was verified in vitro using surface plasmon resonance. By this analysis, TIAR containing two or three RNA recognition domains (TIAR12 and TIAR123) showed low but significant binding to the C-rich sequence. In vivo, insertion of the C-rich motif into a heterologous reporter strongly suppressed its translation in cultured cells. Using this signature motif, an additional approximately 2,209 UniGene targets were identified (2.0% of the total UniGene database). A subset of specific mRNAs were validated by RNP IP analysis. Interestingly, in response to treatment with short-wavelength UV light (UVC), a stress agent causing DNA damage, each of these target mRNAs bearing C-rich motifs dissociated from TIAR. In turn, expression of the encoded proteins was elevated in a TIAR-dependent manner. In sum, we report the identification of a C-rich signature motif present in TIAR target mRNAs whose association with TIAR decreases following exposure to a stress-causing agent.

  10. Ribosomal proteins: toward a next generation standard for prokaryotic systematics?

    PubMed

    Ramulu, Hemalatha Golaconda; Groussin, Mathieu; Talla, Emmanuel; Planel, Remi; Daubin, Vincent; Brochier-Armanet, Céline

    2014-06-01

    The seminal work of Carl Woese and co-workers has contributed to promote the RNA component of the small subunit of the ribosome (SSU rRNA) as a "gold standard" of modern prokaryotic taxonomy and systematics, and an essential tool to explore microbial diversity. Yet, this marker has a limited resolving power, especially at deep phylogenetic depth and can lead to strongly biased trees. The ever-larger number of available complete genomes now calls for a novel standard dataset of robust protein markers that may complement SSU rRNA. In this respect, concatenation of ribosomal proteins (r-proteins) is being growingly used to reconstruct large-scale prokaryotic phylogenies, but their suitability for systematic and/or taxonomic purposes has not been specifically addressed. Using Proteobacteria as a case study, we show that amino acid and nucleic acid r-protein sequences contain a reliable phylogenetic signal at a wide range of taxonomic depths, which has not been totally blurred by mutational saturation or horizontal gene transfer. The use of accurate evolutionary models and reconstruction methods allows overcoming most tree reconstruction artefacts resulting from compositional biases and/or fast evolutionary rates. The inferred phylogenies allow clarifying the relationships among most proteobacterial orders and families, along with the position of several unclassified lineages, suggesting some possible revisions of the current classification. In addition, we investigate the root of the Proteobacteria by considering the time-variation of nucleic acid composition of r-protein sequences and the information carried by horizontal gene transfers, two approaches that do not require the use of an outgroup and limit tree reconstruction artefacts. Altogether, our analyses indicate that r-proteins may represent a promising standard for prokaryotic taxonomy and systematics. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. An inventory of yeast proteins associated with nucleolar and ribosomal components

    PubMed Central

    Staub, Eike; Mackowiak, Sebastian; Vingron, Martin

    2006-01-01

    Background Although baker's yeast is a primary model organism for research on eukaryotic ribosome assembly and nucleoli, the list of its proteins that are functionally associated with nucleoli or ribosomes is still incomplete. We trained a naïve Bayesian classifier to predict novel proteins that are associated with yeast nucleoli or ribosomes based on parts lists of nucleoli in model organisms and large-scale protein interaction data sets. Phylogenetic profiling and gene expression analysis were carried out to shed light on evolutionary and regulatory aspects of nucleoli and ribosome assembly. Results We predict that, in addition to 439 known proteins, a further 62 yeast proteins are associated with components of the nucleolus or the ribosome. The complete set comprises a large core of archaeal-type proteins, several bacterial-type proteins, but mostly eukaryote-specific inventions. Expression of nucleolar and ribosomal genes tends to be strongly co-regulated compared to other yeast genes. Conclusion The number of proteins associated with nucleolar or ribosomal components in yeast is at least 14% higher than known before. The nucleolus probably evolved from an archaeal-type ribosome maturation machinery by recruitment of several bacterial-type and mostly eukaryote-specific factors. Not only expression of ribosomal protein genes, but also expression of genes encoding the 90S processosome, are strongly co-regulated and both regulatory programs are distinct from each other. PMID:17067374

  12. Ribosomal Protein S14 Negatively Regulates c-Myc Activity*

    PubMed Central

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Liao, Peng; Lu, Hua

    2013-01-01

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  13. Structural insights into the interaction of the ribosomal P stalk protein P2 with a type II ribosome-inactivating protein ricin

    PubMed Central

    Fan, Xiaojiao; Zhu, Yuwei; Wang, Chongyuan; Niu, Liwen; Teng, Maikun; Li, Xu

    2016-01-01

    Ricin is a type II ribosome-inactivating protein (RIP) that depurinates A4324 at the sarcin-ricin loop of 28 S ribosomal RNA (rRNA), thus inactivating the ribosome by preventing elongation factors from binding to the GTPase activation centre. Recent studies have disclosed that the conserved C-terminal domain (CTD) of eukaryotic ribosomal P stalk proteins is involved in the process that RIPs target ribosome. However, the details of the molecular interaction between ricin and P stalk proteins remain unknown. Here, we report the structure of ricin-A chain (RTA) in a complex with the CTD of the human ribosomal protein P2. The structure shows that the Phe111, Leu113 and Phe114 residues of P2 insert into a hydrophobic pocket formed by the Tyr183, Arg235, Phe240 and Ile251 residues of RTA, while Asp115 of P2 forms hydrogen bonds with Arg235 of RTA. The key residues in RTA and P2 for complex formation were mutated, and their importance was determined by pull-down assays. The results from cell-free translation assays further confirmed that the interaction with P stalk proteins is essential for the inhibition of protein synthesis by RTA. Taken together, our results provide a structural basis that will improve our understanding of the process by which ricin targets the ribosome, which will benefit the development of effective small-molecule inhibitors for use as therapeutic agents. PMID:27886256

  14. Protein synthesis inhibitors enhance the expression of mRNAs for early inducible inflammatory genes via mRNA stabilization.

    PubMed

    Yamazaki, Soh; Takeshige, Koichiro

    2008-02-01

    Expression of inflammatory genes is regulated at multiple steps, including transcriptional activation and mRNA stabilization. During an investigation into the requirement of de novo protein synthesis for the induction of inflammatory genes, it was revealed that protein synthesis inhibitors unexpectedly potentiated the induction of mRNAs for primary response genes, while the inhibitors suppressed the induction of secondary inducible genes as previously described. Stimulus-induced nuclear translocation and promoter recruitment of NF-kappaB, which is responsible for the transcriptional activation of many inflammatory genes, were largely unaffected by the inhibitors. Instead, these inhibitors prolonged the half-lives of all of the primary inducible mRNAs tested. Thus, these findings emphasize the important contribution of regulated mRNA longevity to gene expression induced by pro-inflammatory stimulation.

  15. Erythromycin- and chloramphenicol-induced ribosomal assembly defects are secondary effects of protein synthesis inhibition.

    PubMed

    Siibak, Triinu; Peil, Lauri; Xiong, Liqun; Mankin, Alexander; Remme, Jaanus; Tenson, Tanel

    2009-02-01

    Several protein synthesis inhibitors are known to inhibit ribosome assembly. This may be a consequence of direct binding of the antibiotic to ribosome precursor particles, or it could result indirectly from loss of coordination in the production of ribosomal components due to the inhibition of protein synthesis. Here we demonstrate that erythromycin and chloramphenicol, inhibitors of the large ribosomal subunit, affect the assembly of both the large and small subunits. Expression of a small erythromycin resistance peptide acting in cis on mature ribosomes relieves the erythromycin-mediated assembly defect for both subunits. Erythromycin treatment of bacteria expressing a mixture of erythromycin-sensitive and -resistant ribosomes produced comparable effects on subunit assembly. These results argue in favor of the view that erythromycin and chloramphenicol affect the assembly of the large ribosomal subunit indirectly.

  16. Histone deacetylase 6 associates with ribosomes and regulates de novo protein translation during arsenite stress.

    PubMed

    Kappeler, Kyle V; Zhang, Jack; Dinh, Thai Nho; Strom, Joshua G; Chen, Qin M

    2012-05-01

    Histone deacetylase 6 (HDAC6) is known as a cytoplasmic enzyme that regulates cell migration, cell adhesion, and degradation of misfolded proteins by deacetylating substrates such as α-tubulin and Hsp90. When HaCaT keratinocytes were exposed to 1-200μM sodium arsenite, we observed perinuclear localization of HDAC6 within 30 min. Although the overall level of HDAC6 protein did not change, sodium arsenite caused an increase of HDAC6 in ribosomal fractions. Separation of ribosomal subunits versus intact ribosomes or polysomes indicated that HDAC6 was mainly detected in 40/43S fractions containing the small ribosomal subunit in untreated cells but was associated with 40/43S and 60/80S ribosomal fractions in arsenite-treated cells. Immunocytochemistry studies revealed that arsenite caused colocalization of HDAC6 with the ribosomal large and small subunit protein L36a and S6. Both L36a and S6 were detected in the immunocomplex of HDAC6 isolated from arsenite-treated cells. The observed physical interaction of HDAC6 with ribosomes pointed to a role of HDAC6 in stress-induced protein translation. Among arsenite stress-induced proteins, de novo Nrf2 protein translation was inhibited by Tubastatin A. These data demonstrate that HDAC6 was recruited to ribosomes, physically interacted with ribosomal proteins, and regulated de novo protein translation in keratinocytes responding to arsenite stress.

  17. Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus

    SciTech Connect

    Inglis, S.C.

    1982-12-01

    Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpes virus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRN As in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.

  18. Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

    PubMed Central

    de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

    2010-01-01

    Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

  19. Specialized ribosomes: a new frontier in gene regulation and organismal biology.

    PubMed

    Xue, Shifeng; Barna, Maria

    2012-05-23

    Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than intrinsic regulatory capacity in mRNA translation. However, emerging studies reveal that ribosome activity may be highly regulated. Heterogeneity in ribosome composition resulting from differential expression and post-translational modifications of ribosomal proteins, ribosomal RNA (rRNA) diversity and the activity of ribosome-associated factors may generate 'specialized ribosomes' that have a substantial impact on how the genomic template is translated into functional proteins. Moreover, constitutive components of the ribosome may also exert more specialized activities by virtue of their interactions with specific mRNA regulatory elements such as internal ribosome entry sites (IRESs) or upstream open reading frames (uORFs). Here we discuss the hypothesis that intrinsic regulation by the ribosome acts to selectively translate subsets of mRNAs harbouring unique cis-regulatory elements, thereby introducing an additional level of regulation in gene expression and the life of an organism.

  20. The Circadian Clock Coordinates Ribosome Biogenesis

    PubMed Central

    Symul, Laura; Martin, Eva; Atger, Florian; Naef, Felix; Gachon, Frédéric

    2013-01-01

    Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. PMID:23300384

  1. Characteristics and clustering of human ribosomal protein genes

    PubMed Central

    Ishii, Kyota; Washio, Takanori; Uechi, Tamayo; Yoshihama, Maki; Kenmochi, Naoya; Tomita, Masaru

    2006-01-01

    Background The ribosome is a central player in the translation system, which in mammals consists of four RNA species and 79 ribosomal proteins (RPs). The control mechanisms of gene expression and the functions of RPs are believed to be identical. Most RP genes have common promoters and were therefore assumed to have a unified gene expression control mechanism. Results We systematically analyzed the homogeneity and heterogeneity of RP genes on the basis of their expression profiles, promoter structures, encoded amino acid compositions, and codon compositions. The results revealed that (1) most RP genes are coordinately expressed at the mRNA level, with higher signals in the spleen, lymph node dissection (LND), and fetal brain. However, 17 genes, including the P protein genes (RPLP0, RPLP1, RPLP2), are expressed in a tissue-specific manner. (2) Most promoters have GC boxes and possible binding sites for nuclear respiratory factor 2, Yin and Yang 1, and/or activator protein 1. However, they do not have canonical TATA boxes. (3) Analysis of the amino acid composition of the encoded proteins indicated a high lysine and arginine content. (4) The major RP genes exhibit a characteristic synonymous codon composition with high rates of G or C in the third-codon position and a high content of AAG, CAG, ATC, GAG, CAC, and CTG. Conclusion Eleven of the RP genes are still identified as being unique and did not exhibit at least some of the above characteristics, indicating that they may have unknown functions not present in other RP genes. Furthermore, we found sequences conserved between human and mouse genes around the transcription start sites and in the intronic regions. This study suggests certain overall trends and characteristic features of human RP genes. PMID:16504170

  2. Ribosomal protein gene mapping and human chromosomal disorders

    SciTech Connect

    Kenmochi, N.; Goodman, N.; Page, D.C.

    1994-09-01

    In Drosophila, the Minute phenotype (reduced body size, diminished viability and fertility, and short, thin bristles) results from heterozygous deficiencies (deletions) at any one of 50 loci scattered about the genome. A handful of these Minute loci have been molecularly characterized, and all have been found to encode ribosomal proteins. Thus, the Minute phenotype appears to result from reduced protein synthetic capacity in flies with one rather than two copies of a given ribosomal protein (rp) gene. We are pursuing the possibility that similar reductions in protein synthetic capacity--again resulting from rp gene deficiencies--might underlie phenotypes associated with certain chromosomal disorders in humans. We and our colleagues have reported findings consistent with a role for RPS4 deficiency in the etiology of certain features of Turner syndrome, a complex human disorder classically associated with an XO karyotype. We are intrigued by the possibility that deficiencies of other human rp genes might cause phenotypic abnormalities similar to those seen in Turner syndrome--just as deficiencies of any of a number of Drosophila rp genes cause the Minute phenotype. We must first learn the chromosomal map position of each of the estimated 83 human rp genes. The task of mapping the functional (intron-containing) rp genes is complicated by the existence of processed pseudogenes elsewhere in the genome. To date, we have assigned (or confirmed the previous assignment of) 38 rp genes to individual human chromosomes by PCR analysis of human-rodent somatic cell hybrids containing subsets of human chromosomes, with all but four chromosomes carrying at least one rp gene. We have also identified more than 100 large-insert human YAC (yeast artificial chromosome) clones that contain individual rp genes. Such screening of YAC libraries will result in precise positioning of the rp genes on the emerging physical map of the human genome.

  3. Inactivation of ribosomes by an inhibitor of protein synthesis from Salmonella enteritidis.

    PubMed

    Brigotti, M; Nanetti, A; Montanaro, L; Sperti, S

    1993-01-01

    Sonic extracts of Salmonella enteritidis contain a factor which inhibits protein synthesis in cell-free systems by irreversibly inactivating ribosomes. The extent of the inactivation of ribosomes depends on the system used to assay protein synthesis, natural mRNA translation being more strongly inhibited than poly(U) translation. The inhibitory power of the Salmonella factor is destroyed by trypsin and by 5% mercaptoethanol. Placental RNase inhibitor is unable to protect ribosomes from inactivation.

  4. Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs

    PubMed Central

    Soheilypour, M.; Mofrad, M. R. K.

    2016-01-01

    Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus. PMID:27805000

  5. Are there proteins between the ribosomal subunits? Hot tritium bombardment experiments.

    PubMed

    Yusupov, M M; Spirin, A S

    1986-03-03

    The hot tritium bombardment technique [(1976) Dokl. Akad. Nauk SSSR 228, 1237-1238] was used for studying the surface localization of ribosomal proteins on Escherichia coli ribosomes. The degree of tritium labeling of proteins was considered as a measure of their exposure (surface localization). Proteins S1, S4, S7, S9 and/or S11, S12 and/or L20, S13, S18, S20, S21, L5, L6, L7/L12, L10, L11, L16, L17, L24, L26 and L27 were shown to be the most exposed on the ribosome surface. The sets of exposed ribosomal proteins on the surface of 70 S ribosomes, on the one hand, and the surfaces of 50 S and 30 S ribosomal subunits in the dissociated state, on the other, were compared. It was found that the dissociation of ribosomes into subunits did not result in exposure of additional ribosomal proteins. The conclusion was drawn that proteins are absent from the contacting surfaces of the ribosomal subunits.

  6. Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    PubMed Central

    Fuchs, Gabriele; Diges, Camille; Kohlstaedt, Lori A.; Wehner, Karen A.; Sarnow, Peter

    2011-01-01

    Ribosomes exist as a heterogenous pool of macromolecular complexes composed of ribosomal RNA molecules, ribosomal proteins and numerous associated, “non-ribosomal” proteins. To identify non-ribosomal proteins that may modulate ribosome activity, we examined the composition of translationally active and inactive ribosomes using a proteomic multidimensional protein identification technology. Notably, the phosphorylated isoform of glycogen synthase, GYS1, was preferentially associated with elongating ribosomes. Depletion of GYS1 affected the translation of a subset of cellular mRNAs, some of which encode proteins that modulate protein biosynthesis. These findings argue that GYS1 abundance, and by virtue of its ribosomal association, provide a feedback loop between the energy state of the cells and the translation machinery. PMID:21570405

  7. Assembly of the 30S ribosomal subunit: positioning ribosomal protein S13 in the S7 assembly branch.

    PubMed

    Grondek, Joel F; Culver, Gloria M

    2004-12-01

    Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 A away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3' major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data.

  8. The P1/P2 proteins of the human ribosomal stalk are required for ribosome binding and depurination by ricin in human cells

    PubMed Central

    May, Kerrie L.; Li, Xiao-Ping; Martínez-Azorín, Francisco; Ballesta, Juan P.G.; Grela, Przemysław; Tchórzewski, Marek; Tumer, Nilgun E.

    2012-01-01

    Ricin A chain (RTA) depurinates the sarcin/ricin loop (SRL) of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1/P2 bound to P0, while the yeast stalk consists of two different heterodimers, P1α/P2β and P2α/P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here we examine whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the SRL. Using siRNA-mediated knockdown of P1/P2 expression in human cells, we demonstrate that the depurination activity of RTA is lower when P1 and P2 protein levels are reduced. Ribosomes from P1/P2-depleted cells have a reduced ability to bind RTA by Biacore analysis, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1/P2 dimer, further demonstrating the importance of the human P1/P2 proteins in enabling RTA to bind and depurinate human ribosomes. PMID:22909382

  9. Alternative patterns of transcription and translation of the ribosomal protein L32 mRNA in somatic and spermatogenic cells in mice.

    PubMed

    Kleene, Kenneth C; Cataldo, Leah; Mastrangelo, Mary-Ann; Tagne, Jean-Bosco

    2003-11-15

    The patterns of transcription and translation of the ribosomal protein L32 (Rpl32) mRNA differ greatly in adult testis and somatic tissues. Northern blots reveal that the levels of Rpl32 mRNA are four- to five-fold higher in prepubertal and adult testes, and purified pachytene spermatocytes and round spermatids than in a variety of nongrowing adult somatic tissues. 5' RACE demonstrates that transcription in 8-day prepubertal testis, which lacks meiotic and haploid cells, strongly prefers the same start site in the 5' terminal oligopyrimidine tract (5' TOP) that is used is somatic cells. The 5' TOP is a cis element that inhibits translation of many mRNAs in nongrowing somatic cells. Although the sizes of deadenylated Rpl32 mRNAs are indistinguishable in somatic and spermatogenic cells, transcription initiates at 11 sites over a 31-nt segment in adult testis and approximately 62% of Rpl32 mRNAs lack a 5' TOP. In agreement with previous studies, low levels of cycloheximide increase the proportions and sizes of polysomes in absorbance profiles, and increase the proportions and sizes of polysomes translating four 5' TOP mRNA species including the Rpl32 mRNA in 8-day seminiferous tubules. In contrast, cycloheximide has little or no effect on the absorbance profiles and distribution of Rpl32 mRNA and 5' TOP mRNAs in adult seminiferous tubules. The failure of cycloheximide to increase the size of polysomes in adult seminiferous tubules implies a block in the pathway by which ribosomes are recruited onto translationally active mRNAs.

  10. Characterization of hibernating ribosomes in mammalian cells

    PubMed Central

    Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A

    2011-01-01

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions. PMID:21768774

  11. Characterization of hibernating ribosomes in mammalian cells.

    PubMed

    Krokowski, Dawid; Gaccioli, Francesca; Majumder, Mithu; Mullins, Michael R; Yuan, Celvie L; Papadopoulou, Barbara; Merrick, William C; Komar, Anton A; Taylor, Derek; Hatzoglou, Maria

    2011-08-15

    Protein synthesis across kingdoms involves the assembly of 70S (prokaryotes) or 80S (eukaryotes) ribosomes on the mRNAs to be translated. 70S ribosomes are protected from degradation in bacteria during stationary growth or stress conditions by forming dimers that migrate in polysome profiles as 100S complexes. Formation of ribosome dimers in Escherichia coli is mediated by proteins, namely the ribosome modulation factor (RMF), which is induced in the stationary phase of cell growth. It is reported here a similar ribosomal complex of 110S in eukaryotic cells, which forms during nutrient starvation. The dynamic nature of the 110S ribosomal complex (mammalian equivalent of the bacterial 100S) was supported by the rapid conversion into polysomes upon nutrient-refeeding via a mechanism sensitive to inhibitors of translation initiation. Several experiments were used to show that the 110S complex is a dimer of nontranslating ribosomes. Cryo-electron microscopy visualization of the 110S complex revealed that two 80S ribosomes are connected by a flexible, albeit localized, interaction. We conclude that, similarly to bacteria, rat cells contain stress-induced ribosomal dimers. The identification of ribosomal dimers in rat cells will bring new insights in our thinking of the ribosome structure and its function during the cellular response to stress conditions.

  12. Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable.

    PubMed

    Vanet, A; Plumbridge, J A; Guérin, M F; Alix, J H

    1994-12-01

    The prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11-methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C-terminus of the protein and plasmids overproducing the wild-type and the two mutant proteins have been constructed. The wild-type PrmA protein could be crosslinked to its radiolabelled substrate, S-adenosyl-L-methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H-labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro-constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.

  13. Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

    PubMed

    Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C; Vanderklish, Peter W; Edelman, Gerald M; Mauro, Vincent P

    2005-02-08

    The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

  14. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues. [Zea mays

    SciTech Connect

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-11-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of ({sup 32}P) ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of {sup 32}P incorporation and the electrophoretic patterns were dependent on {sup 32}P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K{sub m} values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins.

  15. Phosphorylation of Ribosomal Proteins Induced by Auxins in Maize Embryonic Tissues

    PubMed Central

    Pérez, Laura; Aguilar, Raúl; Méndez, Alma Pérez; de Jiménez, Estela Sánchez

    1990-01-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [32P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32P incorporation and the electrophoretic patterns were dependent on 32P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent Km values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins. Images Figure 3 PMID:16667828

  16. Isolation and characterization of ribosome-inactivating proteins from Cucurbitaceae.

    PubMed

    Zhang, Daoning; Halaweish, Fathi T

    2007-03-01

    Due to their RNA-N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in biomedical and agricultural research. We have isolated and characterized two such proteins, foetidissimin II and texanin, from two Cucurbitaceae species. Foetidissimin II, obtained from the roots of Cucurbita foetidissima, was identified as a type-2 RIP, with a molecular weight of 61 kDa, as estimated by gel electrophoresis. It is composed of two chains, a 29-kDa chain A, and a 32-kDa chain B. Texanin, isolated from the fruits of Cucurbita texana, is a type-I RIP, with a single chain of molecular weight 29.7 kDa, as estimated by MALDI-TOF-MS. Both proteins exhibit RNA-N-glycosidase activity, with aniline playing a critical role in rRNA cleavage. The IC50 value of foetidissimin II, determined by cell-free protein-synthesis inhibition, was 0.251 muM. In an in vitro cytotoxicity assay, foetidissimin II exhibited IC50 values of ca. 70 nM to both adenocarcinoma and erythroleukemia cells. Texanin exhibited a weaker anticancer activity against erythroleukemia cells, with an IC50 value of 95 microM, but no activity against adenocarcinoma cells. The N-terminal sequences of both proteins were compared with those of reported RIPs.

  17. Ribosomally encoded antibacterial proteins and peptides from Pseudomonas.

    PubMed

    Ghequire, Maarten G K; De Mot, René

    2014-07-01

    Members of the Pseudomonas genus produce diverse secondary metabolites affecting other bacteria, fungi or predating nematodes and protozoa but are also equipped with the capacity to secrete different types of ribosomally encoded toxic peptides and proteins, ranging from small microcins to large tailocins. Studies with the human pathogen Pseudomonas aeruginosa have revealed that effector proteins of type VI secretion systems are part of the antibacterial armamentarium deployed by pseudomonads. A novel class of antibacterial proteins with structural similarity to plant lectins was discovered by studying antagonism among plant-associated Pseudomonas strains. A genomic perspective on pseudomonad bacteriocinogeny shows that the modular architecture of S pyocins of P. aeruginosa is retained in a large diversified group of bacteriocins, most of which target DNA or RNA. Similar modularity is present in as yet poorly characterized Rhs (recombination hot spot) proteins and CDI (contact-dependent inhibition) proteins. Well-delimited domains for receptor recognition or cytotoxicity enable the design of chimeric toxins with novel functionalities, which has been applied successfully for S and R pyocins. Little is known regarding how these antibacterials are released and ultimately reach their targets. Other remaining issues concern the identification of environmental triggers activating these systems and assessment of their ecological impact in niches populated by pseudomonads. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  18. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis.

    PubMed

    Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E; Gonzalez, Ruben L

    2011-08-21

    Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex during translocation.

  19. Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis.

    PubMed

    Erlacher, Matthias D; Chirkova, Anna; Voegele, Paul; Polacek, Norbert

    2011-05-01

    The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

  20. Dom34 Rescues Ribosomes in 3´ Untranslated Regions

    PubMed Central

    Guydosh, Nicholas R.; Green, Rachel

    2014-01-01

    SUMMARY Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3´ UTRs. These ribosomes appear to gain access to the 3 UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them. PMID:24581494

  1. Dom34 rescues ribosomes in 3' untranslated regions.

    PubMed

    Guydosh, Nicholas R; Green, Rachel

    2014-02-27

    Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here, we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3' UTRs. These ribosomes appear to gain access to the 3' UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

  2. Mutations in ribosomal proteins S4 and S12 influence the higher order structure of 16 S ribosomal RNA.

    PubMed

    Allen, P N; Noller, H F

    1989-08-05

    We have studied the effects of protein mutations on the higher order structure of 16 S rRNA in Escherichia coli ribosomes, using a set of structure-sensitive chemical probes. Ten mutant strains were studied, which contained alterations in ribosomal proteins S4 and S12, including double mutants containing both altered S4 and S12. Two ribosomal ambiguity (ram) S4 mutant strains, four streptomycin resistant (SmR) S12 mutant strains, one streptomycin pseudodependent (SmP) S12 mutant strain, one streptomycin dependent (SmD) S12 mutant strain and two streptomycin independent (Sm1) double mutants (containing both-SmD and ram mutations) were probed and compared to an isogenic wild-type strain. In ribosomes from strains containing S4 ram mutations, nucleotides A8 and A26 become more reactive to dimethyl sulfate (DMS) at their N-1 positions. In ribosomes from strains bearing the SmD allele, A908, A909, A1413 and G1487 are significantly less reactive to chemical probes. These same effects are observed when the S4 and S12 mutations are present simultaneously in the double mutants. An interesting correlation is found between the reactivity of A908 and the miscoding potential of SmR, SmD, SmP and wild-type ribosomes; the reactivity of A908 increases as the translational error frequency of the ribosomes increases. In the case of ram ribosomes, the reactivity of A908 resembles that of wild-type, unless tRNA is bound, in which case it becomes hyper-reactive. Similarly, streptomycin has little effect on A908 in wild-type ribosomes unless tRNA is bound, in which case its reactivity increases to resemble that of ram ribosomes with bound tRNA. Finally, interaction of streptomycin with SmP and SmD ribosomes causes the reactivity of A908 to increase to near-wild-type levels. A simple model is proposed, in which the reactivity of A908 reflects the position of an equilibrium between two conformational states of the 30 S subunit, one of which is DMS-reactive, and the other DMS

  3. The Ribosome Filter Redux

    PubMed Central

    Mauro, Vincent P.; Edelman, Gerald M.

    2010-01-01

    The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it. PMID:17890902

  4. Autogenous Regulation of Splicing of the Transcript of a Yeast Ribosomal Protein Gene

    NASA Astrophysics Data System (ADS)

    Dabeva, Mariana D.; Post-Beittenmiller, Martha A.; Warner, Jonathan R.

    1986-08-01

    The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.

  5. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  6. The integrity of the G2421-C2395 base pair in the ribosomal E-site is crucial for protein synthesis

    PubMed Central

    Koch, Miriam; Clementi, Nina; Rusca, Nicola; Vögele, Paul; Erlacher, Matthias; Polacek, Norbert

    2015-01-01

    During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis. PMID:25826414

  7. Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

    PubMed Central

    Zhu, Yiping; Chen, Guifang; Lv, Fengxiang; Wang, Xinlu; Ji, Xin; Xu, Yihui; Sun, Jing; Wu, Li; Zheng, Yong-Tang; Gao, Guangxia

    2011-01-01

    The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3′ end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5′ end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5′ and 3′ mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs. PMID:21876179

  8. Rhn1, a nuclear protein, is required for suppression of meiotic mRNAs in mitotically dividing fission yeast.

    PubMed

    Sugiyama, Tomoyasu; Sugioka-Sugiyama, Rie; Hada, Kazumasa; Niwa, Ryusuke

    2012-01-01

    In the fission yeast Schizosaccharomyces pombe, many meiotic mRNAs are transcribed during mitosis and meiosis and selectively eliminated in mitotic cells. However, this pathway for mRNA decay, called the determinant of selective removal (DSR)-Mmi1 system, targets only some of the numerous meiotic mRNAs that are transcribed in mitotic cells. Here we describe Rhn1, a nuclear protein involved in meiotic mRNA suppression in vegetative fission yeast. Rhn1 is homologous to budding yeast Rtt103 and localizes to one or a few discrete nuclear dots in growing vegetative cells. Rhn1 colocalizes with a pre-mRNA 3'-end processing factor, Pcf11, and with the 5'-3' exoribonuclease, Dhp1; moreover, Rhn1 coimmunoprecipitates with Pcf11. Loss of rhn1 results in elevated sensitivity to high temperature, to thiabendazole (TBZ), and to UV. Interestingly, meiotic mRNAs--including moa1(+), mcp5(+), and mug96(+)--accumulate in mitotic rhn1Δ cells. Accumulation of meiotic mRNAs also occurs in strains lacking Lsk1, a kinase that phosphorylates serine 2 (Ser-2) in the C-terminal domain (CTD) of RNA polymerase II (Pol II), and in strains lacking Sen1, an ATP-dependent 5'-3' RNA/DNA helicase: notably, both Lsk1 and Sen1 have been implicated in termination of Pol II-dependent transcription. Furthermore, RNAi knockdown of cids-2, a Caenorhabditis elegans ortholog of rhn1(+), leads to elevated expression of a germline-specific gene, pgl-1, in somatic cells. These results indicate that Rhn1 contributes to the suppression of meiotic mRNAs in vegetative fission yeast and that the mechanism by which Rhn1 downregulates germline-specific transcripts may be conserved in unicellular and multicellular organisms.

  9. Two classes of proteins and mRNAs in Lilium longiflorum L. indentified by human vitronectin probes.

    PubMed Central

    Wang, C S; Walling, L L; Gu, Y Q; Ware, C F; Lord, E M

    1994-01-01

    Vitronectin (VN) is a substrate adhesion molecule, an extracellular matrix glycoprotein that facilitates cell adhesion and cell movement in animals. We have reported the cross-reactivity of a 55-kD protein in plants with rabbit anti-human VN antibodies and the presence of VN-like sequences in plant genomes using a human VN cDNA probe. We have extended these studies by using human VN riboprobes to detect VN-like mRNAs in lily (Lilium longiflorum L.) and soybean. In both species, two mRNAs were detected. We have also identified a new cross-reactive protein (41 kD) using a different preparation of human VN antiserum. In lily roots five 41-kD isoforms were observed, whereas only three of these isoforms accumulated in leaves. Monospecific antibodies prepared against the plant proteins cross-reacted with the human VN protein and vice versa. We have purified the 41-kD protein using two-dimensional gel electrophoresis, and amino acid composition analysis indicates that it is similar in composition to human VN. PMID:7512737

  10. Organization of proteins in mammalian mitochondrial ribosomes: accessibility to lactoperoxidase-catalyzed radioiodination

    SciTech Connect

    Denslow, N.D.; O'Brien, T.W.

    1984-08-10

    To assess the relative exposure of individual ribosomal proteins (r-proteins) in the large and small subunits of the bovine mitochondrial ribosome, double label iodination technique was used. Regions of r-proteins exposed in purified ribosomal subunits were labeled with /sup 131/I using the lactoperoxidase-catalyzed iodination system, and additional reactive groups available upon denaturing the r-proteins in urea were labeled with /sup 125/I using the chloramine-T mediated reaction. The ratio of /sup 131/I to /sup 125/I incorporated into individual proteins under these conditions is representative of the degree of exposure for each of the proteins in the subunits. In this manner, the r-proteins have been grouped into 3 classes depending on their degree of exposure: high exposure, intermediate exposure, and essentially buried. While both subunits have a few proteins in the highly exposed group, and a large number of proteins in the intermediate exposure group, only the large ribosomal subunit has an appreciable number of proteins which appear essentially buried. The more buried proteins may serve mainly structural roles, perhaps acting as assembly proteins, since many from this group bind to ribosomal RNA. The more superficially disposed proteins may comprise binding sites for macromolecules that interact with ribosomes during protein synthesis, as well as stabilizing the association of the large and small subribosomal particles.

  11. A ribosome-inactivating protein in a Drosophila defensive symbiont

    PubMed Central

    Hamilton, Phineas T.; Peng, Fangni; Boulanger, Martin J.; Perlman, Steve J.

    2016-01-01

    Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense. PMID:26712000

  12. Phosphorylated proteins of the mammalian mitochondrial ribosome: implications in protein synthesis

    PubMed Central

    Miller, Jennifer L.; Cimen, Huseyin; Koc, Hasan; Koc, Emine C.

    2009-01-01

    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, PKCδ, 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. PMID:19702336

  13. LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection

    PubMed Central

    Mattijssen, Sandy; Arimbasseri, Aneeshkumar G; Iben, James R; Gaidamakov, Sergei; Lee, Joowon; Hafner, Markus

    2017-01-01

    Messenger RNA function is controlled by the 3' poly(A) tail (PAT) and poly(A)-binding protein (PABP). La-related protein-4 (LARP4) binds poly(A) and PABP. LARP4 mRNA contains a translation-dependent, coding region determinant (CRD) of instability that limits its expression. Although the CRD comprises <10% of LARP4 codons, the mRNA levels vary >20 fold with synonymous CRD substitutions that accommodate tRNA dynamics. Separately, overexpression of the most limiting tRNA increases LARP4 levels and reveals its functional activity, net lengthening of the PATs of heterologous mRNAs with concomitant stabilization, including ribosomal protein (RP) mRNAs. Genetic deletion of cellular LARP4 decreases PAT length and RPmRNA stability. This LARP4 activity requires its PABP-interaction domain and the RNA-binding module which we show is sensitive to poly(A) 3'-termini, consistent with protection from deadenylation. The results indicate that LARP4 is a posttranscriptional regulator of ribosomal protein production in mammalian cells and suggest that this activity can be controlled by tRNA levels. PMID:28895529

  14. Effects of chronic ethanol feeding on the protein composition of mitochondrial ribosomes.

    PubMed

    Cahill, A; Cunningham, C C

    2000-10-01

    Chronic ethanol feeding has been shown to decrease the number of functionally active mitochondrial ribosomes by 55%. In this work, 55S mitochondrial ribosomes were isolated from rat liver and their constitutive proteins characterized by two-dimensional polyacrylamide gel electrophoresis and quantified by densitometry. A total of 86 proteins were found to be associated with the mitochondrial ribosome. This compares with 70 isolated from cytoplasmic ribosomes. In addition, mitochondrial ribosomal proteins were found to be significantly less basic than their cytoplasmic counterparts. Chronic ethanol feeding was found to significantly decrease the levels of a number of constitutive proteins of the mitochondrial ribosome when compared to those isolated from pair-fed controls. Sucrose density gradient analyses revealed a significant decrease in the number of intact 55S ribosomes. It is suggested that ethanol-elicited alterations in specific constitutive proteins of the mitochondrial ribosome may lead to impaired assembly of the monosome and that this may result in lower levels of those displaying functional activity.

  15. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  16. Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction.

    PubMed

    McDermott, Suzanne M; Yang, Lu; Halstead, James M; Hamilton, Russell S; Meignin, Carine; Davis, Ilan

    2014-10-01

    Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

  17. Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes.

    PubMed

    Abeysirigunawardena, Sanjaya C; Kim, Hajin; Lai, Jonathan; Ragunathan, Kaushik; Rappé, Mollie C; Luthey-Schulten, Zaida; Ha, Taekjip; Woodson, Sarah A

    2017-09-08

    Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5' domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure.Ribosomes assemble through the hierarchical addition of proteins to a ribosomal RNA scaffold. Here the authors use three-color single-molecule FRET to show how the dynamics of the rRNA dictate the order in which multiple proteins assemble on the 5' domain of the E. coli 16S rRNA.

  18. Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

    PubMed Central

    Ghosh, Arnab; Komar, Anton A

    2015-01-01

    High-resolution structures of yeast ribosomes have improved our understanding of the architecture and organization of eukaryotic rRNA and proteins, as well as eukaryote-specific extensions present in some conserved ribosomal proteins. Despite this progress, assignment of specific functions to individual proteins and/or eukaryote-specific protein extensions remains challenging. It has been suggested that eukaryote-specific extensions of conserved proteins from the small ribosomal subunit may facilitate eukaryote-specific reactions in the initiation phase of protein synthesis. This review summarizes emerging data describing the structural and functional significance of eukaryote-specific extensions of conserved small ribosomal subunit proteins, particularly their possible roles in recruitment and spatial organization of eukaryote-specific initiation factors. PMID:26779416

  19. Analysis of Ribosome Inactivating Protein (RIP): A Bioinformatics Approach

    NASA Astrophysics Data System (ADS)

    Jothi, G. Edward Gnana; Majilla, G. Sahaya Jose; Subhashini, D.; Deivasigamani, B.

    2012-10-01

    In spite of the medical advances in recent years, the world is in need of different sources to encounter certain health issues.Ribosome Inactivating Proteins (RIPs) were found to be one among them. In order to get easy access about RIPs, there is a need to analyse RIPs towards constructing a database on RIPs. Also, multiple sequence alignment was done towards screening for homologues of significant RIPs from rare sources against RIPs from easily available sources in terms of similarity. Protein sequences were retrieved from SWISS-PROT and are further analysed using pair wise and multiple sequence alignment.Analysis shows that, 151 RIPs have been characterized to date. Amongst them, there are 87 type I, 37 type II, 1 type III and 25 unknown RIPs. The sequence length information of various RIPs about the availability of full or partial sequence was also found. The multiple sequence alignment of 37 type I RIP using the online server Multalin, indicates the presence of 20 conserved residues. Pairwise alignment and multiple sequence alignment of certain selected RIPs in two groups namely Group I and Group II were carried out and the consensus level was found to be 98%, 98% and 90% respectively.

  20. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    PubMed

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-04-16

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well.

  1. Removal of protein S1 from Escherichia coli ribosomes without the use of affinity chromatography.

    PubMed

    Baranovskaya, Marianna D; Ugarov, Victor I; Chetverina, Helena V; Chetverin, Alexander B

    2017-01-15

    The paper reports an inexpensive and efficient procedure for the removal of protein S1 from E. coli ribosomes. It comprises incubation of ribosomes in a pyrimidine polyribonucleotide solution followed by centrifugation of the sample through a sucrose cushion. To avoid co-sedimentation of the S1-bound polypyrimidine with the ribosomes, its length should not exceed several hundred nucleotides. Unlike popular affinity chromatography through a poly(U) Sepharose or poly(U) cellulose column, the method tolerates limited polyribonucleotide degradation by eventual traces of ribonucleases, and can readily be incorporated into standard protocols for the isolation of ribosomes by centrifugation. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

    PubMed Central

    Girbés, T; Barbieri, L; Ferreras, M; Arias, F J; Rojo, M A; Iglesias, R; Alegre, C; Escarmis, C; Stirpe, F

    1993-01-01

    The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations. Images PMID:8407849

  3. Functional link between ribosome formation and biogenesis of iron-sulfur proteins.

    PubMed

    Yarunin, Alexander; Panse, Vikram Govind; Petfalski, Elisabeth; Dez, Christophe; Tollervey, David; Hurt, Eduard C

    2005-02-09

    In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.

  4. Functional link between ribosome formation and biogenesis of iron–sulfur proteins

    PubMed Central

    Yarunin, Alexander; Panse, Vikram Govind; Petfalski, Elisabeth; Dez, Christophe; Tollervey, David; Hurt, Ed

    2005-01-01

    In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron–sulfur (Fe–S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe–S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe–S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe–S proteins. PMID:15660135

  5. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory

    PubMed Central

    Suarez, Stéphanie; Ferroni, Agnès; Lotz, Aurélie; Jolley, Keith A.; Guérin, Philippe; Leto, Julie; Dauphin, Brunhilde; Jamet, Anne; Maiden, Martin C.J.; Nassif, Xavier; Armengaud, Jean

    2014-01-01

    Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. PMID:23916798

  6. Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

    PubMed

    di Toppi, L S; Gorini, P; Properzi, G; Barbieri, L; Spanò, L

    1996-09-01

    Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein(-1) and a molecular mass of 26-28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.

  7. Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors

    PubMed Central

    1984-01-01

    Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA. PMID:6202701

  8. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    PubMed

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  9. Protein quality control at the ribosome: focus on RAC, NAC and RQC.

    PubMed

    Gamerdinger, Martin

    2016-10-15

    The biogenesis of new polypeptides by ribosomes and their subsequent correct folding and localization to the appropriate cellular compartments are essential key processes to maintain protein homoeostasis. These complex mechanisms are governed by a repertoire of protein biogenesis factors that directly bind to the ribosome and chaperone nascent polypeptide chains as soon as they emerge from the ribosomal tunnel exit. This nascent chain 'welcoming committee' regulates multiple co-translational processes including protein modifications, folding, targeting and degradation. Acting at the front of the protein production line, these ribosome-associated protein biogenesis factors lead the way in the cellular proteostasis network to ensure proteome integrity. In this article, I focus on three different systems in eukaryotes that are critical for the maintenance of protein homoeostasis by controlling the birth, life and death of nascent polypeptide chains.

  10. Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly.

    PubMed

    Mayerle, Megan; Woodson, Sarah A

    2013-04-01

    Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5' domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

  11. The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

    PubMed Central

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Ramajo, Jorge; Martinez-Salas, Encarnación

    2016-01-01

    RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation. PMID:27507887

  12. Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane.

    PubMed

    MacKenzie, James A; Payne, R Mark

    2004-03-12

    The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.

  13. The Saccharomyces cerevisiae protein Stm1p facilitates ribosome preservation during quiescence

    SciTech Connect

    Van Dyke, Natalya; Chanchorn, Ekkawit; Van Dyke, Michael W.

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer Stm1p confers increased resistance to the macrolide starvation-mimic rapamycin. Black-Right-Pointing-Pointer Stm1p maintains 80S ribosome integrity during stationary phase-induced quiescence. Black-Right-Pointing-Pointer Stm1p facilitates polysome formation following quiescence exit. Black-Right-Pointing-Pointer Stm1p facilitates protein synthesis following quiescence exit. Black-Right-Pointing-Pointer Stm1p is a ribosome preservation factor under conditions of nutrient deprivation. -- Abstract: Once cells exhaust nutrients from their environment, they enter an alternative resting state known as quiescence, whereby proliferation ceases and essential nutrients are obtained through internal stores and through the catabolism of existing macromolecules and organelles. One example of this is ribophagy, the degradation of ribosomes through the process of autophagy. However, some ribosomes need to be preserved for an anticipated recovery from nutrient deprivation. We found that the ribosome-associated protein Stm1p greatly increases the quantity of 80S ribosomes present in quiescent yeast cells and that these ribosomes facilitate increased protein synthesis rates once nutrients are restored. These findings suggest that Stm1p can act as a ribosome preservation factor under conditions of nutrient deprivation and restoration.

  14. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

    PubMed Central

    Yerlikaya, Seda; Meusburger, Madeleine; Kumari, Romika; Huber, Alexandre; Anrather, Dorothea; Costanzo, Michael; Boone, Charles; Ammerer, Gustav; Baranov, Pavel V.; Loewith, Robbie

    2016-01-01

    Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously. PMID:26582391

  15. Identifying eIF4E-binding protein translationally-controlled transcripts reveals links to mRNAs bound by specific PUF proteins

    PubMed Central

    Cridge, Andrew G.; Castelli, Lydia M.; Smirnova, Julia B.; Selley, Julian N.; Rowe, William; Hubbard, Simon J.; McCarthy, John E.G.; Ashe, Mark P.; Grant, Christopher M.; Pavitt, Graham D.

    2010-01-01

    eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought. PMID:20705650

  16. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy

    PubMed Central

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2014-01-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knockin mice expressing non-phosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knockin mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knockin mice compared to their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knockin mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knockin and wild type mice, indicating that mTORC1 was still activated in the knockin mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knockin mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth. PMID:25229342

  17. Plants Producing Ribosome-Inactivating Proteins in Traditional Medicine.

    PubMed

    Polito, Letizia; Bortolotti, Massimo; Maiello, Stefania; Battelli, Maria Giulia; Bolognesi, Andrea

    2016-11-18

    Ribosome-inactivating proteins (RIPs) are enzymes that deadenylate nucleic acids and are broadly distributed in the plant kingdom. Many plants that contain RIPs are listed in the pharmacopoeias of folk medicine all over the world, mostly because of their toxicity. This review analyses the position occupied in traditional medicine by plants from which RIPs have been isolated. The overview starts from the antique age of the Mediterranean area with ancient Egypt, followed by the Greek and Roman classic period. Then, the ancient oriental civilizations of China and India are evaluated. More recently, Unani medicine and European folk medicine are examined. Finally, the African and American folk medicines are taken into consideration. In conclusion, a list of RIP-expressing plants, which have been used in folk medicine, is provided with the geographical distribution and the prescriptions that are recommended by traditional healers. Some final considerations are provided on the present utilization of such herbal treatments, both in developing and developed countries, often in the absence of scientific validation. The most promising prospect for the medicinal use of RIP-expressing plants is the conjugation of purified RIPs to antibodies that recognise tumour antigens for cancer therapy.

  18. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22.

    PubMed

    Moore, Sean D; Sauer, Robert T

    2008-11-25

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (DeltaMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that DeltaMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which DeltaMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.

  19. Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

    PubMed Central

    Moore, Sean D.; Sauer, Robert T.

    2008-01-01

    Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (ΔMKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ΔMKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ΔMKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels. PMID:19015512

  20. Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

    PubMed Central

    Shi, Wei-Wei; Tang, Yun-Sang; Sze, See-Yuen; Zhu, Zhen-Ning; Wong, Kam-Bo; Shaw, Pang-Chui

    2016-01-01

    Ricin is a type 2 ribosome-inactivating protein (RIP), containing a catalytic A chain and a lectin-like B chain. It inhibits protein synthesis by depurinating the N-glycosidic bond at α-sarcin/ricin loop (SRL) of the 28S rRNA, which thereby prevents the binding of elongation factors to the GTPase activation center of the ribosome. Here, we present the 1.6 Å crystal structure of Ricin A chain (RTA) complexed to the C-terminal peptide of the ribosomal stalk protein P2, which plays a crucial role in specific recognition of elongation factors and recruitment of eukaryote-specific RIPs to the ribosomes. Our structure reveals that the C-terminal GFGLFD motif of P2 peptide is inserted into a hydrophobic pocket of RTA, while the interaction assays demonstrate the structurally untraced SDDDM motif of P2 peptide contributes to the interaction with RTA. This interaction mode of RTA and P protein is in contrast to that with trichosanthin (TCS), Shiga-toxin (Stx) and the active form of maize RIP (MOD), implying the flexibility of the P2 peptide-RIP interaction, for the latter to gain access to ribosome. PMID:27754366

  1. Massively convergent evolution for ribosomal protein gene content in plastid and mitochondrial genomes.

    PubMed

    Maier, Uwe-G; Zauner, Stefan; Woehle, Christian; Bolte, Kathrin; Hempel, Franziska; Allen, John F; Martin, William F

    2013-01-01

    Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force.

  2. Massively Convergent Evolution for Ribosomal Protein Gene Content in Plastid and Mitochondrial Genomes

    PubMed Central

    Maier, Uwe-G; Zauner, Stefan; Woehle, Christian; Bolte, Kathrin; Hempel, Franziska; Allen, John F.; Martin, William F.

    2013-01-01

    Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force. PMID:24259312

  3. Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

    PubMed

    Zinoviev, Alexandra; Hellen, Christopher U T; Pestova, Tatyana V

    2015-03-19

    Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs

    PubMed Central

    Zinoviev, Alexandra; Hellen, Christopher U. T.; Pestova, Tatyana V.

    2015-01-01

    Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long ORFs on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG designated “termination upstream ribosomal binding site” (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2*. To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes, and diminishes dependence on eIF3 of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes. PMID:25794616

  5. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

    PubMed

    Dauber, Bianca; Poon, David; Dos Santos, Theodore; Duguay, Brett A; Mehta, Ninad; Saffran, Holly A; Smiley, James R

    2016-07-01

    The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects

  6. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation

    PubMed Central

    Dauber, Bianca; Poon, David; dos Santos, Theodore; Duguay, Brett A.; Mehta, Ninad; Saffran, Holly A.

    2016-01-01

    ABSTRACT The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. IMPORTANCE The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least

  7. Studies on the Assembly Characteristics of Large Subunit Ribosomal Proteins in S. cerevisae

    PubMed Central

    Ohmayer, Uli; Gamalinda, Michael; Sauert, Martina; Ossowski, Julius; Pöll, Gisela; Linnemann, Jan; Hierlmeier, Thomas; Perez-Fernandez, Jorge; Kumcuoglu, Beril; Leger-Silvestre, Isabelle; Faubladier, Marlène; Griesenbeck, Joachim; Woolford, John; Tschochner, Herbert; Milkereit, Philipp

    2013-01-01

    During the assembly process of ribosomal subunits, their structural components, the ribosomal RNAs (rRNAs) and the ribosomal proteins (r-proteins) have to join together in a highly dynamic and defined manner to enable the efficient formation of functional ribosomes. In this work, the assembly of large ribosomal subunit (LSU) r-proteins from the eukaryote S. cerevisiae was systematically investigated. Groups of LSU r-proteins with specific assembly characteristics were detected by comparing the protein composition of affinity purified early, middle, late or mature LSU (precursor) particles by semi-quantitative mass spectrometry. The impact of yeast LSU r-proteins rpL25, rpL2, rpL43, and rpL21 on the composition of intermediate to late nuclear LSU precursors was analyzed in more detail. Effects of these proteins on the assembly states of other r-proteins and on the transient LSU precursor association of several ribosome biogenesis factors, including Nog2, Rsa4 and Nop53, are discussed. PMID:23874617

  8. Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

    PubMed

    Wienen, B; Ehrlich, R; Stöffler-Meilicke, M; Stöffler, G; Smith, I; Weiss, D; Vince, R; Pestka, S

    1979-08-25

    Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

  9. The ribosomal protein genes and Minute loci of Drosophila melanogaster

    PubMed Central

    Marygold, Steven J; Roote, John; Reuter, Gunter; Lambertsson, Andrew; Ashburner, Michael; Millburn, Gillian H; Harrison, Paul M; Yu, Zhan; Kenmochi, Naoya; Kaufman, Thomas C; Leevers, Sally J; Cook, Kevin R

    2007-01-01

    Background Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes. Results We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci. Conclusion This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome. PMID:17927810

  10. Alternative Ways to Think about Cellular Internal Ribosome Entry*

    PubMed Central

    Gilbert, Wendy V.

    2010-01-01

    Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of “cap-dependent” and “IRES-dependent” should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation. PMID:20576611

  11. Trans-translation in Helicobacter pylori: essentiality of ribosome rescue and requirement of protein tagging for stress resistance and competence.

    PubMed

    Thibonnier, Marie; Thiberge, Jean-Michel; De Reuse, Hilde

    2008-01-01

    The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans. We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori. We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics.

  12. Trans-Translation in Helicobacter pylori: Essentiality of Ribosome Rescue and Requirement of Protein Tagging for Stress Resistance and Competence

    PubMed Central

    Thibonnier, Marie; Thiberge, Jean-Michel; De Reuse, Hilde

    2008-01-01

    Background The ubiquitous bacterial trans-translation is one of the most studied quality control mechanisms. Trans-translation requires two specific factors, a small RNA SsrA (tmRNA) and a protein co-factor SmpB, to promote the release of ribosomes stalled on defective mRNAs and to add a specific tag sequence to aberrant polypeptides to direct them to degradation pathways. Helicobacter pylori is a pathogen persistently colonizing a hostile niche, the stomach of humans. Principal Findings We investigated the role of trans-translation in this bacterium well fitted to resist stressful conditions and found that both smpB and ssrA were essential genes. Five mutant versions of ssrA were generated in H. pylori in order to investigate the function of trans-translation in this organism. Mutation of the resume codon that allows the switch of template of the ribosome required for its release was essential in vivo, however a mutant in which this codon was followed by stop codons interrupting the tag sequence was viable. Therefore one round of translation is sufficient to promote the rescue of stalled ribosomes. A mutant expressing a truncated SsrA tag was viable in H. pylori, but affected in competence and tolerance to both oxidative and antibiotic stresses. This demonstrates that control of protein degradation through trans-translation is by itself central in the management of stress conditions and of competence and supports a regulatory role of trans-translation-dependent protein tagging. In addition, the expression of smpB and ssrA was found to be induced upon acid exposure of H. pylori. Conclusions We conclude to a central role of trans-translation in H. pylori both for ribosome rescue possibly due to more severe stalling and for protein degradation to recover from stress conditions frequently encountered in the gastric environment. Finally, the essential trans-translation machinery of H. pylori is an excellent specific target for the development of novel antibiotics. PMID

  13. The ribosome and its role in protein folding: looking through a magnifying glass.

    PubMed

    Javed, Abid; Christodoulou, John; Cabrita, Lisa D; Orlova, Elena V

    2017-06-01

    Protein folding, a process that underpins cellular activity, begins co-translationally on the ribosome. During translation, a newly synthesized polypeptide chain enters the ribosomal exit tunnel and actively interacts with the ribosome elements - the r-proteins and rRNA that line the tunnel - prior to emerging into the cellular milieu. While understanding of the structure and function of the ribosome has advanced significantly, little is known about the process of folding of the emerging nascent chain (NC). Advances in cryo-electron microscopy are enabling visualization of NCs within the exit tunnel, allowing early glimpses of the interplay between the NC and the ribosome. Once it has emerged from the exit tunnel into the cytosol, the NC (still attached to its parent ribosome) can acquire a range of conformations, which can be characterized by NMR spectroscopy. Using experimental restraints within molecular-dynamics simulations, the ensemble of NC structures can be described. In order to delineate the process of co-translational protein folding, a hybrid structural biology approach is foreseeable, potentially offering a complete atomic description of protein folding as it occurs on the ribosome.

  14. YsxC, an essential protein in Staphylococcus aureus crucial for ribosome assembly/stability.

    PubMed

    Cooper, Elizabeth L; García-Lara, Jorge; Foster, Simon J

    2009-12-18

    Bacterial growth and division requires a core set of essential proteins, several of which are still of unknown function. They are also attractive targets for the development of new antibiotics. YsxC is a member of a family of GTPases highly conserved across eubacteria with a possible ribosome associated function. Here, we demonstrate by the creation of a conditional lethal mutant that ysxC is apparently essential for growth in S. aureus. To begin to elucidate YsxC function, a translational fusion of YsxC to the CBP-ProteinA tag in the staphylococcal chromosome was made, enabling Tandem Affinity Purification (TAP) of YsxC-interacting partners. These included the ribosomal proteins S2, S10 and L17, as well as the beta' subunit of the RNA polymerase. YsxC was then shown to copurify with ribosomes as an accessory protein specifically localizing to the 50 S subunit. YsxC depletion led to a decrease in the presence of mature ribosomes, indicating a role in ribosome assembly and/or stability in S. aureus. In this study we demonstrate that YsxC of S. aureus localizes to the ribosomes, is crucial for ribosomal stability and is apparently essential for the life of S. aureus.

  15. Isolation of a 5S RNA-Protein Complex from Mammalian Ribosomes

    PubMed Central

    Blobel, Günter

    1971-01-01

    5S RNA is removed from the large ribosomal subunit of both rat liver and rabbit reticulocyte ribosomes by treatment with EDTA to remove magnesium ions. The RNA is removed not as a naked molecule, but in association with a single ribosomal protein (molecular weight about 35,000) as a ribonucleoprotein, sedimenting at about 7 S. Conditions for the exclusive removal of the 5S RNA-protein complex have been established. Both ribosomal subunits undergo distinct changes in their sedimentation rate, concomitant with the loss of their biological activity, when sedimented into sucrose gradients containing high concentrations of monovalent ions and low concentrations of magnesium. Under these conditions the large subunit loses 5S RNA, the 5S RNA-associated protein, and several more proteins. Images PMID:5001943

  16. The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control.

    PubMed

    Guillier, Maude; Allemand, Frédéric; Graffe, Monique; Raibaud, Sophie; Dardel, Frédéric; Springer, Mathias; Chiaruttini, Claude

    2005-05-01

    The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.

  17. The rotaviral NSP3 protein stimulates translation of polyadenylated target mRNAs independently of its RNA-binding domain

    SciTech Connect

    Keryer-Bibens, Cecile; Legagneux, Vincent; Namanda-Vanderbeken, Allen; Cosson, Bertrand; Paillard, Luc; Poncet, Didier; Osborne, H. Beverley

    2009-12-11

    The non-structural protein 3 (NSP3) of rotaviruses is an RNA-binding protein that specifically recognises a 4 nucleotide sequence at the 3' extremity of the non-polyadenylated viral mRNAs. NSP3 also has a high affinity for eIF4G. These two functions are clearly delimited in separate domains the structures of which have been determined. They are joined by a central domain implicated in the dimerisation of the full length protein. The bridging function of NSP3 between the 3' end of the viral mRNA and eIF4G has been proposed to enhance the synthesis of viral proteins. However, this role has been questioned as knock-down of NSP3 did not impair viral protein synthesis. We show here using a MS2/MS2-CP tethering assay that a C-terminal fragment of NSP3 containing the eIF4G binding domain and the dimerisation domain can increase the expression of a protein encoded by a target reporter mRNA in HEK 293 cells. The amount of reporter mRNA in the cells is not significantly affected by the presence of the NSP3 derived fusion protein showing that the enhanced protein expression is due to increased translation. These results show that NSP3 can act as a translational enhancer even on a polyadenylated mRNA that should be a substrate for PABP1.

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

    PubMed

    Zurdo, J; Parada, P; van den Berg, A; Nusspaumer, G; Jimenez-Diaz, A; Remacha, M; Ballesta, J P

    2000-08-01

    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.

  19. [Protein L16 of the Escherichia coli ribosome: possible role in protein biosynthesis].

    PubMed

    Maĭmets, T O; Ustav, M B; Remme, Ia L; Villems, R L

    1984-01-01

    We show that Escherichia coli 50S ribosomal subunits depleted of protein L16 can nevertheless catalyze the transfer of the peptide moiety from fMet-tRNA to puromycin, being, however, unable to use a fragment CACCA-Phe as an acceptor substrate. On the other hand, we found that protein L16 as well as its large fragment (amino acids 10-136) both interact with tRNA in solution (Kd approximately 10(-7) M). Moreover, L16 interacts with CACCA-Phe in solution as well as protects 3' end of tRNA from the enzymatic degradation. We suggest that L16, although not being the peptidyl transferase as such, is involved in the binding of the 3' end cytidines of tRNA into the ribosomal A site.

  20. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation*

    PubMed Central

    Timpano, Sara; Uniacke, James

    2016-01-01

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5′ cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or “normoxia,” is far from physiological or “normal.” In fact, oxygen in human tissues ranges from 1–11% or “physioxia.” Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1–11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. PMID:27002144

  1. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation.

    PubMed

    Timpano, Sara; Uniacke, James

    2016-05-13

    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Epstein-Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs.

    PubMed

    Juillard, Franceline; Bazot, Quentin; Mure, Fabrice; Tafforeau, Lionel; Macri, Christophe; Rabourdin-Combe, Chantal; Lotteau, Vincent; Manet, Evelyne; Gruffat, Henri

    2012-08-01

    The Epstein-Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20's labile interactions with the RNA.

  3. Epstein–Barr virus protein EB2 stimulates cytoplasmic mRNA accumulation by counteracting the deleterious effects of SRp20 on viral mRNAs

    PubMed Central

    Juillard, Franceline; Bazot, Quentin; Mure, Fabrice; Tafforeau, Lionel; Macri, Christophe; Rabourdin-Combe, Chantal; Lotteau, Vincent; Manet, Evelyne; Gruffat, Henri

    2012-01-01

    The Epstein–Barr Virus (EBV) protein EB2 (also called Mta, SM and BMLF1), is an essential nuclear protein produced during the replicative cycle of EBV. EB2 is required for the efficient cytoplasmic accumulation of viral mRNAs derived from intronless genes. EB2 is an RNA-binding protein whose expression has been shown to influence RNA stability, splicing, nuclear export and translation. Using a yeast two-hybrid screen, we have identified three SR proteins, SF2/ASF, 9G8 and SRp20, as cellular partners of EB2. Then, by using siRNA to deplete cells of specific SR proteins, we found that SRp20 plays an essential role in the processing of several model mRNAs: the Renilla luciferase reporter mRNA, the human β-globin cDNA transcript and two EBV late mRNAs. These four mRNAs were previously found to be highly dependent on EB2 for their efficient cytoplasmic accumulation. Here, we show that SRp20 depletion results in an increase in the accumulation of these mRNAs, which correlates with an absence of additive effect of EB2, suggesting that EB2 functions by antagonizing SRp20. Moreover, by using RNA-immunoprecipitation assays we found that EB2 enhances the association of SRp20 with the β-globin transcript suggesting that EB2 acts by stabilizing SRp20’s labile interactions with the RNA. PMID:22505578

  4. Conditionally lethal ribosomal protein mutants: characterization of a locus required for modification of 50S subunit proteins.

    PubMed Central

    Kushner, S R; Maples, V F; Champney, W S

    1977-01-01

    Mutagenized P1 bacteriophage were used to transduce a marker (aroE) adjacent to the cluster or ribosomal protein genes located at 72 min on the Escherichia coli chromosome. Linked temperature-sensitive transductants were isolated and characterized. A mutant unable to grow at 44 degrees was found to be defective in protein synthesis both in vivo and in vitro. At the restrictive temperature mutant cells lost all polyribosomes. Analysis of the ribosomal proteins revealed alterations in at least four 50S subunit proteins. The mutation (called rimE1, ribosomal protein modification) mapped between rpsE and aroE. It is suggested that the rimE locus is the structural gene for an activity that modifies a selected number of ribosomal proteins. Images PMID:322127

  5. In vivo assembling of bacterial ribosomal protein L11 into yeast ribosomes makes the particles sensitive to the prokaryotic specific antibiotic thiostrepton.

    PubMed

    García-Marcos, Alberto; Morreale, Antonio; Guarinos, Esther; Briones, Elisa; Remacha, Miguel; Ortiz, Angel R; Ballesta, Juan P G

    2007-01-01

    Eukaryotic ribosomal stalk protein L12 and its bacterial orthologue L11 play a central role on ribosomal conformational changes during translocation. Deletion of the two genes encoding L12 in Saccharomyces cerevisiae resulted in a very slow-growth phenotype. Gene RPL12B, but not the RPL12A, cloned in centromeric plasmids fully restored control protein level and the growth rate when expressed in a L12-deprived strain. The same strain has been transformed to express Escherichia coli protein EcL11 under the control of yeast RPL12B promoter. The bacterial protein has been found in similar amounts in washed ribosomes from the transformed yeast strain and from control E. coli cells, however, EcL11 was unable to restore the defective acidic protein stalk composition caused by the absence of ScL12 in the yeast ribosome. Protein EcL11 induced a 10% increase in L12-defective cell growth rate, although the in vitro polymerizing capacity of the EcL11-containing ribosomes is restored in a higher proportion, and, moreover, the particles became partially sensitive to the prokaryotic specific antibiotic thiostrepton. Molecular dynamic simulations using modelled complexes support the correct assembly of bacterial L11 into the yeast ribosome and confirm its direct implication of its CTD in the binding of thiostrepton to ribosomes.

  6. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    PubMed

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  7. Structural basis for the interaction of protein S1 with the Escherichia coli ribosome

    PubMed Central

    Byrgazov, Konstantin; Grishkovskaya, Irina; Arenz, Stefan; Coudevylle, Nicolas; Temmel, Hannes; Wilson, Daniel N.; Djinovic-Carugo, Kristina; Moll, Isabella

    2015-01-01

    In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1–ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the ′ribosome puzzle′, namely the detailed molecular insight into the topology of the S1–ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome. PMID:25510494

  8. The human Shwachman-Diamond syndrome protein, SBDS, associates with ribosomal RNA.

    PubMed

    Ganapathi, Karthik A; Austin, Karyn M; Lee, Chung-Sheng; Dias, Anusha; Malsch, Maggie M; Reed, Robin; Shimamura, Akiko

    2007-09-01

    Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic dysfunction, and leukemia predisposition. Mutations in the SBDS gene are identified in most patients with SDS. SBDS encodes a highly conserved protein of unknown function. Data from SBDS orthologs suggest that SBDS may play a role in ribosome biogenesis or RNA processing. Human SBDS is enriched in the nucleolus, the major cellular site of ribosome biogenesis. Here we report that SBDS nucleolar localization is dependent on active rRNA transcription. Cells from patients with SDS or Diamond-Blackfan anemia are hypersensitive to low doses of actinomycin D, an inhibitor of rRNA transcription. The addition of wild-type SBDS complements the actinomycin D hypersensitivity of SDS patient cells. SBDS migrates together with the 60S large ribosomal subunit in sucrose gradients and coprecipitates with 28S ribosomal RNA (rRNA). Loss of SBDS is not associated with a discrete block in rRNA maturation or with decreased levels of the 60S ribosomal subunit. SBDS forms a protein complex with nucleophosmin, a multifunctional protein implicated in ribosome biogenesis and leukemogenesis. Our studies support the addition of SDS to the growing list of human bone marrow failure syndromes involving the ribosome.

  9. Structural basis for the interaction of protein S1 with the Escherichia coli ribosome.

    PubMed

    Byrgazov, Konstantin; Grishkovskaya, Irina; Arenz, Stefan; Coudevylle, Nicolas; Temmel, Hannes; Wilson, Daniel N; Djinovic-Carugo, Kristina; Moll, Isabella

    2015-01-01

    In Gram-negative bacteria, the multi-domain protein S1 is essential for translation initiation, as it recruits the mRNA and facilitates its localization in the decoding centre. In sharp contrast to its functional importance, S1 is still lacking from the high-resolution structures available for Escherichia coli and Thermus thermophilus ribosomes and thus the molecular mechanism governing the S1-ribosome interaction has still remained elusive. Here, we present the structure of the N-terminal S1 domain D1 when bound to the ribosome at atomic resolution by using a combination of NMR, X-ray crystallography and cryo-electron microscopy. Together with biochemical assays, the structure reveals that S1 is anchored to the ribosome primarily via a stabilizing π-stacking interaction within the short but conserved N-terminal segment that is flexibly connected to domain D1. This interaction is further stabilized by salt bridges involving the zinc binding pocket of protein S2. Overall, this work provides one hitherto enigmatic piece in the 'ribosome puzzle', namely the detailed molecular insight into the topology of the S1-ribosome interface. Moreover, our data suggest novel mechanisms that have the potential to modulate protein synthesis in response to environmental cues by changing the affinity of S1 for the ribosome. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Sequential domain assembly of ribosomal protein S3 drives 40S subunit maturation

    PubMed Central

    Mitterer, Valentin; Murat, Guillaume; Réty, Stéphane; Blaud, Magali; Delbos, Lila; Stanborough, Tamsyn; Bergler, Helmut; Leulliot, Nicolas; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    Eukaryotic ribosomes assemble by association of ribosomal RNA with ribosomal proteins into nuclear precursor particles, which undergo a complex maturation pathway coordinated by non-ribosomal assembly factors. Here, we provide functional insights into how successive structural re-arrangements in ribosomal protein S3 promote maturation of the 40S ribosomal subunit. We show that S3 dimerizes and is imported into the nucleus with its N-domain in a rotated conformation and associated with the chaperone Yar1. Initial assembly of S3 with 40S precursors occurs via its C-domain, while the N-domain protrudes from the 40S surface. Yar1 is replaced by the assembly factor Ltv1, thereby fixing the S3 N-domain in the rotated orientation and preventing its 40S association. Finally, Ltv1 release, triggered by phosphorylation, and flipping of the S3 N-domain into its final position results in the stable integration of S3. Such a stepwise assembly may represent a new paradigm for the incorporation of ribosomal proteins. PMID:26831757

  11. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

    PubMed

    Nouws, Jessica; Goswami, Arvind V; Bestwick, Megan; McCann, Beverly Jo; Surovtseva, Yulia V; Shadel, Gerald S

    2016-01-08

    To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Molecular basis for protection of ribosomal protein L4 from cellular degradation

    PubMed Central

    Huber, Ferdinand M.; Hoelz, André

    2017-01-01

    Eukaryotic ribosome biogenesis requires the nuclear import of ∼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone–substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery. PMID:28148929

  13. Involvement of ribosomal protein L6 in assembly of functional 50S ribosomal subunit in Escherichia coli cells

    SciTech Connect

    Shigeno, Yuta; Uchiumi, Toshio; Nomura, Takaomi

    2016-04-22

    Ribosomal protein L6, an essential component of the large (50S) subunit, primarily binds to helix 97 of 23S rRNA and locates near the sarcin/ricin loop of helix 95 that directly interacts with GTPase translation factors. Although L6 is believed to play important roles in factor-dependent ribosomal function, crucial biochemical evidence for this hypothesis has not been obtained. We constructed and characterized an Escherichia coli mutant bearing a chromosomal L6 gene (rplF) disruption and carrying a plasmid with an arabinose-inducible L6 gene. Although this ΔL6 mutant grew more slowly than its wild-type parent, it proliferated in the presence of arabinose. Interestingly, cell growth in the absence of arabinose was biphasic. Early growth lasted only a few generations (LI-phase) and was followed by a suspension of growth for several hours (S-phase). This suspension was followed by a second growth phase (LII-phase). Cells harvested at both LI- and S-phases contained ribosomes with reduced factor-dependent GTPase activity and accumulated 50S subunit precursors (45S particles). The 45S particles completely lacked L6. Complete 50S subunits containing L6 were observed in all growth phases regardless of the L6-depleted condition, implying that the ΔL6 mutant escaped death because of a leaky expression of L6 from the complementing plasmid. We conclude that L6 is essential for the assembly of functional 50S subunits at the late stage. We thus established conditions for the isolation of L6-depleted 50S subunits, which are essential to study the role of L6 in translation. - Highlights: • We constructed an in vivo functional assay system for Escherichia coli ribosomal protein L6. • Growth of an E. coli ΔL6 mutant was biphasic when L6 levels were depleted. • The ΔL6 mutant accumulated 50S ribosomal subunit precursors that sedimented at 45S. • L6 is a key player in the late stage of E. coli 50S subunit assembly.

  14. A 40 kd protein binds specifically to the 5'-untranslated regions of yeast mitochondrial mRNAs.

    PubMed Central

    Papadopoulou, B; Dekker, P; Blom, J; Grivell, L A

    1990-01-01

    Using a gel mobility shift assay we show that a 40 kd protein (p40), present in extracts of yeast mitochondria, binds specifically to the 5'-untranslated leader of cytochrome c oxidase subunit II mRNA. Binding of p40 to coxII RNA protects an 8-10 nucleotide segment from diethylpyocarbonate modification, indicating that the protein interacts with only a restricted region of the 5'-leader. This segment is located at position -12 with respect to the initiation AUG. Deletion of 10 nucleotides encompassing this site completely abolishes protein binding. Nevertheless, Bal31 deletion analysis within the coxII leader shows that a major part of the leader is essential for p40 binding, suggesting that binding of the protein is also dependent on secondary structural features. p40 binds to other mitochondrial leader mRNAs including those for coxI, coxIII and cyt b. p40 is present in a cytoplasmic (rho0) petite mutant lacking mitochondrial protein synthesis. It is therefore presumably nuclear encoded. The possible biological function of the protein is discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 8. Fig. 9. PMID:1701144

  15. Identification of antituberculosis agents that target ribosomal protein interactions using a yeast two-hybrid system.

    PubMed

    Lin, Yuan; Li, Yan; Zhu, Yuanjun; Zhang, Jing; Li, Yongzhen; Liu, Xiao; Jiang, Wei; Yu, Shishan; You, Xue-Fu; Xiao, Chunling; Hong, Bin; Wang, Yanchang; Jiang, Jian-Dong; Si, Shuyi

    2012-10-23

    Mycobacterium tuberculosis kills about 2 million people annually and antibiotic resistance is a cause of increased mortality. Therefore, development of new antituberculosis drugs is urgent for the control of widespread tuberculosis infections. For this purpose, we performed an innovative screen to identify new agents that disrupt the function of ribosomes in M. tuberculosis. Two bacterial ribosomal proteins L12 and L10 interact with each other and constitute the stalk of the 50S ribosomal subunit, which recruits initiation and elongation factors (EFs) during translation. Therefore, the L12-L10 interaction should be essential for ribosomal function and protein synthesis. We established a yeast two-hybrid system to identify small molecules that block the interaction between L12 and L10 proteins from M. tuberculosis. Using this system, we identified two compounds T766 and T054 that show strong bactericidal activity against tuberculosis but with low toxicity to mice and other bacterial strains. Moreover, using surface plasmon resonance (SPR) assay, we have demonstrated that these compounds bind specifically to L12 to disrupt L12-L10 interaction. Overproduction of L12 protein, but not L10, lowers the antibacterial activity of T766 and T054, indicating that the ribosome is likely the cellular target. Therefore, our data demonstrate that this yeast two-hybrid system is a useful tool to identify unique antituberculosis agents targeting the ribosomal protein L12-L10 interaction.

  16. Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1

    PubMed Central

    Mitterer, Valentin; Gantenbein, Nadine; Birner-Gruenberger, Ruth; Murat, Guillaume; Bergler, Helmut; Kressler, Dieter; Pertschy, Brigitte

    2016-01-01

    After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein. PMID:27819319

  17. Regulatory elements of Caenorhabditis elegans ribosomal protein genes

    PubMed Central

    2012-01-01

    Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species

  18. Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae

    PubMed Central

    Casolari, Jason M.; Thompson, Michael A.; Salzman, Julia; Champion, Lowry M.; Moerner, W. E.; Brown, Patrick O.

    2012-01-01

    Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe her e a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches. PMID:22359641

  19. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

    PubMed Central

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

    2014-01-01

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

  20. Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex.

    PubMed

    Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B; Webb, Kristofor; Bennett, Eric J; Vinterbo, Staal; Potter, Clinton S; Carragher, Bridget; Joazeiro, Claudio A P

    2014-11-11

    All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

  1. Multiple binding of repressed mRNAs by the P-body protein Rck/p54

    PubMed Central

    Ernoult-Lange, Michèle; Baconnais, Sonia; Harper, Maryannick; Minshall, Nicola; Souquere, Sylvie; Boudier, Thomas; Bénard, Marianne; Andrey, Philippe; Pierron, Gérard; Kress, Michel; Standart, Nancy; le Cam, Eric; Weil, Dominique

    2012-01-01

    Translational repression is achieved by protein complexes that typically bind 3′ UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5′ extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation. PMID:22836354

  2. Structural insights into the role of rRNA modifications in protein synthesis and ribosome assembly.

    PubMed

    Polikanov, Yury S; Melnikov, Sergey V; Söll, Dieter; Steitz, Thomas A

    2015-04-01

    We report crystal structures of the Thermus thermophilus ribosome at 2.3- to 2.5-Å resolution, which have enabled modeling of rRNA modifications. The structures reveal contacts of modified nucleotides with mRNA and tRNAs or protein pY, and contacts within the ribosome interior stabilizing the functional fold of rRNA. Our work provides a resource to explore the roles of rRNA modifications and yields a more comprehensive atomic model of a bacterial ribosome.

  3. Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein.

    PubMed

    Park, Keun-Yong; Kim, Min-Suk; Choi, Sie-Young

    2005-04-15

    A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.

  4. Ribosomal analysis of rapid rates of protein synthesis in the Antarctic sea urchin Sterechinus neumayeri.

    PubMed

    Pace, Douglas A; Maxson, Robert; Manahan, Donal T

    2010-02-01

    Previous research has shown that developing stages of the Antarctic sea urchin Sterechinus neumayeri have high rates of protein synthesis that are comparable to those of similar species living in much warmer waters. Direct measurements of the biosynthetic capacities of isolated ribosomes have not been reported for marine organisms living in the extreme-cold environment of Antarctica. Such measurements are required for a mechanistic understanding of how the critical and highly complex processes involved in protein synthesis are regulated in animals living in the coldest marine environment on Earth (< -1 degrees C). We tested the hypothesis that high rates of protein synthesis in the cold are a direct result of high biosynthetic capacities of ribosomes engaged in protein synthesis. Our results show that the rate at which ribosomes manufacture proteins (i.e., the peptide elongation rate) at -1 degrees C is surprisingly similar to rates measured in other sea urchin species at temperatures that are over 15 degrees C warmer. Average peptide elongation rates for a range of developmental stages of the Antarctic sea urchin were 0.36 codons s(-1) (+/- 0.05, SE). On the basis of subcellular rate determinations of ribosomal activity, we calculated stage-specific rates of protein synthesis for blastulae and gastrulae to be 3.7 and 6.5 ng protein h(-1), respectively. These findings support the conclusion that the high rates of biosynthesis previously reported for the Antarctic sea urchin are an outcome of high ribosomal activities.

  5. Expression of transforming growth factor-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo.

    PubMed

    Jakowlew, S B; Ciment, G; Tuan, R S; Sporn, M B; Roberts, A B

    1994-01-01

    Specific cDNA probes and antibodies for chicken transforming growth factor (TGF)-beta 2 and beta 3 were used to study expression of TGF-beta 2 and beta 3 mRNAs and proteins in the developing chicken embryo. Expression of the mRNAs for both TGF-beta isoforms was detected by day 1.5 of incubation (Hamburger and Hamilton stage 10) by RNA Northern blot analysis and increased with developmental age. Expression of TGF-beta 2 and beta 3 mRNAs was detected in every embryonic tissue examined, with the level of expression of both isoforms being high in heart, brain and muscle and low in kidney and liver. Coordinate unidirectional upregulation of expression of TGF-beta 2 and beta 3 mRNAs occurred in most embryonic tissues with development except the heart, where the steady-state level of expression of TGF-beta 2 mRNA decreased with age, while that of TGF-beta 3 mRNA increased. In situ hybridization analysis detected TGF-beta 2 and beta 3 mRNAs as early as the definitive primitive streak stage (stage 4). During neurulation (stage 10), TGF-beta 2 and beta 3 mRNAs were detected in cells of all three germ layers; TGF-beta 3 mRNA was detected in neurectoderm as well. Following neurulation, TGF-beta 3 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome and dermomyotome at stage 16; expression of TGF-beta 2 mRNA was not as prominent as TGF-beta 3 mRNA in these structures. By stage 29, both TGF-beta 2 and beta 3 mRNAs were localized in several tissues including heart, lung, gizzard and feathers. Immunohistochemical staining analysis detected immunoreactive TGF-beta 2 and beta 3 proteins in all three germ layers of stage 4 embryos. Staining for TGF-beta 2 and beta 3 proteins was detected in several cell types and tissues in the early developing embryo frequently in the same locations as TGF-beta 2 and beta 3 mRNAs, with staining for TGF-beta 2 being less intense than TGF-beta 3. However, in some cases, localization of TGF-beta 2 and beta 3 proteins was

  6. In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation.

    PubMed Central

    Yates, J L; Arfsten, A E; Nomura, M

    1980-01-01

    Escherichia coli ribosomal protein L1 (0.5 micro M) was found to inhibit the synthesis of both proteins of the L11 operon, L11 and L1, but not the synthesis of other proteins directed by lambda rifd 18 DNA. Similarly, S4 (1 micro M) selectively inhibited the synthesis of three proteins of the alpha operon, S13, S11, and S4, directed by lambda spcI DNA or a restriction enzyme fragment obtained from this DNA. S8 (3.6 micro M) also showed preferential inhibitory effects on the synthesis of some proteins encoded in the spc operon, L24 and L5 (and probably S14 and S8), directed by lambda spcl DNA or a restriction enzyme fragment carrying the genes for these proteins. The inhibitory effect of L1 was observed only with L1 and not with other proteins examined, including S4 and S8. Similarly, the effect of S4 was not observed with L1 or S8, and that of S8 was not seen with L1 or S4. Inhibition was shown to take place at the level of translation rather than transcription. Thus, at least some ribosomal proteins (L1 S4, and S8) have the ability to cause selective translational inhibition of the synthesis of certain ribosomal proteins whose genes are in the same operon as their own. These results support the hypothesis that certain free ribosomal proteins not assembled into ribosomes act as "autogenous" feedback inhibitors to regulate the synthesis of ribosomal proteins. Images PMID:6445562

  7. Differential Phosphorylation of Ribosomal Proteins in Arabidopsis thaliana Plants during Day and Night

    PubMed Central

    Turkina, Maria V.; Klang Årstrand, Hanna; Vener, Alexander V.

    2011-01-01

    Protein synthesis in plants is characterized by increase in the translation rates for numerous proteins and central metabolic enzymes during the day phase of the photoperiod. The detailed molecular mechanisms of this diurnal regulation are unknown, while eukaryotic protein translation is mainly controlled at the level of ribosomal initiation complexes, which also involves multiple events of protein phosphorylation. We characterized the extent of protein phosphorylation in cytosolic ribosomes isolated from leaves of the model plant Arabidopsis thaliana harvested during day or night. Proteomic analyses of preparations corresponding to both phases of the photoperiod detected phosphorylation at eight serine residues in the C-termini of six ribosomal proteins: S2-3, S6-1, S6-2, P0-2, P1 and L29-1. This included previously unknown phosphorylation of the 40S ribosomal protein S6 at Ser-231. Relative quantification of the phosphorylated peptides using stable isotope labeling and mass spectrometry revealed a 2.2 times increase in the day/night phosphorylation ratio at this site. Phosphorylation of the S6-1 and S6-2 variants of the same protein at Ser-240 increased by the factors of 4.2 and 1.8, respectively. The 1.6 increase in phosphorylation during the day was also found at Ser-58 of the 60S ribosomal protein L29-1. It is suggested that differential phosphorylation of the ribosomal proteins S6-1, S6-2 and L29-1 may contribute to modulation of the diurnal protein synthesis in plants. PMID:22195043

  8. Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons.

    PubMed

    Court, Felipe A; Midha, Rajiv; Cisterna, Bruno A; Grochmal, Joey; Shakhbazau, Antos; Hendriks, William T; Van Minnen, Jan

    2011-10-01

    Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

  9. SRY interacts with ribosomal proteins S7 and L13a in nuclear speckles.

    PubMed

    Sato, Youichi; Yano, Shojiro; Ewis, Ashraf A; Nakahori, Yutaka

    2011-05-01

    The SRY (sex-determining region on the Y chromosome) is essential for male development; however, the molecular mechanism by which the SRY induces testis development is still unclear. To elucidate the mechanism of testis development, we identified SRY-interacting proteins using a yeast two-hybrid system. We found two ribosomal proteins, RPS7 (ribosomal protein S7) and RPL13a (ribosomal protein L13a) that interact with the HMG (high-mobility group) box domain of SRY. Furthermore, we confirmed the intracellular distributions of RPS7, RPL13a and SRY and found that the three proteins were co-expressed in COS1 cells. SRY, RPS7 and RPL13a were co-localized in nuclear speckles. These findings suggest that SRY plays an important role in activities associated with nuclear speckles via an unknown mechanism.

  10. Cotranslational Protein Folding within the Ribosome Tunnel Influences Trigger-Factor Recruitment

    PubMed Central

    Lin, Ku-Feng; Sun, Chia-Sui; Huang, Yi-Chen; Chan, Sunney I.; Koubek, Jiri; Wu, Tzong-Huah; Huang, Joseph J.-T.

    2012-01-01

    In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting. PMID:22735532

  11. A Novel Method to Evaluate Ribosomal Performance in Cell-Free Protein Synthesis Systems.

    PubMed

    Kempf, Noémie; Remes, Cristina; Ledesch, Ralph; Züchner, Tina; Höfig, Henning; Ritter, Ilona; Katranidis, Alexandros; Fitter, Jörg

    2017-04-24

    Cell-free protein synthesis (CFPS) systems were designed to produce proteins with a minimal set of purified components, thus offering the possibility to follow translation as well as protein folding. In order to characterize the performance of the ribosomes in such a system, it is crucial to separately quantify the two main components of productivity, namely the fraction of active ribosomes and the number of synthesizing cycles. Here, we provide a direct and highly reliable measure of ribosomal activity in any given CFPS system, introducing an enhanced-arrest peptide variant. We observe an almost complete stalling of ribosomes that produce GFPem (~95%), as determined by common centrifugation techniques and fluorescence correlation spectroscopy (FCS). Moreover, we thoroughly study the effect of different ribosomal modifications independently on activity and number of synthesizing cycles. Finally, employing two-colour coincidence detection and two-colour colocalisation microscopy, we demonstrate real-time access to key productivity parameters with minimal sample consumption on a single ribosome level.

  12. The nuclear import of ribosomal proteins is regulated by mTOR

    PubMed Central

    Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

    2014-01-01

    Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

  13. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis

    PubMed Central

    Fei, Jingyi; Richard, Arianne C.; Bronson, Jonathan E.; Gonzalez, & Ruben L.

    2011-01-01

    Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during translocation. PMID:21857664

  14. Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

    PubMed Central

    Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R.; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

    2014-01-01

    Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. PMID:24835311

  15. Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia.

    PubMed

    Aspesi, Anna; Pavesi, Elisa; Robotti, Elisa; Crescitelli, Rossella; Boria, Ilenia; Avondo, Federica; Moniz, Hélène; Da Costa, Lydie; Mohandas, Narla; Roncaglia, Paola; Ramenghi, Ugo; Ronchi, Antonella; Gustincich, Stefano; Merlin, Simone; Marengo, Emilio; Ellis, Steven R; Follenzi, Antonia; Santoro, Claudio; Dianzani, Irma

    2014-07-25

    Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA. Copyright © 2014. Published by Elsevier B.V.

  16. Dosage Sensitivity of RPL9 and Concerted Evolution of Ribosomal Protein Genes in Plants

    PubMed Central

    Devis, Deborah; Firth, Sue M.; Liang, Zhe; Byrne, Mary E.

    2015-01-01

    The ribosome in higher eukaryotes is a large macromolecular complex composed of four rRNAs and eighty different ribosomal proteins. In plants, each ribosomal protein is encoded by multiple genes. Duplicate genes within a family are often necessary to provide a threshold dose of a ribosomal protein but in some instances appear to have non-redundant functions. Here, we addressed whether divergent members of the RPL9 gene family are dosage sensitive or whether these genes have non-overlapping functions. The RPL9 family in Arabidopsis thaliana comprises two nearly identical members, RPL9B and RPL9C, and a more divergent member, RPL9D. Mutations in RPL9C and RPL9D genes lead to delayed growth early in development, and loss of both genes is embryo lethal, indicating that these are dosage-sensitive and redundant genes. Phylogenetic analysis of RPL9 as well as RPL4, RPL5, RPL27a, RPL36a, and RPS6 family genes in the Brassicaceae indicated that multicopy ribosomal protein genes have been largely retained following whole genome duplication. However, these gene families also show instances of tandem duplication, small scale deletion, and evidence of gene conversion. Furthermore, phylogenetic analysis of RPL9 genes in angiosperm species showed that genes within a species are more closely related to each other than to RPL9 genes in other species, suggesting ribosomal protein genes undergo convergent evolution. Our analysis indicates that ribosomal protein gene retention following whole genome duplication contributes to the number of genes in a family. However, small scale rearrangements influence copy number and likely drive concerted evolution of these dosage-sensitive genes. PMID:26734020

  17. The Ribosomal Protein uL22 Modulates the Shape of the Protein Exit Tunnel.

    PubMed

    Wekselman, Itai; Zimmerman, Ella; Davidovich, Chen; Belousoff, Matthew; Matzov, Donna; Krupkin, Miri; Rozenberg, Haim; Bashan, Anat; Friedlander, Gilgi; Kjeldgaard, Jette; Ingmer, Hanne; Lindahl, Lasse; Zengel, Janice M; Yonath, Ada

    2017-08-01

    Erythromycin is a clinically useful antibiotic that binds to an rRNA pocket in the ribosomal exit tunnel. Commonly, resistance to erythromycin is acquired by alterations of rRNA nucleotides that interact with the drug. Mutations in the β hairpin of ribosomal protein uL22, which is rather distal to the erythromycin binding site, also generate resistance to the antibiotic. We have determined the crystal structure of the large ribosomal subunit from Deinococcus radiodurans with a three amino acid insertion within the β hairpin of uL22 that renders resistance to erythromycin. The structure reveals a shift of the β hairpin of the mutated uL22 toward the interior of the exit tunnel, triggering a cascade of structural alterations of rRNA nucleotides that propagate to the erythromycin binding pocket. Our findings support recent studies showing that the interactions between uL22 and specific sequences within nascent chains trigger conformational rearrangements in the exit tunnel. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile

    PubMed Central

    Bonnin, Pierre; Kern, Norbert; Young, Neil T.; Stansfield, Ian

    2017-01-01

    The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5’ to the 3’ end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling studies. PMID

  19. Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes

    PubMed Central

    Zhang, Jingyu; Harnpicharnchai, Piyanun; Jakovljevic, Jelena; Tang, Lan; Guo, Yurong; Oeffinger, Marlene; Rout, Michael P.; Hiley, Shawna L.; Hughes, Timothy; Woolford, John L.

    2007-01-01

    More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors—e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs—remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm. PMID:17938242

  20. Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes.

    PubMed

    Zhang, Jingyu; Harnpicharnchai, Piyanun; Jakovljevic, Jelena; Tang, Lan; Guo, Yurong; Oeffinger, Marlene; Rout, Michael P; Hiley, Shawna L; Hughes, Timothy; Woolford, John L

    2007-10-15

    More than 170 proteins are necessary for assembly of ribosomes in eukaryotes. However, cofactors that function with each of these proteins, substrates on which they act, and the precise functions of assembly factors--e.g., recruiting other molecules into preribosomes or triggering structural rearrangements of pre-rRNPs--remain mostly unknown. Here we investigated the recruitment of two ribosomal proteins and 5S ribosomal RNA (rRNA) into nascent ribosomes. We identified a ribonucleoprotein neighborhood in preribosomes that contains two yeast ribosome assembly factors, Rpf2 and Rrs1, two ribosomal proteins, rpL5 and rpL11, and 5S rRNA. Interactions between each of these four proteins have been confirmed by binding assays in vitro. These molecules assemble into 90S preribosomal particles containing 35S rRNA precursor (pre-rRNA). Rpf2 and Rrs1 are required for recruiting rpL5, rpL11, and 5S rRNA into preribosomes. In the absence of association of these molecules with pre-rRNPs, processing of 27SB pre-rRNA is blocked. Consequently, the abortive 66S pre-rRNPs are prematurely released from the nucleolus to the nucleoplasm, and cannot be exported to the cytoplasm.

  1. The essential polysome-associated RNA-binding protein RBP42 targets mRNAs involved in Trypanosoma brucei energy metabolism

    PubMed Central

    Das, Anish; Morales, Rachel; Banday, Mahrukh; Garcia, Stacey; Hao, Li; Cross, George A.M.; Estevez, Antonio M.; Bellofatto, Vivian

    2012-01-01

    RNA-binding proteins that target mRNA coding regions are emerging as regulators of post-transcriptional processes in eukaryotes. Here we describe a newly identified RNA-binding protein, RBP42, which targets the coding region of mRNAs in the insect form of the African trypanosome, Trypanosoma brucei. RBP42 is an essential protein and associates with polysome-bound mRNAs in the cytoplasm. A global survey of RBP42-bound mRNAs was performed by applying HITS-CLIP technology, which captures protein–RNA interactions in vivo using UV light. Specific RBP42–mRNA interactions, as well as mRNA interactions with a known RNA-binding protein, were purified using specific antibodies. Target RNA sequences were identified and quantified using high-throughput RNA sequencing. Analysis revealed that RBP42 bound mainly within the coding region of mRNAs that encode proteins involved in cellular energy metabolism. Although the mechanism of RBP42's function is unclear at present, we speculate that RBP42 plays a critical role in modulating T. brucei energy metabolism. PMID:22966087

  2. Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics.

    PubMed

    Pakula, Kevin K; Hansen, Lykke H; Vester, Birte

    2017-09-01

    Several groups of antibiotics inhibit bacterial growth by binding to bacterial ribosomes. Mutations in ribosomal protein L3 have been associated with resistance to linezolid and tiamulin, which both bind at the peptidyl transferase center in the ribosome. Resistance to these and other antibiotics also occurs through methylation of 23S rRNA at position A2503 by the methyltransferase Cfr. The mutations in L3 and the cfr gene have been found together in clinical isolates, raising the question of whether they have a combined effect on antibiotic resistance or growth. We transformed a plasmid-borne cfr gene into a uL3-depleted Escherichia coli strain containing either wild-type L3 or L3 with one of seven mutations, G147R, Q148F, N149S, N149D, N149R, Q150L, or T151P, expressed from plasmid-carried rplC genes. The L3 mutations are well tolerated, with small to moderate growth rate decreases. The presence of Cfr has a very minor influence on the growth rate. The resistance of the transformants to linezolid, tiamulin, florfenicol, and Synercid (a combination of quinupristin and dalfopristin [Q-D]) was measured by MIC assays. The resistance from Cfr was, in all cases, stronger than the effects of the L3 mutations, but various effects were obtained with the combinations of Cfr and L3 mutations ranging from a synergistic to an antagonistic effect. Linezolid and tiamulin susceptibility varied greatly among the L3 mutations, while no significant effects on florfenicol and Q-D susceptibility were seen. This study underscores the complex interplay between various resistance mechanisms and cross-resistance, even from antibiotics with overlapping binding sites. Copyright © 2017 American Society for Microbiology.

  3. A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.

    PubMed

    Sebastian, Sarah; Brochet, Mathieu; Collins, Mark O; Schwach, Frank; Jones, Matthew L; Goulding, David; Rayner, Julian C; Choudhary, Jyoti S; Billker, Oliver

    2012-07-19

    Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3' and 5'UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. A Plasmodium Calcium-Dependent Protein Kinase Controls Zygote Development and Transmission by Translationally Activating Repressed mRNAs

    PubMed Central

    Sebastian, Sarah; Brochet, Mathieu; Collins, Mark O.; Schwach, Frank; Jones, Matthew L.; Goulding, David; Rayner, Julian C.; Choudhary, Jyoti S.; Billker, Oliver

    2012-01-01

    Summary Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3′ and 5′UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression. PMID:22817984

  5. SR proteins SRp20 and 9G8 contribute to efficient export of herpes simplex virus 1 mRNAs

    SciTech Connect

    Escudero-Paunetto, Laurimar; Li Ling; Hernandez, Felicia P.; Sandri-Goldin, Rozanne M.

    2010-06-05

    Herpes simplex virus 1 (HSV-1) mRNAs are exported to the cytoplasm through the export receptor TAP/NFX1. HSV-1 multifunctional protein ICP27 interacts with TAP/NXF1, binds viral RNAs, and is required for efficient viral RNA export. In ICP27 mutant infections, viral RNA export is reduced but not ablated, indicating that other export adaptors can aid in viral RNA export. Export adaptor protein Aly/REF is recruited to viral replication compartments, however, Aly/REF knockdown has little effect on viral RNA export. SR proteins SRp20 and 9G8 interact with TAP/NXF1 and mediate export of some cellular RNAs. We report that siRNA knockdown of SRp20 or 9G8 resulted in about a 10 fold decrease in virus yields and in nuclear accumulation of poly(A+) RNA. In infected cells depleted of SRp20, newly transcribed Bromouridine-labeled RNA also accumulated in the nucleus. We conclude that SRp20 and 9G8 contribute to HSV-1 RNA export.

  6. The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

    PubMed Central

    Tutuncuoglu, Beril; Jakovljevic, Jelena; Wu, Shan; Gao, Ning

    2016-01-01

    Assaying effects on pre-rRNA processing and ribosome assembly upon depleting individual ribosomal proteins (r-proteins) provided an initial paradigm for assembly of eukaryotic ribosomes in vivo—that each structural domain of ribosomal subunits assembles in a hierarchical fashion. However, two features suggest that a more complex pathway may exist: (i) Some r-proteins contain extensions that reach long distances across ribosomes to interact with multiple rRNA domains as well as with other r-proteins. (ii) Individual r-proteins may assemble in a stepwise fashion. For example, the globular domain of an r-protein might assemble separately from its extensions. Thus, these extensions might play roles in assembly that could not be revealed by depleting the entire protein. Here, we show that deleting or mutating extensions of r-proteins L7 (uL30) and L35 (uL29) from yeast reveal important roles in early and middle steps during 60S ribosomal subunit biogenesis. Detailed analysis of the N-terminal terminal extension of L8 (eL8) showed that it is necessary for late nuclear stages of 60S subunit assembly involving two major remodeling events: removal of the ITS2 spacer; and reorganization of the central protuberance (CP) containing 5S rRNA and r-proteins L5 (uL18) and L11 (uL5). Mutations in the L8 extension block processing of 7S pre-rRNA, prevent release of assembly factors Rpf2 and Rrs1 from pre-ribosomes, which is required for rotation of the CP, and block association of Sda1, the Rix1 complex, and the Rea1 ATPase involved in late steps of remodeling. PMID:27390266

  7. The Contributions of the Ribosome Biogenesis Protein Utp5/WDR43 to Craniofacial Development.

    PubMed

    Sondalle, S B; Baserga, S J; Yelick, P C

    2016-10-01

    Fairly recently, it was recognized that human ribosomopathies-developmental defects caused by mutations in ribosome biogenesis proteins-can exhibit tissue-specific defects rather than the expected global defects. This apparent anomaly-that seemingly ubiquitously expressed and required ribosomal proteins can have distinct functions in cell and tissue differentiation-has spurred new areas of research focused on better understanding translational mechanisms, biogenesis, and function in diverse cell types. This renewed appreciation for, and need to better understand, roles for ribosomal proteins in human development and disease has identified surprising similarities and differences in a variety of human ribosomopathies. Here, we discuss ribosomal protein functions in health and disease, focusing on the ribosome biogenesis protein Utp5/WDR43. New and exciting research in this field is anticipated to provide insight into a variety of previously understudied craniofacial dysostoses and result in significantly improved knowledge and understanding of roles for translational machinery in human craniofacial development and disease. © International & American Associations for Dental Research 2016.

  8. Molecular cloning and characterization of a cDNA encoding the Paracoccidioides brasiliensis 135 ribosomal protein.

    PubMed

    Jesuino, Rosália S A; Pereira, Maristela; Felipe, M Sueli S; Azevedo, Maristella O; Soares, Célia M A

    2004-06-01

    A 630 bp cDNA encoding an L35 ribosomal protein of Paracoccidioides brasiliensis, designated as Pbl35, was cloned from a yeast expression library. Pbl35 encodes a polypeptide of 125 amino acids, with a predicted molecular mass of 14.5 kDa and a pI of 11.0. The deduced PbL35 shows significant conservation in respect to other described ribosomal L35 proteins from eukaryotes and prokaryotes. Motifs of ribosomal proteins are present in PbL35, including a bipartite nuclear localization signal (NLS) that could be related to the protein addressing to the nucleolus for the ribosomal assembly. The mRNA for PbL35, about 700 nucleotides in length, is expressed at a high level in P. brasiliensis. The PbL35 and the deduced amino acid sequence constitute the first description of a ribosomal protein in P. brasiliensis. The cDNA was deposited in GenBank under accession number AF416509.

  9. Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly.

    PubMed

    Cameron, Dale M; Gregory, Steven T; Thompson, Jill; Suh, Moo-Jin; Limbach, Patrick A; Dahlberg, Albert E

    2004-09-01

    The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. MALDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.

  10. Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

    PubMed Central

    Ingolia, Nicholas T.; Brar, Gloria A.; Stern-Ginossar, Noam; Harris, Michael S.; Talhouarne, Gaëlle J. S.; Jackson, Sarah E.; Wills, Mark R.; Weissman, Jonathan S.

    2014-01-01

    SUMMARY Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be non-coding, including 5′ UTRs and lncRNAs. Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here we show hallmarks of translation in these footprints: co-purification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts to understand how cells manage and exploit its consequences. PMID:25159147

  11. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

    PubMed

    Ingolia, Nicholas T; Brar, Gloria A; Stern-Ginossar, Noam; Harris, Michael S; Talhouarne, Gaëlle J S; Jackson, Sarah E; Wills, Mark R; Weissman, Jonathan S

    2014-09-11

    Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences.

  12. Peptide Chain Termination: Effect of Protein S on Ribosomal Binding of Release Factors

    PubMed Central

    Goldstein, J. L.; Caskey, C. T.

    1970-01-01

    The protein factor S, previously shown to stimulate polypeptide chain termination in bacterial extracts, has two effects upon the complex formed between ribosomes, release factor, and terminator (trinucleotide) codon: (1) in the absence of GTP or GDP, S stimulates formation of an [R·UAA·ribosome] intermediate, and (2) in the presence of GTP or GDP, S participates in dissociation of this intermediate. Factor S can stimulate fMet release from [fMet-tRNAf·AUG·ribosome] intermediates in either the presence or absence of GTP or GDP. A model is proposed which relates the in vitro effects of S ± GTP (or GDP) on fMet release to the effects of S ± GTP (or GDP) on the binding and dissociation of R factor from ribosomes. PMID:5289007

  13. Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly.

    PubMed

    Baker, Kris Ann; Lamichhane, Rajan; Lamichhane, Tek; Rueda, David; Cunningham, Philip R

    2016-09-11

    Interactions between ribosomal proteins (rproteins) and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, forming the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21, and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved among the Bacteria. To identify functionally important motifs within the junction, we subjected nucleotides 652-654 and 752-754 to saturation mutagenesis and selected and analyzed functional mutants. Only 64 mutants with greater than 10% ribosome function in vivo were isolated. S15 overexpression complemented mutations in the junction loop in each of the partially active mutants, although mutations that produced inactive ribosomes were not complemented by overexpression of S15. Single-molecule Förster or fluorescence resonance energy transfer (smFRET) was used to study the Mg(2+)- and S15-induced conformational dynamics of selected junction mutants. Comparison of the structural dynamics of these mutants with the wild type in the presence and absence of S15 revealed specific sequence and structural motifs in the central junction that are important in ribosome function. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Specific N-terminal cleavage of ribosomal protein L27 in Staphylococcus aureus and related bacteria

    PubMed Central

    Wall, Erin A.; Caufield, J. Harry; Lyons, Charles E.; Manning, Keith A.; Dokland, Terje; Christie, Gail E.

    2015-01-01

    Summary Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center. In this report we demonstrate that L27 in Staphylococcus aureus and other Firmicutes is encoded with an N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. We have identified a cysteine protease, conserved among bacteria containing the L27 N-terminal extension, which performs post-translational cleavage of L27. Ribosomal biology in eubacteria has largely been studied in the Gram negative bacterium Escherichia coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of the E. coli ribosome. This research lays the foundation for the development of new therapeutic approaches that target this novel pathway. PMID:25388641

  15. Expression of APP pathway mRNAs and proteins in Alzheimer's disease.

    PubMed

    Matsui, Toshifumi; Ingelsson, Martin; Fukumoto, Hiroaki; Ramasamy, Karunya; Kowa, Hisatomo; Frosch, Matthew P; Irizarry, Michael C; Hyman, Bradley T

    2007-08-03

    In both trisomy 21 and rare cases of triplication of amyloid precursor protein (APP) Alzheimer's disease (AD) pathological changes are believed to be secondary to increased expression of APP. We hypothesized that sporadic AD may also be associated with changes in transcription of APP or its metabolic partners. To address this issue, temporal neocortex of 27 AD and 21 non-demented control brains was examined to assess mRNA levels of APP isoforms (total APP, APP containing the Kunitz protease inhibitor domain [APP-KPI] and APP770) and APP metabolic enzymatic partners (the APP cleaving enzymes beta-secretase [BACE] and presenilin-1 [PS-1], and putative clearance molecules, low-density lipoprotein receptor protein [LRP] and apolipoprotein E [apoE]). Furthermore, we evaluated how changes in APP at the mRNA level affect the amount of Tris buffer extractable APP protein and Abeta40 and 42 peptides in AD and control brains. As assessed by quantitative PCR, APP-KPI (p=0.007), APP770 (p=0.004), PS-1 (p=0.004), LRP (p=0.003), apoE (p=0.0002) and GFAP (p<0.0001) mRNA levels all increased in AD, and there was a shift from APP695 (a neuronal isoform) towards KPI containing isoforms that are present in glia as well. APP-KPI mRNA levels correlated with soluble APPalpha-KPI protein (sAPPalpha-KPI) levels measured by ELISA (tau=0.33, p=0.015 by Kendall's rank correlation); in turn, soluble APPalpha-KPI protein levels positively correlated with Tris-extractable, soluble Abeta40 (p=0.046) and 42 levels (p=0.007). The ratio of soluble APPalpha-KPI protein levels to total APP protein increased in AD, and also correlated with GFAP protein levels in AD. These results suggest that altered transcription of APP in AD is proportionately associated with Abeta peptide, may occur in the context of gliosis, and may contribute to Abeta deposition in sporadic AD.

  16. Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress

    PubMed Central

    Bursać, Sladana; Brdovčak, Maja Cokarić; Pfannkuchen, Martin; Orsolić, Ines; Golomb, Lior; Zhu, Yan; Katz, Chen; Daftuar, Lilyn; Grabušić, Kristina; Vukelić, Iva; Filić, Vedrana; Oren, Moshe; Prives, Carol; Volarević, Siniša

    2012-01-01

    Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis. PMID:23169665

  17. Ribosomal protein deficiency causes Tp53-independent erythropoiesis failure in zebrafish.

    PubMed

    Yadav, Gnaneshwar V; Chakraborty, Anirban; Uechi, Tamayo; Kenmochi, Naoya

    2014-04-01

    Diamond-Blackfan anemia is an inherited genetic disease caused by mutations in ribosomal protein genes. The disease is characterized by bone marrow failure, congenital anomalies, and a severe erythroid defect. The activation of the TP53 pathway has been suggested to be critical for the pathophysiology of Diamond-Blackfan anemia. While this pathway plays a role in the morphological defects that associate with ribosomal protein loss-of-function in animal models, its role in the erythroid defects has not been clearly established. To understand the specificity of erythroid defects in Diamond-Blackfan anemia, we knocked down five RP genes (two Diamond-Blackfan anemia-associated and three non-Diamond-Blackfan anemia-associated) in zebrafish and analyzed the effects on the developmental and erythroid phenotypes in the presence and absence of Tp53. The co-inhibition of Tp53 activity rescued the morphological deformities but did not alleviate the erythroid aplasia indicating that ribosomal protein deficiency causes erythroid failure in a Tp53-independent manner. Interestingly, treatment with L-Leucine or L-Arginine, amino acids that augment mRNA translation via mTOR pathway, rescued the morphological defects and resulted in a substantial recovery of erythroid cells. Our results suggest that altered translation because of impaired ribosome function could be responsible for the morphological and erythroid defects in ribosomal protein-deficient zebrafish. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Cut in translation: ribosome-dependent mRNA decay.

    PubMed

    Lalaouna, David; Massé, Eric

    2017-05-02

    Transcription and translation are two complex mechanisms that are tightly coupled in prokaryotic cells. Even before the completion of transcription, ribosomes attach to the nascent mRNA and initiate protein synthesis. Remarkably, recent publications have indicated an association between translation and decay of certain mRNAs. In this issue of The EMBO Journal, Leroy et al (2017) depicts a fascinating mechanism of mRNA degradation, which involves the ribosome-associated ribonuclease Rae1 in Bacillus subtilis In a translation-dependent manner, Rae1 binds the ribosomal aminoacylation (A)-site and cleaves between specific codons of the targeted mRNA. © 2017 The Authors.

  19. Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis.

    PubMed

    Potter, Matthew D; Nicchitta, Christopher V

    2002-06-28

    In current views, translation-coupled ribosome binding to the endoplasmic reticulum (ER) membrane is transient, with association occurring via the signal recognition particle pathway and dissociation occurring upon the termination of protein synthesis. Recent studies indicate, however, that ribosomal subunits remain membrane-bound following the termination of protein synthesis. To define the mechanism of post-termination ribosome association with the ER membrane, membrane-bound ribosomes were detergent-solubilized from tissue culture cells at different stages of the protein synthesis cycle, and the composition of the ribosome-associated membrane protein fraction was determined. We report that ribosomes reside in stable association with the Sec61alpha-translocon following the termination stage of protein synthesis. Additionally, in vitro experiments revealed that solubilized, gradient-purified ribosome-translocon complexes were able to initiate the translation of secretory and cytosolic proteins and were functional in assays of signal sequence recognition. Using this experimental system, synthesis of signal sequence-bearing polypeptides yielded a tight ribosome-translocon junction; synthesis of nascent polypeptides lacking a signal sequence resulted in a disruption of this junction. On the basis of these data, we propose that in situ, ribosomes reside in association with the translocon throughout the cycle of protein synthesis, with membrane release occurring upon translation of proteins lacking topogenic signals.

  20. Gene clusters for ribosomal proteins in the mitochondrial genome of a liverwort, Marchantia polymorpha.

    PubMed Central

    Takemura, M; Oda, K; Yamato, K; Ohta, E; Nakamura, Y; Nozato, N; Akashi, K; Ohyama, K

    1992-01-01

    We detected 16 genes for ribosomal proteins in the complete sequence of the mitochondrial DNA from a liverwort, Marchantia polymorpha. The genes formed two major clusters, rps12-rps7 and rps10-rpl2-rps19-rps3-rpl16-rpl5- rps14-rps8- rpl6-rps13-rps11-rps1, very similar in organization to Escherichia coli ribosomal protein operons (str and S10-spc-alpha operons, respectively). In contrast, rps2 and rps4 genes were located separately in the liverwort mitochondrial genome (the latter was part of the alpha operon in E. coli). Furthermore, several ribosomal proteins encoded by the liverwort mitochondrial genome differed substantially in size from their counterparts in E. coli and liverwort chloroplast. PMID:1620617

  1. Pokeweed Antiviral Protein, a Ribosome Inactivating Protein: Activity, Inhibition and Prospects

    PubMed Central

    Domashevskiy, Artem V.; Goss, Dixie J.

    2015-01-01

    Viruses employ an array of elaborate strategies to overcome plant defense mechanisms and must adapt to the requirements of the host translational systems. Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an RNA N-glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of large rRNA, arresting protein synthesis at the translocation step. PAP is thought to play an important role in the plant’s defense mechanism against foreign pathogens. This review focuses on the structure, function, and the relationship of PAP to other RIPs, discusses molecular aspects of PAP antiviral activity, the novel inhibition of this plant toxin by a virus counteraction—a peptide linked to the viral genome (VPg), and possible applications of RIP-conjugated immunotoxins in cancer therapeutics. PMID:25635465

  2. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. The CELF1 RNA-Binding Protein Regulates Decay of Signal Recognition Particle mRNAs and Limits Secretion in Mouse Myoblasts

    PubMed Central

    Russo, Joseph; Lee, Jerome E.; López, Carolina M.; Anderson, John; Nguyen, Thuy-mi P.; Heck, Adam M.; Wilusz, Jeffrey

    2017-01-01

    We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and in vitro binding assays using recombinant CELF1, we now provide evidence that CELF1 directly binds the mRNAs encoding each of the subunits of the SRP. Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion. PMID:28129347

  4. Promoter sequences direct cytoplasmic localization and translation of mRNAs during starvation in yeast.

    PubMed

    Zid, Brian M; O'Shea, Erin K

    2014-10-02

    A universal feature of the response to stress and nutrient limitation is transcriptional upregulation of genes that encode proteins important for survival. Under many such conditions, the overall protein synthesis level is reduced, thereby dampening the stress response at the level of protein expression. For example, during glucose starvation in Saccharomyces cerevisiae (yeast), translation is rapidly repressed, yet the transcription of many stress- and glucose-repressed genes is increased. Here we show, using ribosomal profiling and microscopy, that this transcriptionally upregulated gene set consists of two classes: one class produces messenger RNAs that are translated during glucose starvation and are diffusely localized in the cytoplasm, including many heat-shock protein mRNAs; and the other class produces mRNAs that are not efficiently translated during glucose starvation and are concentrated in foci that co-localize with P bodies and stress granules, a class that is enriched for mRNAs involved in glucose metabolism. Surprisingly, the information specifying the differential localization and protein production of these two classes of mRNA is encoded in the promoter sequence: promoter responsiveness to heat-shock factor 1 (Hsf1) specifies diffuse cytoplasmic localization and higher protein production on glucose starvation. Thus, promoter sequences can influence not only the levels of mRNAs but also the subcellular localization of mRNAs and the efficiency with which they are translated, enabling cells to tailor protein production to the environmental conditions.

  5. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA

    PubMed Central

    Wang, Minshi; Parshin, Andrey V.; Shcherbik, Natalia; Pestov, Dimitri G.

    2015-01-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5′-truncated 5.8S rRNA, which we named 5.8SC. The 5′ exoribonuclease Xrn2 is involved in the generation of both 5.8SC and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8SC rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes. PMID:25995445

  6. Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

    PubMed

    Wang, Minshi; Parshin, Andrey V; Shcherbik, Natalia; Pestov, Dimitri G

    2015-07-01

    Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

  7. Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation

    PubMed Central

    Haubenreisser, Olaf; Wimmer, Bjoern; Weber, Manuela; Karl, Thomas; Klausegger, Alfred; Breitenbach, Michael; Hintner, Helmut; von der Haar, Tobias; Tuite, Mick F.; Breitenbach-Koller, Lore

    2013-01-01

    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered. PMID:23861776

  8. Specialized yeast ribosomes: a customized tool for selective mRNA translation.

    PubMed

    Bauer, Johann W; Brandl, Clemens; Haubenreisser, Olaf; Wimmer, Bjoern; Weber, Manuela; Karl, Thomas; Klausegger, Alfred; Breitenbach, Michael; Hintner, Helmut; von der Haar, Tobias; Tuite, Mick F; Breitenbach-Koller, Lore

    2013-01-01

    Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered 'specialized' ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.

  9. Ribosomal RNA and protein transcripts persist in the cysts of Entamoeba invadens.

    PubMed

    Ojha, Sandeep; Ahamad, Jamaluddin; Bhattacharya, Alok; Bhattacharya, Sudha

    2014-06-01

    In most organisms rDNA transcription ceases under conditions of growth stress. However, we have earlier shown that pre-rRNA accumulates during encystation in Entamoeba invadens. We labeled newly-synthesized rRNA during encystation, with [methyl-(3)H] methionine in the presence of chitinase to enable uptake of isotope. Incorporation rate reduced after 24h, and then increased to reach levels comparable with normal cells. The label was rapidly chased to the ribosomal pellet in dividing cells, while at late stages of encystation the ratio of counts going to the pellet dropped 3-fold. The transcript levels of selected ribosomal protein genes also went down initially but went up again at later stages of encystation. This suggested that rRNA and ribosomal protein transcription may be coordinately regulated. Our data shows that encysting E. invadens cells accumulate transcripts of both the RNA and protein components of the ribosome, which may ensure rapid synthesis of new ribosomes when growth resumes. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Protein Folding Activity of Ribosomal RNA Is a Selective Target of Two Unrelated Antiprion Drugs

    PubMed Central

    Tribouillard-Tanvier, Déborah; Dos Reis, Suzana; Gug, Fabienne; Voisset, Cécile; Béringue, Vincent; Sabate, Raimon; Kikovska, Ema; Talarek, Nicolas; Bach, Stéphane; Huang, Chenhui; Desban, Nathalie; Saupe, Sven J.; Supattapone, Surachai; Thuret, Jean-Yves; Chédin, Stéphane; Vilette, Didier; Galons, Hervé; Sanyal, Suparna; Blondel, Marc

    2008-01-01

    Background 6-Aminophenanthridine (6AP) and Guanabenz (GA, a drug currently in use for the treatment of hypertension) were isolated as antiprion drugs using a yeast-based assay. These structurally unrelated molecules are also active against mammalian prion in several cell-based assays and in vivo in a mouse model for prion-based diseases. Methodology/Principal Findings Here we report the identification of cellular targets of these drugs. Using affinity chromatography matrices for both drugs, we demonstrate an RNA-dependent interaction of 6AP and GA with the ribosome. These specific interactions have no effect on the peptidyl transferase activity of the ribosome or on global translation. In contrast, 6AP and GA specifically inhibit the ribosomal RNA-mediated protein folding activity of the ribosome. Conclusion/Significance 6AP and GA are therefore the first compounds to selectively inhibit the protein folding activity of the ribosome. They thus constitute precious tools to study the yet largely unexplored biological role of this protein folding activity. PMID:18478094

  11. Recent advances in the uses and applications of ribosome-inactivating proteins from plants.

    PubMed

    Girbés, T; Ferreras, J M; Iglesias, R; Citores, L; De Torre, C; Carbajales, M L; Jiménez, P; De Benito, F M; Muñoz, R

    1996-06-01

    Plant ribosome-inactivating proteins (RIPs) are inhibitors present in all parts of plants that irreversibly inactivate eukaryotic ribosomes, thus impairing protein synthesis. RIPs are enzymes with N-glycosidase activity on the large rRNA. Their powerful inhibitory activity has been made use of advantageously to construct conjugates with suitable carriers targeted to altered specific cells. RIPs may be used to inhibit replication of both animal and plant viruses. The introduction of genes coding for RIPs into the genome of plants leads to an increase in resistance towards fungal pathogens and viruses. RIPs are important tools for the treatment of cancer and AIDS and for the protection of crop production.

  12. E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

    PubMed Central

    Shasmal, Manidip; Dey, Sandip; Shaikh, Tanvir R.; Bhakta, Sayan; Sengupta, Jayati

    2016-01-01

    It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome. PMID:26822933

  13. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway

    PubMed Central

    Coyle, John H.; Bor, Yeou-Cherng; Rekosh, David; Hammarskjold, Marie-Louise

    2011-01-01

    Post-transcriptional regulation of mRNA includes restriction mechanisms to prevent export and expression of mRNAs that are incompletely spliced. Here we present evidence that the mammalian protein Tpr is involved in this restriction. To study the role of Tpr in export of mRNA with retained introns, we used reporters in which the mRNA was exported either via the Nxf1/Nxt1 pathway using a CTE or via the Crm1 pathway using Rev/RRE. Our data show that even modest knockdown of Tpr using RNAi leads to a significant increase in export and translation from the mRNA containing the CTE. In contrast, Tpr perturbation has no effect on export of mRNA containing the RRE, either in the absence or presence of Rev. Also, no effects were observed on export of a completely spliced mRNA. Taken together, our results indicate that Tpr plays an important role in quality control of mRNA trafficked on the Nxf1 pathway. PMID:21613532

  14. RNA editing in six mitochondrial ribosomal protein genes of Didymium iridis.

    PubMed

    Hendrickson, Peter G; Silliker, Margaret E

    2010-06-01

    Similarity searches with Didymium iridis mitochondrial genomic DNA identified six possible ribosomal protein-coding regions, however, each region contained stop codons that would need to be removed by RNA editing to produce functional transcripts. RT-PCR was used to amplify these regions from total RNA for cloning and sequencing. Six functional transcripts were verified for the following ribosomal protein genes: rpS12, rpS7, rpL2, rpS19, rpS3, and rpL16. The editing events observed, such as single C and U nucleotide insertions and a dinucleotide insertion, were consistent with previously observed editing patterns seen in D. iridis. Additionally, a new form of insertional editing, a single A insertion, was observed in a conserved region of the rpL16 gene. While the majority of codons created by editing specify hydrophobic amino acids, a greater proportion of the codons created in these hydrophilic ribosomal proteins called for positively charged amino acids in comparison to the previously characterized hydrophobic respiratory protein genes. This first report of edited soluble mitochondrial ribosomal proteins in myxomycetes expands upon the RNA editing patterns previously seen; there was: a greater proportion of created codons specifying positively charged amino acids, a shift in the codon position edited, and the insertion of single A nucleotides.

  15. Cis-regulatory RNA elements that regulate specialized ribosome activity

    PubMed Central

    Xue, Shifeng; Barna, Maria

    2015-01-01

    Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5′UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution. PMID:26327194

  16. Time-Resolved Proteomics Extends Ribosome Profiling-Based Measurements of Protein Synthesis Dynamics.

    PubMed

    Liu, Tzu-Yu; Huang, Hector H; Wheeler, Diamond; Xu, Yichen; Wells, James A; Song, Yun S; Wiita, Arun P

    2017-06-28

    Ribosome profiling is a widespread tool for studying translational dynamics in human cells. Its central assumption is that ribosome footprint density on a transcript quantitatively reflects protein synthesis. Here, we test this assumption using pulsed-SILAC (pSILAC) high-accuracy targeted proteomics. We focus on multiple myeloma cells exposed to bortezomib, a first-line chemotherapy and proteasome inhibitor. In the absence of drug effects, we found that direct measurement of protein synthesis by pSILAC correlated well with indirect measurement of synthesis from ribosome footprint density. This correlation, however, broke down under bortezomib-induced stress. By developing a statistical model integrating longitudinal proteomic and mRNA-sequencing measurements, we found that proteomics could directly detect global alterations in translational rate caused by bortezomib; these changes are not detectable by ribosomal profiling alone. Further, by incorporating pSILAC data into a gene expression model, we predict cell-stress specific proteome remodeling events. These results demonstrate that pSILAC provides an important complement to ribosome profiling in measuring proteome dynamics. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Ribosomal Proteins and Human Diseases: Pathogenesis, Molecular Mechanisms, and Therapeutic Implications

    PubMed Central

    Wang, Wei; Nag, Subhasree; Zhang, Xu; Wang, Ming-Hai; Wang, Hui; Zhou, Jianwei; Zhang, Ruiwen

    2014-01-01

    Ribosomes are essential components of the protein synthesis machinery. The process of ribosome biogenesis is well organized and tightly regulated. Recent studies have shown that ribosomal proteins (RPs) have extraribosomal functions that are involved in cell proliferation, differentiation, apoptosis, DNA repair, and other cellular processes. The dysfunction of RPs has been linked to the development and progression of hematological, metabolic, and cardiovascular diseases and cancer. Perturbation of ribosome biogenesis results in ribosomal stress, which triggers activation of the p53 signaling pathway through RPs-MDM2 interactions, resulting in p53-dependent cell cycle arrest and apoptosis. RPs also regulate cellular functions through p53-independent mechanisms. We herein review the recent advances in several forefronts of RP research, including the understanding of their biological features and roles in regulating cellular functions, maintaining cell homeostasis, and their involvement in the pathogenesis of human diseases. We also highlight the translational potential of this research for the identification of molecular biomarkers, and in the discovery and development of novel treatments for human diseases. PMID:25164622

  18. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling.

    PubMed

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein-EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here.

  19. Molecular characterization of a human gene for S28 ribosomal binding protein

    SciTech Connect

    Wong, P.; Borst, D.E.; Chader, G.J.

    1994-09-01

    The mechanism of ribosome action and the ribosomal binding proteins which cooperatively interact in the working of this structure are not completely understood. Theoretically, mutations in genes that encode these proteins may compromise the efficiency of protein synthesis and therefore lead to a functional disorder. In the course of our search for human genes which show homology to the C. elegans CED-4 death gene, we have serendipitously identified one of the human S28 ribosomal binding protein genes as a random fragment fused to the end of one of our putative CED-4 positive homologue clones. The cloned S28 fragment consists of 381 nucleotides with a putative open reading frame of 113 amino acids. Sequence comparisons to GenBank revealed significant homologies to ribosomal binding protein genes in other species (including the rat S28 ribosomal binding protein gene) indicating that the S28 gene sequence is highly conserved. This finding is confirmed by zooblot analysis. Significant homologies also exist to two human expressed tagged sites (HUMRIBPROB; L05091 and HSAFIF072; Z21908). Analysis of the putative S28 peptide sequence allows insights into possible functional regions of the protein. The identification of 8 distinct bands upon Southern analysis of the S28 fragments suggests that there are multiple copies of the S28 gene in the human genome. Mapping of the S28 fragment on somatic cell hybrid panels identified distinct S28 gene loci on chromosomes 1, 2, 7, 10, 11, 12, 17 expression in adult tissues (pancreas, kidney, muscle, liver, lung, placenta, brain, heart, and retina) as well as in fetal tissues (kidney, liver, lung, brain, and heart).

  20. Analysis of plant ribosomes with asymmetric flow field-flow fractionation.

    PubMed

    Pitkänen, Leena; Tuomainen, Päivi; Eskelin, Katri

    2014-02-01

    Ribosome profiling is a technique used to separate ribosomal subunits, 80S ribosomes (monosomes), and polyribosomes (polysomes) from other RNA-protein complexes. It is traditionally performed in sucrose gradients. In this study, we used asymmetric flow field-flow fractionation (AsFlFFF) to characterize ribosome profiles of Nicotiana benthamiana plants. With the optimized running conditions, we were able to separate free molecules from ribosomal subunits and intact ribosomes. We used various chemical and enzymatic treatments to validate the positions of subunits, monosomes, and polysomes in the AsFlFFF fractograms. We also characterized the protein and RNA content of AsFlFFF fractions by gel electrophoresis and western blotting. The reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ribosomes remained bound to messenger RNAs (mRNAs) during the analysis. Therefore, we conclude that AsFlFFF can be used for ribosome profiling to study the mRNAs that are being translated. It can also be used to study the protein composition of ribosomes that are active in translation at that particular moment.

  1. Recognition of Ribosomal Protein L11 by the Protein Trimethyltransferase PrmA

    SciTech Connect

    Demirci,H.; Gregory, S.; Dahlberg, A.; Jogl, G.

    2007-01-01

    Bacterial ribosomal protein L11 is post-translationally trimethylated at multiple residues by a single methyltransferase, PrmA. Here, we describe four structures of PrmA from the extreme thermophile Thermus thermophilus. Two apo-PrmA structures at 1.59 and 2.3 {angstrom} resolution and a third with bound cofactor S-adenosyl-L-methionine at 1.75 {angstrom} each exhibit distinct relative positions of the substrate recognition and catalytic domains, revealing how PrmA can position the L11 substrate for multiple, consecutive side-chain methylation reactions. The fourth structure, the PrmA-L11 enzyme-substrate complex at 2.4 {angstrom} resolution, illustrates the highly specific interaction of the N-terminal domain with its substrate and places Lys39 in the PrmA active site. The presence of a unique flexible loop in the cofactor-binding site suggests how exchange of AdoMet with the reaction product S-adenosyl-L-homocysteine can occur without necessitating the dissociation of PrmA from L11. Finally, the mode of interaction of PrmA with L11 explains its observed preference for L11 as substrate before its assembly into the 50S ribosomal subunit.

  2. Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis.

    PubMed

    Jensen, Liselotte E; Whitehead, Alexander S

    2003-08-01

    Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

  3. The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

    PubMed Central

    Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

    2016-01-01

    Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27kip1 and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27kip1 mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. PMID:26926106

  4. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes

    PubMed Central

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-01

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality. DOI: http://dx.doi.org/10.7554/eLife.10415.001 PMID:26760529

  5. Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes.

    PubMed

    Katz, Zachary B; English, Brian P; Lionnet, Timothée; Yoon, Young J; Monnier, Nilah; Ovryn, Ben; Bathe, Mark; Singer, Robert H

    2016-01-13

    Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.

  6. NAD+-dependent Deacetylase SIRT3 Regulates Mitochondrial Protein Synthesis by Deacetylation of the Ribosomal Protein MRPL10*

    PubMed Central

    Yang, Yongjie; Cimen, Huseyin; Han, Min-Joon; Shi, Tong; Deng, Jian-Hong; Koc, Hasan; Palacios, Orsolya M.; Montier, Laura; Bai, Yidong; Tong, Qiang; Koc, Emine C.

    2010-01-01

    A member of the sirtuin family of NAD+-dependent deacetylases, SIRT3, is located in mammalian mitochondria and is important for regulation of mitochondrial metabolism, cell survival, and longevity. In this study, MRPL10 (mitochondrial ribosomal protein L10) was identified as the major acetylated protein in the mitochondrial ribosome. Ribosome-associated SIRT3 was found to be responsible for deacetylation of MRPL10 in an NAD+-dependent manner. We mapped the acetylated Lys residues by tandem mass spectrometry and determined the role of these residues in acetylation of MRPL10 by site-directed mutagenesis. Furthermore, we observed that the increased acetylation of MRPL10 led to an increase in translational activity of mitochondrial ribosomes in Sirt3−/− mice. In a similar manner, ectopic expression and knockdown of SIRT3 in C2C12 cells resulted in the suppression and enhancement of mitochondrial protein synthesis, respectively. Our findings constitute the first evidence for the regulation of mitochondrial protein synthesis by the reversible acetylation of the mitochondrial ribosome and characterize MRPL10 as a novel substrate of the NAD+-dependent deacetylase, SIRT3. PMID:20042612

  7. Tempo and mode of gene duplication in mammalian ribosomal protein evolution.

    PubMed

    Dharia, Asav P; Obla, Ajay; Gajdosik, Matthew D; Simon, Amanda; Nelson, Craig E

    2014-01-01

    Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism.

  8. Protein Folding Activity of the Ribosome (PFAR) –– A Target for Antiprion Compounds

    PubMed Central

    Banerjee, Debapriya; Sanyal, Suparna

    2014-01-01

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools. PMID:25341659

  9. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  10. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  11. Transcription by moonlight: structural basis of an extraribosomal activity of ribosomal protein S10.

    PubMed

    Weisberg, Robert A

    2008-12-26

    In this issue of Molecular Cell, Luo et al. (2008) show that S10 protein can function in the ribosome or the transcript elongation complex with minimal structural change, providing new insights into the roles of S10 and NusB in transcript elongation.

  12. Enhanced pest resistance of maize leaves expressing monocot crop plant derived ribosome inactivating protein and agglutinin

    USDA-ARS?s Scientific Manuscript database

    Although many insect resistance genes have been identified, the number of studies examining their effects in combination using transgenic systems is limited. We introduced a construct into maize containing the coding sequence for maize ribosome inactivating protein (MRIP), wheat germ agglutinin (WGA...

  13. Tempo and Mode of Gene Duplication in Mammalian Ribosomal Protein Evolution

    PubMed Central

    Gajdosik, Matthew D.; Simon, Amanda; Nelson, Craig E.

    2014-01-01

    Gene duplication has been widely recognized as a major driver of evolutionary change and organismal complexity through the generation of multi-gene families. Therefore, understanding the forces that govern the evolution of gene families through the retention or loss of duplicated genes is fundamentally important in our efforts to study genome evolution. Previous work from our lab has shown that ribosomal protein (RP) genes constitute one of the largest classes of conserved duplicated genes in mammals. This result was surprising due to the fact that ribosomal protein genes evolve slowly and transcript levels are very tightly regulated. In our present study, we identified and characterized all RP duplicates in eight mammalian genomes in order to investigate the tempo and mode of ribosomal protein family evolution. We show that a sizable number of duplicates are transcriptionally active and are very highly conserved. Furthermore, we conclude that existing gene duplication models do not readily account for the preservation of a very large number of intact retroduplicated ribosomal protein (RT-RP) genes observed in mammalian genomes. We suggest that selection against dominant-negative mutations may underlie the unexpected retention and conservation of duplicated RP genes, and may shape the fate of newly duplicated genes, regardless of duplication mechanism. PMID:25369106

  14. A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins

    PubMed Central

    Sung, Min-Kyung; Porras-Yakushi, Tanya R; Reitsma, Justin M; Huber, Ferdinand M; Sweredoski, Michael J; Hoelz, André; Hess, Sonja; Deshaies, Raymond J

    2016-01-01

    Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001 PMID:27552055

  15. A Possible Role of the Full-Length Nascent Protein in Post-Translational Ribosome Recycling

    PubMed Central

    Das, Debasis; Samanta, Dibyendu; Bhattacharya, Arpita; Basu, Arunima; Das, Anindita; Ghosh, Jaydip; Chakrabarti, Abhijit; Das Gupta, Chanchal

    2017-01-01

    Each cycle of translation initiation in bacterial cell requires free 50S and 30S ribosomal subunits originating from the post-translational dissociation of 70S ribosome from the previous cycle. Literature shows stable dissociation of 70S from model post-termination complexes by the concerted action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G) that interact with the rRNA bridge B2a/B2b joining 50S to 30S. In such experimental models, the role of full-length nascent protein was never considered seriously. We observed relatively slow release of full-length nascent protein from 50Sof post translation ribosome, and in that process, its toe prints on the rRNA in vivo and in in vitro translation with E.coli S30 extract. We reported earlier that a number of chemically unfolded proteins like bovine carbonic anhydrase (BCA), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), lysozyme, ovalbumin etc., when added to free 70Sin lieu of the full length nascent proteins, also interact with identical RNA regions of the 23S rRNA. Interestingly the rRNA nucleotides that slow down release of the C-terminus of full-length unfolded protein were found in close proximity to the B2a/B2b bridge. It indicated a potentially important chemical reaction conserved throughout the evolution. Here we set out to probe that conserved role of unfolded protein conformation in splitting the free or post-termination 70S. How both the RRF-EFG dependent and the plausible nascent protein–EFG dependent ribosome recycling pathways might be relevant in bacteria is discussed here. PMID:28099529

  16. Inhibition of Protein Synthesis on the Ribosome by Tildipirosin Compared with Other Veterinary Macrolides

    PubMed Central

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf

    2012-01-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC50], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site. PMID:22926570

  17. Inhibition of protein synthesis on the ribosome by tildipirosin compared with other veterinary macrolides.

    PubMed

    Andersen, Niels Møller; Poehlsgaard, Jacob; Warrass, Ralf; Douthwaite, Stephen

    2012-11-01

    Tildipirosin is a 16-membered-ring macrolide developed to treat bacterial pathogens, including Mannheimia haemolytica and Pasteurella multocida, that cause respiratory tract infections in cattle and swine. Here we evaluated the efficacy of tildipirosin at inhibiting protein synthesis on the ribosome (50% inhibitory concentration [IC(50)], 0.23 ± 0.01 μM) and compared it with the established veterinary macrolides tylosin, tilmicosin, and tulathromycin. Mutation and methylation at key rRNA nucleotides revealed differences in the interactions of these macrolides within their common ribosomal binding site.

  18. Ribosomal protein L16 binds to the 3'-end of transfer RNA.

    PubMed

    Maimets, T; Remme, J; Villems, R

    1984-01-23

    Escherichia coli 50 S ribosomal subunits were reconstituted with and without protein L16 present. The latter particles, although active in puromycin reaction, were unable to use CACCA-Phe as an acceptor substrate. We also found that L16 interacts directly with this oligonucleotide and, in the complex with tRNA, protects its 3'-end from pancreatic ribonuclease digestion. We suggest that the role of L16 is in the fixation of the aminoacyl stem of tRNA to the ribosome at its A-site.

  19. Dietary protein intake and skeletal-muscle protein metabolism in rats. Studies with salt-washed ribosomes and transfer factors

    PubMed Central

    Alexis, S. D.; Basta, S.; Young, Vernon R.

    1972-01-01

    1. Aspects of skeletal muscle protein synthesis in vitro were studied in young rats given a low-protein diet for up to 10 days and during re-feeding with an adequate diet. 2. Partially purified muscle transfer factors (transferases I and II), crude and purified (NH4Cl-washed) ribosomes and a pH5 enzyme fraction were prepared for this purpose. 3. A marked decrease in the capacity of crude ribosomes to carry out cell-free polypeptide synthesis occurred within 4 days of feeding the low-protein diet. 4. The capacity of salt-washed ribosomes to promote amino acid polymerization, in the presence of added transfer factors and aminoacyl-tRNA, was only slightly decreased by the dietary treatment. 5. However, the capacity of salt-washed ribosomes to bind 14C-labelled aminoacyl-tRNA was decreased by feeding the low-protein diet. 6. The capacity of the pH5 enzyme fraction to promote amino acid incorporation in a complete cell-free system was decreased within 2 days of feeding the low-protein diet. There is no evidence that the change is associated with aminoacyl-tRNA synthetase or binding enzyme activities of the pH5 fractions. 7. These changes are discussed in relation to the diminished rate of protein synthesis in the intact muscle cell when rats are given a low-protein diet. PMID:4634827

  20. The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control

    PubMed Central

    ZENGEL, JANICE M.; JERAULD, ADAM; WALKER, ANDRE; WAHL, MARKUS C.; LINDAHL, LASSE

    2003-01-01

    Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon. PMID:13130133

  1. The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control.

    PubMed

    Zengel, Janice M; Jerauld, Adam; Walker, Andre; Wahl, Markus C; Lindahl, Lasse

    2003-10-01

    Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.

  2. Varicella-Zoster Virus IE4 Protein Interacts with SR Proteins and Exports mRNAs through the TAP/NXF1 Pathway

    PubMed Central

    Ote, Isabelle; Lebrun, Marielle; Vandevenne, Patricia; Bontems, Sébastien; Medina-Palazon, Cahora; Manet, Evelyne; Piette, Jacques; Sadzot-Delvaux, Catherine

    2009-01-01

    Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export. PMID:19924249

  3. Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway.

    PubMed

    Ote, Isabelle; Lebrun, Marielle; Vandevenne, Patricia; Bontems, Sébastien; Medina-Palazon, Cahora; Manet, Evelyne; Piette, Jacques; Sadzot-Delvaux, Catherine

    2009-11-18

    Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

  4. Translation elongation can control translation initiation on eukaryotic mRNAs

    PubMed Central

    Chu, Dominique; Kazana, Eleanna; Bellanger, Noémie; Singh, Tarun; Tuite, Mick F; von der Haar, Tobias

    2014-01-01

    Synonymous codons encode the same amino acid, but differ in other biophysical properties. The evolutionary selection of codons whose properties are optimal for a cell generates the phenomenon of codon bias. Although recent studies have shown strong effects of codon usage changes on protein expression levels and cellular physiology, no translational control mechanism is known that links codon usage to protein expression levels. Here, we demonstrate a novel translational control mechanism that responds to the speed of ribosome movement immediately after the start codon. High initiation rates are only possible if start codons are liberated sufficiently fast, thus accounting for the observation that fast codons are overrepresented in highly expressed proteins. In contrast, slow codons lead to slow liberation of the start codon by initiating ribosomes, thereby interfering with efficient translation initiation. Codon usage thus evolved as a means to optimise translation on individual mRNAs, as well as global optimisation of ribosome availability. PMID:24357599

  5. A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding.

    PubMed

    Cabrita, Lisa D; Cassaignau, AnaÏs M E; Launay, Helene M M; Waudby, Christopher A; Wlodarski, Tomasz; Camilloni, Carlo; Karyadi, Maria-Evangelia; Robertson, Amy L; Wang, Xiaolin; Wentink, Anne S; Goodsell, Luke; Woolhead, Cheryl A; Vendruscolo, Michele; Dobson, Christopher M; Christodoulou, John

    2016-04-01

    Although detailed pictures of ribosome structures are emerging, little is known about the structural and cotranslational folding properties of nascent polypeptide chains at the atomic level. Here we used solution-state NMR spectroscopy to define a structural ensemble of a ribosome-nascent chain complex (RNC) formed during protein biosynthesis in Escherichia coli, in which a pair of immunoglobulin-like domains adopts a folded N-terminal domain (FLN5) and a disordered but compact C-terminal domain (FLN6). To study how FLN5 acquires its native structure cotranslationally, we progressively shortened the RNC constructs. We found that the ribosome modulates the folding process, because the complete sequence of FLN5 emerged well beyond the tunnel before acquiring native structure, whereas FLN5 in isolation folded spontaneously, even when truncated. This finding suggests that regulating structure acquisition during biosynthesis can reduce the probability of misfolding, particularly of homologous domains.

  6. Mechanism of tetracycline resistance by ribosomal protection protein Tet(O).

    PubMed

    Li, Wen; Atkinson, Gemma C; Thakor, Nehal S; Allas, Ular; Lu, Chuao-chao; Chan, Kwok-Yan; Tenson, Tanel; Schulten, Klaus; Wilson, Kevin S; Hauryliuk, Vasili; Frank, Joachim

    2013-01-01

    Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6-Å resolution. This atomic model allowed us to identify the Tet(O)-ribosome binding sites, which involve three characteristic loops in domain 4 of Tet(O). Replacements of the three amino-acid tips of these loops by a single glycine residue result in loss of Tet(O)-mediated tetracycline resistance. On the basis of these findings, the mechanism of Tet(O)-mediated tetracycline resistance can be explained in molecular detail.

  7. Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication

    PubMed Central

    Gérus, Marie; Hoareau-Aveilla, Coralie; Kiss, Tamás; Caizergues-Ferrer, Michèle; Henry, Yves; Henras, Anthony K.

    2012-01-01

    Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes. PMID:22434888

  8. Fluctuations in protein synthesis from a single RNA template: Stochastic kinetics of ribosomes

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T. V.

    2009-01-01

    Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them—namely, the dwell time distribution—has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

  9. Systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change.

    PubMed

    Xu, Qin; Song, Zhihong; Zhu, Caiyun; Tao, Chengcheng; Kang, Lifang; Liu, Wei; He, Fei; Yan, Juan; Sang, Tao

    2017-02-13

    Long non-coding RNA (lncRNA) is a class of non-coding RNA with important regulatory roles in biological process of organisms. The systematic comparison of lncRNAs with protein coding mRNAs in population expression and their response to environmental change are still poorly understood. Here we identified 17,610 lncRNAs and calculated their expression levels based on RNA-seq of 80 individuals of Miscanthus lutarioriparius from two environments, the nearly native habitats and transplanted field, respectively. LncRNAs had significantly higher expression diversity and lower expression frequency in population than protein coding mRNAs in both environments, which suggested that lncRNAs may experience more relaxed selection or divergent evolution in population compared with protein coding RNAs. In addition, the increase of expression diversity for lncRNAs was always significantly higher and the magnitude of fold change of expression in new stress environment was significantly larger than protein-coding mRNAs. These results suggested that lncRNAs may be more sensitive to environmental change than protein-coding mRNAs. Analysis of environment-robust and environment-specific lncRNA-mRNA co-expression network between two environments revealed the characterization of lncRNAs in response to environmental change. Furthermore, candidate lncRNAs contributing to water use efficiency (WUE) identified based on the WUE-lncRNA-mRNA co-expression network suggested the roles of lncRNAs in response to environmental change. Our study provided a comprehensive understanding of expression characterization of lncRNAs in population for M. lutarioriparius under field condition, which would be useful to explore the roles of lncRNAs and could accelerate the process of adaptation in new environment for many plants.

  10. YqjD is an inner membrane protein associated with stationary-phase ribosomes in Escherichia coli.

    PubMed

    Yoshida, Hideji; Maki, Yasushi; Furuike, Shou; Sakai, Akiko; Ueta, Masami; Wada, Akira

    2012-08-01

    Here, we provide evidence that YqjD, a hypothetical protein of Escherichia coli, is an inner membrane and ribosome binding protein. This protein is expressed during the stationary growth phase, and expression is regulated by stress response sigma factor RpoS. YqjD possesses a transmembrane motif in the C-terminal region and associates with 70S and 100S ribosomes at the N-terminal region. Interestingly, E. coli possesses two paralogous proteins of YqjD, ElaB and YgaM, which are expressed and bind to ribosomes in a similar manner to YqjD. Overexpression of YqjD leads to inhibition of cell growth. It has been suggested that YqjD loses ribosomal activity and localizes ribosomes to the membrane during the stationary phase.

  11. A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis.

    PubMed

    Singh, Jaspal; Tyers, Mike

    2009-08-15

    The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1-Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis.

  12. Yeast ribosomal protein L10 helps coordinate tRNA movement through the large subunit

    PubMed Central

    Petrov, Alexey N.; Meskauskas, Arturas; Roshwalb, Sara C.; Dinman, Jonathan D.

    2008-01-01

    Yeast ribosomal protein L10 (E. coli L16) is located at the center of a topological nexus that connects many functional regions of the large subunit. This essential protein has previously been implicated in processes as diverse as ribosome biogenesis, translational fidelity and mRNA stability. Here, the inability to maintain the yeast Killer virus was used as a proxy for large subunit defects to identify a series of L10 mutants. These mapped to roughly four discrete regions of the protein. A detailed analysis of mutants located in the N-terminal ‘hook’ of L10, which inserts into the bulge of 25S rRNA helix 89, revealed strong effects on rRNA structure corresponding to the entire path taken by the tRNA 3′ end as it moves through the large subunit during the elongation cycle. The mutant-induced structural changes are wide-ranging, affecting ribosome biogenesis, elongation factor binding, drug resistance/hypersensitivity, translational fidelity and virus maintenance. The importance of L10 as a potential transducer of information through the ribosome, and of a possible role of its N-terminal domain in switching between the pre- and post-translocational states are discussed. PMID:18824477

  13. A Rab escort protein integrates the secretion system with TOR signaling and ribosome biogenesis

    PubMed Central

    Singh, Jaspal; Tyers, Mike

    2009-01-01

    The coupling of environmental conditions to cell growth and division is integral to cell fitness. In Saccharomyces cerevisiae, the transcription factor Sfp1 couples nutrient status to cell growth rate by controlling the expression of ribosome biogenesis (Ribi) and ribosomal protein (RP) genes. Sfp1 is localized to the nucleus in rich nutrients, but upon nutrient limitation or target of rapamycin (TOR) pathway inhibition by rapamycin, Sfp1 rapidly exits the nucleus, leading to repression of the Ribi/RP regulons. Through systematic cell-based screens we found that many components of the secretory system influence Sfp1 localization. Notably, the essential Rab escort protein Mrs6 exhibited a nutrient-sensitive interaction with Sfp1. Overexpression of Mrs6 prevented nuclear localization of Sfp1 in rich nutrients, whereas loss of Mrs6 resulted in nuclear Sfp1 localization in poor nutrients. These effects were specific to Sfp1 and independent of the protein kinase C (PKC) pathway, suggesting that Mrs6 lies in a distinct branch of TOR and ribosome biogenesis regulation. Rapamycin-resistant alleles of MRS6 were defective in the cytoplasmic retention of Sfp1, the control of cell size, and in the repression of the Ribi/RP regulons. The Sfp1–Mrs6 interaction is a nexus for growth regulation that links the secretory system and TOR-dependent nutrient signaling to ribosome biogenesis. PMID:19684114

  14. Coexistence of sense and anti-sense mRNAs of variant surface protein in Giardia lamblia trophozoites.

    PubMed

    Guo, Junli; Zheng, Wenyu; Wang, Yuehua; Li, Yao; Lu, Siqi; Feng, Xianmin

    2014-02-14

    A strategy of the parasitic protozoan Giardia lamblia to evade attack from the host immune system is periodic changes of its surface antigen, a member of the variant surface protein (VSP) family. A post-transcriptional gene silencing mechanism has been proposed to explain the presence of only one among many possible VSPs at any time. To investigate this phenomenon further, we extracted total RNA from cultured trophozoites of the G. lamblia C2 isolate, and cDNA was reverse-transcribed from the RNA. Sense and anti-sense VSPs were amplified from the total cDNA using nested PCR with primers designed from the 3'-conserved region and the known 5' or 3' end of the cDNA library. Sequence analyses of the amplified products revealed more than 34 full-length antisense VSPs and a smear of sense VSPs. Sequence alignments and comparisons revealed that these VSPs contained variable N-termini and conserved C-termini, and could be classified into 5 clades based on the sizes and variations of the N-terminal sequence. All antisense VSPs existed in the sense forms, but no corresponding antisense VSP existed for sense RNA (snsRNA) 16. The coexistence of sense and antisense VSP mRNAs in cultured G. lamblia supports the post-transcriptional regulation of VSP expression. We propose that VSPs transcribed simultaneously in the sense and antisense forms form double-stranded RNAs (dsRNAs) which are degraded by the Dicer endonuclease, while a VSP without an antisense transcription (e.g., snsRNA16) will be expressed on the surface of Giardia. In addition, in the course of this investigation VSPs were identified that were previously not known. PCR-based amplification of specific sense and antisense VSP cDNAs can be used to identify the specific VSP on G. lamblia trophozoites, which is easier than using specific monoclonal antibody approaches. Copyright © 2014. Published by Elsevier Inc.

  15. Exploring human 40S ribosomal proteins binding to the 18S rRNA fragment containing major 3'-terminal domain.

    PubMed

    Gopanenko, Alexander V; Malygin, Alexey A; Karpova, Galina G

    2015-02-01

    Association of ribosomal proteins with rRNA during assembly of ribosomal subunits is an intricate process, which is strictly regulated in vivo. As for the assembly in vitro, it was reported so far only for prokaryotic subunits. Bacterial ribosomal proteins are capable of selective binding to 16S rRNA as well as to its separate morphological domains. In this work, we explored binding of total protein of human 40S ribosomal subunit to the RNA transcript corresponding to the major 3'-domain of 18S rRNA. We showed that the resulting ribonucleoprotein particles contained almost all of the expected ribosomal proteins, whose binding sites are located in this 18S rRNA domain in the 40S subunit, together with several nonspecific proteins. The binding in solution was accompanied with aggregation of the RNA-protein complexes. Ribosomal proteins bound to the RNA transcript protected from chemical modification mostly those 18S rRNA nucleotides that are known to be involved in binding with the proteins in the 40S subunit and thereby demonstrated their ability to selectively bind to the rRNA in vitro. The possible implication of unstructured extensions of eukaryotic ribosomal proteins in their nonspecific binding with rRNA and in subsequent aggregation of the resulting complexes is discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons.

    PubMed

    Biever, Anne; Puighermanal, Emma; Nishi, Akinori; David, Alexandre; Panciatici, Claire; Longueville, Sophie; Xirodimas, Dimitris; Gangarossa, Giuseppe; Meyuhas, Oded; Hervé, Denis; Girault, Jean-Antoine; Valjent, Emmanuel

    2015-03-11

    Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

  17. Tumor Suppressor PDCD4 Represses Internal Ribosome Entry Site-Mediated Translation of Antiapoptotic Proteins and Is Regulated by S6 Kinase 2

    PubMed Central

    Liwak, Urszula; Thakor, Nehal; Jordan, Lindsay E.; Roy, Rajat; Lewis, Stephen M.; Pardo, Olivier E.; Seckl, Michael

    2012-01-01

    Apoptosis can be regulated by extracellular signals that are communicated by peptides such as fibroblast growth factor 2 (FGF-2) that have important roles in tumor cell proliferation. The prosurvival effects of FGF-2 are transduced by the activation of the ribosomal protein S6 kinase 2 (S6K2), which increases the expression of the antiapoptotic proteins X chromosome-linked Inhibitor of Apoptosis (XIAP) and Bcl-xL. We now show that the FGF-2–S6K2 prosurvival signaling is mediated by the tumor suppressor programmed cell death 4 (PDCD4). We demonstrate that PDCD4 specifically binds to the internal ribosome entry site (IRES) elements of both the XIAP and Bcl-xL messenger RNAs and represses their translation by inhibiting the formation of the 48S translation initiation complex. Phosphorylation of PDCD4 by activated S6K2 leads to the degradation of PDCD4 and thus the subsequent derepression of XIAP and Bcl-xL translation. Our results identify PDCD4 as a specific repressor of the IRES-dependent translation of cellular mRNAs (such as XIAP and Bcl-xL) that mediate FGF-2–S6K2 prosurvival signaling and provide further insight into the role of PDCD4 in tumor suppression. PMID:22431522

  18. Protein Folding Activity of the Ribosome is involved in Yeast Prion Propagation

    PubMed Central

    Blondel, Marc; Soubigou, Flavie; Evrard, Justine; Nguyen, Phu hai; Hasin, Naushaba; Chédin, Stéphane; Gillet, Reynald; Contesse, Marie-Astrid; Friocourt, Gaëlle; Stahl, Guillaume; Jones, Gary W.; Voisset, Cécile

    2016-01-01

    6AP and GA are potent inhibitors of yeast and mammalian prions and also specific inhibitors of PFAR, the protein-folding activity borne by domain V of the large rRNA of the large subunit of the ribosome. We therefore explored the link between PFAR and yeast prion [PSI+] using both PFAR-enriched mutants and site-directed methylation. We demonstrate that PFAR is involved in propagation and de novo formation of [PSI+]. PFAR and the yeast heat-shock protein Hsp104 partially compensate each other for [PSI+] propagation. Our data also provide insight into new functions for the ribosome in basal thermotolerance and heat-shocked protein refolding. PFAR is thus an evolutionarily conserved cell component implicated in the prion life cycle, and we propose that it could be a potential therapeutic target for human protein misfolding diseases. PMID:27633137

  19. Dimorphism of ribosomal protein L8 among different laboratory strains of Saccharomyces cerevisiae.

    PubMed

    Ishiguro, J; Ono, B

    1980-12-01

    Two-dimensional gel electrophoresis was used in a comparative study of ribosomal proteins from various strains of Saccharomyces cerevisiae. The results demonstrated a case of dimorphism of L8 protein of 60S ribosomal subunit. Of eight strains examined, two strains were one type and six were the other type. The former, which was tentatively designated as altered (A) type, was more acidic than the latter, common (C) type, as shown by mobility difference in pH gradient gel. Heterozygous (A/C) diploid cells contained both types of L8 protein and gave rise to tetrads of 2:2 segregation for A and C types, indicating that the difference of mobility was reflection of the allelic difference of the gene coding for L8 protein.

  20. Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression

    PubMed Central

    Salis, Howard M.; Mirsky, Ethan A.; Voigt, Christopher A.

    2009-01-01

    Microbial engineering often requires fine control over protein expression; for example, to connect genetic circuits 1-7 or control flux through a metabolic pathway 8-13. We have developed a predictive design method for synthetic ribosome binding sites that enables the rational control of a protein's production rate on a proportional scale. Experimental validation of over 100 predictions in Escherichia coli shows that the method is accurate to within a factor of 2.3 over a range of 100,000-fold. The design method also correctly predicts that reusing a ribosome binding site sequence in different genetic contexts can result in different protein expression levels. We demonstrate the method's utility by rationally optimizing a protein's expression level to connect a genetic sensor to a synthetic circuit. The proposed forward engineering approach will accelerate the construction and systematic optimization of large genetic systems. PMID:19801975

  1. Mitochondrial Ribosomal Protein L10 Associates with Cyclin B1/Cdk1 Activity and Mitochondrial Function

    PubMed Central

    Li, Hai-Bo; Wang, Ruo-Xi; Jiang, Hai-Bo; Zhang, En-dong; Tan, Jie-Qiong; Xu, Hui-Zhuo

    2016-01-01

    Mitochondrial ribosomal proteins are important for mitochondrial-encoded protein synthesis and mitochondrial function. In addition to their roles in mitoribosome assembly, several mitochondrial ribosome proteins are also implicated in cellular processes like cell cycle regulation, apoptosis, and mitochondrial homeostasis regulation. Here, we demonstrate that MRPL10 regulates cyclin B1/Cdk1 (cyclin-dependent kinase 1) activity and mitochondrial protein synthesis in mammalian cells. In Drosophila, inactivation of mRpL10 (the Drosophila ortholog of mammalian MRPL10) in eyes results in abnormal eye development. Furthermore, expression of human cyclin B1 suppresses eye phenotypes and mitochondrial abnormality of mRpL10 knockdown Drosophila. This study identified that the physiological regulatory pathway of MRPL10 and providing new insights into the role of MRPL10 in growth control and mitochondrial function. PMID:27726420

  2. Ribosome hibernation factor promotes Staphylococcal survival and differentially represses translation

    PubMed Central

    Basu, Arnab; Yap, Mee-Ngan F.

    2016-01-01

    In opportunistic Gram-positive Staphylococcus aureus, a small protein called hibernation-promoting factor (HPFSa) is sufficient to dimerize 2.5-MDa 70S ribosomes into a translationally inactive 100S complex. Although the 100S dimer is observed in only the stationary phase in Gram-negative gammaproteobacteria, it is ubiquitous throughout all growth phases in S. aureus. The biological significance of the 100S ribosome is poorly understood. Here, we reveal an important role of HPFSa in preserving ribosome integrity and poising cells for translational restart, a process that has significant clinical implications for relapsed staphylococcal infections. We found that the hpf null strain is severely impaired in long-term viability concomitant with a dramatic loss of intact ribosomes. Genome-wide ribosome profiling shows that eliminating HPFSa drastically increased ribosome occupancy at the 5′ end of specific mRNAs under nutrient-limited conditions, suggesting that HPFSa may suppress translation initiation. The protective function of HPFSa on ribosomes resides at the N-terminal conserved basic residues and the extended C-terminal segment, which are critical for dimerization and ribosome binding, respectively. These data provide significant insight into the functional consequences of 100S ribosome loss for protein synthesis and stress adaptation. PMID:27001516

  3. The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome.

    PubMed Central

    Nakagawa, A; Nakashima, T; Taniguchi, M; Hosaka, H; Kimura, M; Tanaka, I

    1999-01-01

    Ribosomal protein L2 is the largest protein component in the ribosome. It is located at or near the peptidyl transferase center and has been a prime candidate for the peptidyl transferase activity. It binds directly to 23S rRNA and plays a crucial role in its assembly. The three-dimensional structure of the RNA-binding domain of L2 from Bacillus stearothermophilus has been determined at 2.3 A resolution by X-ray crystallography using the selenomethionyl MAD method. The RNA-binding domain of L2 consists of two recurring motifs of approximately 70 residues each. The N-terminal domain (positions 60-130) is homologous to the OB-fold, and the C-terminal domain (positions 131-201) is homologous to the SH3-like barrel. Residues Arg86 and Arg155, which have been identified by mutation experiments to be involved in the 23S rRNA binding, are located at the gate of the interface region between the two domains. The molecular architecture suggests how this important protein has evolved from the ancient nucleic acid-binding proteins to create a 23S rRNA-binding domain in the very remote past. PMID:10075918

  4. Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus.

    PubMed

    Cook, Denise; Sanchez-Carbente, Maria del Rayo; Lachance, Claude; Radzioch, Danuta; Tremblay, Sandra; Khandjian, Edouard W; DesGroseillers, Luc; Murai, Keith K

    2011-01-01

    The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.

  5. Mutations of ribosomal protein S5 suppress a defect in late-30S ribosomal subunit biogenesis caused by lack of the RbfA biogenesis factor

    PubMed Central

    Nord, Stefan; Bhatt, Monika J.; Tükenmez, Hasan; Farabaugh, Philip J.; Wikström, P. Mikael

    2015-01-01

    The in vivo assembly of ribosomal subunits requires assistance by maturation proteins that are not part of mature ribosomes. One such protein, RbfA, associates with the 30S ribosomal subunits. Loss of RbfA causes cold sensitivity and defects of the 30S subunit biogenesis and its overexpression partially suppresses the dominant cold sensitivity caused by a C23U mutation in the central pseudoknot of 16S rRNA, a structure essential for ribosome function. We have isolated suppressor mutations that restore partially the growth of an RbfA-lacking strain. Most of the strongest suppressor mutations alter one out of three distinct positions in the carboxy-terminal domain of ribosomal protein S5 (S5) in direct contact with helix 1 and helix 2 of the central pseudoknot. Their effect is to increase the translational capacity of the RbfA-lacking strain as evidenced by an increase in polysomes in the suppressed strains. Overexpression of RimP, a protein factor that along with RbfA regulates formation of the ribosome's central pseudoknot, was lethal to the RbfA-lacking strain but not to a wild-type strain and this lethality was suppressed by the alterations in S5. The S5 mutants alter translational fidelity but these changes do not explain consistently their effect on the RbfA-lacking strain. Our genetic results support a role for the region of S5 modified in the suppressors in the formation of the central pseudoknot in 16S rRNA. PMID:26089326

  6. Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

    PubMed

    Mohamed, J Shaik; Benninghoff, Abby D; Holt, G Joan; Khan, Izhar A

    2007-02-01

    The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

  7. Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid.

    PubMed

    Sanamrad, Arash; Persson, Fredrik; Lundius, Ebba G; Fange, David; Gynnå, Arvid H; Elf, Johan

    2014-08-05

    Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.

  8. Partial purification of mitochondrial ribosomes from broad bean and identification of proteins encoded by the mitochondrial genome.

    PubMed

    Maffey, L; Degand, H; Boutry, M

    1997-04-28

    An approach towards the identification at the protein level of the ribosomal proteins encoded by the mitochondrial genome of broad bean (Vicia faba) has been developed. After Triton X-100 treatment of isolated mitochondria, a fraction enriched in mitochondrial ribosomes was obtained by successive centrifugation, first onto a sucrose cushion, and then in a sucrose gradient. Mitochondrial translation products were labelled in isolated mitochondria with [35S]methionine and added to the enriched mitochondrial ribosomal proteins before separation by two-dimensional gel electrophoresis. Six spots, identified both by Coomassie blue staining and autoradiography, were analysed by protein micro-sequencing. Two of these were shown to correspond to ribosomal proteins S10 and S12. We conclude that these two proteins are encoded by the mitochondrial genome of broad bean and that the method described here can be used to identify other proteins encoded by the mitochondrial genome.

  9. Crystal structure of bacillus subtilis YdaF protein : a putative ribosomal N-acetyltransferase.

    SciTech Connect

    Brunzelle, J. S.; Wu, R.; Korolev, S. V.; Collart, F. R.; Joachimiak, A.; Anderson, W. F.; Biosciences Division; Northwestern Univ.; Saint Louis Univ. School of Medicine

    2004-12-01

    Comparative sequence analysis suggests that the ydaF gene encodes a protein (YdaF) that functions as an N-acetyltransferase, more specifically, a ribosomal N-acetyltransferase. Sequence analysis using basic local alignment search tool (BLAST) suggests that YdaF belongs to a large family of proteins (199 proteins found in 88 unique species of bacteria, archaea, and eukaryotes). YdaF also belongs to the COG1670, which includes the Escherichia coli RimL protein that is known to acetylate ribosomal protein L12. N-acetylation (NAT) has been found in all kingdoms. NAT enzymes catalyze the transfer of an acetyl group from acetyl-CoA (AcCoA) to a primary amino group. For example, NATs can acetylate the N-terminal {alpha}-amino group, the {epsilon}-amino group of lysine residues, aminoglycoside antibiotics, spermine/speridine, or arylalkylamines such as serotonin. The crystal structure of the alleged ribosomal NAT protein, YdaF, from Bacillus subtilis presented here was determined as a part of the Midwest Center for Structural Genomics. The structure maintains the conserved tertiary structure of other known NATs and a high sequence similarity in the presumed AcCoA binding pocket in spite of a very low overall level of sequence identity to other NATs of known structure.

  10. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  11. The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.

    PubMed

    De Silva, Dasmanthie; Fontanesi, Flavia; Barrientos, Antoni

    2013-11-05

    Proteins in a cell are universally synthesized by ribosomes. Mitochondria contain their own ribosomes, which specialize in the synthesis of a handful of proteins required for oxidative phosphorylation. The pathway of mitoribosomal biogenesis and factors involved are poorly characterized. An example is the DEAD box proteins, widely known to participate in the biogenesis of bacterial and cytoplasmic eukaryotic ribosomes as either RNA helicases or RNA chaperones, whose mitochondrial counterparts remain completely unknown. Here, we have identified the Saccharomyces cerevisiae mitochondrial DEAD box protein Mrh4 as essential for large mitoribosome subunit biogenesis. Mrh4 interacts with the 21S rRNA, mitoribosome subassemblies, and fully assembled mitoribosomes. In the absence of Mrh4, the 21S rRNA is matured and forms part of a large on-pathway assembly intermediate missing proteins Mrpl16 and Mrpl39. We conclude that Mrh4 plays an essential role during the late stages of mitoribosome assembly by promoting remodeling of the 21S rRNA-protein interactions. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs.

    PubMed

    Flint, S J; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  13. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late