The Experts below are selected from a list of 264 Experts worldwide ranked by ideXlab platform
Nilgun E. Tumer - One of the best experts on this subject based on the ideXlab platform.
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ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress induced hac1 mrna splicing on the ribosome
Journal of Biological Chemistry, 2019Co-Authors: Michael Pierce, Diana Vengsarkar, John E Mclaughlin, Jennifer Nielsen Kahn, Nilgun E. TumerAbstract:Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound Ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to Ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that Ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with Ribosomes and does not get processed on depurinated Ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with Ribosomes.
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the ribosomal stalk is required for ribosome binding depurination of the rrna and cytotoxicity of ricin a chain in saccharomyces cerevisiae
Molecular Microbiology, 2008Co-Authors: Jiachi Chiou, Miguel Remacha, Juan P G Ballesta, Xiaoping Li, Nilgun E. TumerAbstract:: Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic Ribosomes. Ricin depurinates the eukaryotic Ribosomes more efficiently than the prokaryotic Ribosomes, while PAP can depurinate both types of Ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type Ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
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generation of pokeweed antiviral protein mutations in saccharomyces cerevisiae evidence that ribosome depurination is not sufficient for cytotoxicity
Nucleic Acids Research, 2004Co-Authors: Katalin A. Hudak, Bijal A Parikh, Rong Di, Marianne Baricevic, Mirjana Seskar, Maria Santana, Nilgun E. TumerAbstract:Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved α-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5′ and the 3′ ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate Ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate Ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although Ribosomes were depurinated. These mutants depurinated Ribosomes only during their translation and could not depurinate Ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate Ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate Ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.
Jean-jacques Diaz - One of the best experts on this subject based on the ideXlab platform.
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Emerging Role of Eukaryote Ribosomes in Translational Control
International Journal of Molecular Sciences, 2019Co-Authors: Nicole Dalla Venezia, Frédéric Catez, Virginie Marcel, Anne Vincent, Jean-jacques DiazAbstract:Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that Ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of Ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of "specialized Ribosomes", which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.
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Emerging Role of Eukaryote Ribosomes in Translational Control
International Journal of Molecular Sciences, 2019Co-Authors: Nicole Dalla Venezia, Frédéric Catez, Virginie Marcel, Anne Vincent, Jean-jacques DiazAbstract:Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that Ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of Ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of "specialized Ribosomes", which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today.
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Ribosome biogenesis: an emerging druggable pathway for cancer therapeutics
Biochemical Pharmacology, 2019Co-Authors: Frédéric Catez, Nicole Dalla Venezia, Virginie Marcel, Christiane Zorbas, Denis L.j. Lafontaine, Jean-jacques DiazAbstract:Ribosomes are nanomachines essential for protein production in all living cells. Ribosome synthesis increases in cancer cells to cope with a rise in protein synthesis and sustain unrestricted growth. This increase in ribosome biogenesis is reflected by severe morphological alterations of the nucleolus, the cell compartment where the initial steps of ribosome biogenesis take place. Ribosome biogenesis has recently emerged as an effective target in cancer therapy, and several compounds that inhibit ribosome production or function, killing preferentially cancer cells, have entered clinical trials. Recent research indicates that cells express heterogeneous populations of Ribosomes and that the composition of Ribosomes may play a key role in tumorigenesis, exposing novel therapeutic opportunities. Here, we review recent data demonstrating that ribosome biogenesis is a promising druggable pathway in cancer therapy, and discuss future research perspectives.
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Purification of Ribosomes from human cell lines.
Curr Protoc Cell Biol, 2010Co-Authors: Stéphane Belin, Sabine Hacot, Lionel Daudignon, Gabriel Therizols, Stéphane Pourpe, Hichem C Mertani, Manuel Rosa-calatrava, Jean-jacques DiazAbstract:Highly conserved during evolution, the ribosome is the central effector of protein synthesis. In mammalian cells, the ribosome is a macromolecular complex composed of four different ribosomal RNAs (rRNA) and about 80 ribosomal proteins. Requiring more than 200 factors, ribosome biogenesis is a highly complex process that takes place mainly within the nucleoli of eukaryotic cells. Crystallographic data suggest that the ribosome is a ribozyme, in which the rRNA catalyses the peptide bond formation and ensures quality control of the translation. Ribosomal proteins are involved in this molecular mechanism; nonetheless, their role is still not fully characterized. Recent studies suggest that Ribosomes themselves and/or the mechanisms underlying their synthesis, processing, and assembly play a key role in the establishment and progression of several human pathologies. The protocol described here is simple, efficient, and robust, and allows one to purify high-quality Ribosomes from human cultured cell lines. Ribosomes purified with this protocol are adequate for most of the subsequent analyses of their RNA and protein content.
Akira Wada - One of the best experts on this subject based on the ideXlab platform.
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rmf inactivates Ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel
Genes to Cells, 2004Co-Authors: Hideji Yoshida, Hiroshi Yamamoto, Toshio Uchiumi, Akira WadaAbstract:In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S Ribosomes under stress conditions. The 100S ribosome, a dimer of 70S Ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S Ribosomes. During the stationary phase, most of the 70S Ribosomes turn to 100S Ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S Ribosomes immediately go back to active 70S Ribosomes, showing that inactive 100S active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates Ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.
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The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.
Journal of biochemistry, 2002Co-Authors: Hideji Yoshida, Yasushi Maki, Hisako Kato, Hisao Fujisawa, Kaori Izutsu, Chieko Wada, Akira WadaAbstract:During the stationary growth phase, Escherichia coli 70S Ribosomes are converted to 100S Ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S Ribosomes. In order to elucidate the mechanisms by which 100S Ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S Ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, Ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S Ribosomes are stored as 100S Ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S Ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.
James C. Weisshaar - One of the best experts on this subject based on the ideXlab platform.
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Functional mapping of the E. coli translational machinery using single-molecule tracking.
Molecular Microbiology, 2018Co-Authors: Sonisilpa Mohapatra, James C. WeisshaarAbstract:: The organization of the chromosomal DNA and Ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow' (147 min doubling time). Superresolution fluorescence microscopy reveals strong DNA-ribosome segregation in both cases. In both fast and slow growth, free ribosomal subunits evidently must circulate between the nucleoid (where they initiate co-transcriptional translation) and ribosome-rich regions (where most translation occurs). Single-molecule diffusive behavior dissects the ribosome copies into translating 70S polysomes and free 30S subunits, providing separate spatial distributions for each. In slow growth, ~21,000 total 30S copies/cell comprise ~65% translating 70S Ribosomes and ~35% free 30S subunits. The ratio of 70S Ribosomes to free 30S subunits is ~2.5 outside the nucleoid and ~0.50 inside the nucleoid. This new level of quantitative detail may motivate development of comprehensive, three-dimensional reaction-diffusion models of ribosome, DNA, mRNA and RNAP spatial distributions and dynamics within the E. coli cytoplasm.
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Super-Resolution Study of the Dynamics and Spatial Distribution of Ribosomes in Live E. Coli Cells
Biophysical Journal, 2012Co-Authors: Somenath Bakshi, James C. WeisshaarAbstract:Little is known about the spatial organization and dynamics of the translational machinery in bacteria. We have examined the distribution and diffusion of Ribosomes in live E. coli cells by localizing and tracking single Ribosomes labeled by an S2-eYFP construct expressed from the chromosome. Fast reversible photobleaching of eYFP was used to image single ribosome molecules and obtain the time-averaged spatial distribution of Ribosomes with ∼30-nm resolution. In medium growth conditions (doubling time 62 minutes) the Ribosomes are highly segregated from the nucleoid. In DNA rich regions the concentrations of Ribosomes is less than 20% of that in the ribosome-rich regions in the end caps and space between the two nucleoid lobes. This is in reasonable agreement with a Monte Carlo model using realistic parameters for the number of Ribosomes and the amount of plectonemic DNA. We have studied dynamics of single Ribosomes by using time lapse imaging. The mean diffusion constant is Dribo = 0.04 μm2/s. Halting of transcription with Rifampicin resulted in about 10 fold increase in the diffusion constant and a homogeneous distribution of labels throughout the cytoplasm. The drastic change in dynamics can be explained in part by conversion of polysomes to monomeric Ribosomes and 30S subunits in the absence of new mRNA synthesis. Expansion of the nucleoid after rifampicin treatment might be responsible for eliminating ribosome/DNA segregation and may further enhance diffusion. Halting of translation by Chloramphenicol treatment increased the ribosome-DNA segregation as the DNA compacted. The diffusion constant remained the same. Studies of the spatial distribution of different size fluorescent proteins in the presence and absence of drugs suggest that the degree of segregation from DNA is largely size dependent.
Hideji Yoshida - One of the best experts on this subject based on the ideXlab platform.
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rmf inactivates Ribosomes by covering the peptidyl transferase centre and entrance of peptide exit tunnel
Genes to Cells, 2004Co-Authors: Hideji Yoshida, Hiroshi Yamamoto, Toshio Uchiumi, Akira WadaAbstract:In gram-negative bacteria such as Escherichia coli, protein synthesis is suppressed by the formation of 100S Ribosomes under stress conditions. The 100S ribosome, a dimer of 70S Ribosomes, is formed by ribosome modulation factor (RMF) binding to the 70S Ribosomes. During the stationary phase, most of the 70S Ribosomes turn to 100S Ribosomes, which have lost translational activity. This 100S formation is called the hibernation process in the ribosome cycle of the stationary phase. If stationary phase cells are transferred to fresh medium, the 100S Ribosomes immediately go back to active 70S Ribosomes, showing that inactive 100S active 70S interconversion is a major system regulating translation activity in stationary phase cells. To elucidate the mechanisms of translational inactivation, the binding sites of RMF on 23S rRNA in 100S ribosome of E. coli were examined by a chemical probing method using dimethyl sulphate (DMS). As the results, the nine bases in 23S rRNA were protected from DMS modifications and the modification of one base was enhanced. Interestingly A2451 is included among the protected bases, which is thought to be directly involved in peptidyl transferase activity. We conclude that RMF inactivates Ribosomes by covering the peptidyl transferase (PTase) centre and the entrance of peptide exit tunnel. It is surprising that the cell itself produces a protein that seems to inhibit protein synthesis in a similar manner to antibiotics and that it can reversibly bind to and release from the ribosome in response to environmental conditions.
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The ribosome modulation factor (RMF) binding site on the 100S ribosome of Escherichia coli.
Journal of biochemistry, 2002Co-Authors: Hideji Yoshida, Yasushi Maki, Hisako Kato, Hisao Fujisawa, Kaori Izutsu, Chieko Wada, Akira WadaAbstract:During the stationary growth phase, Escherichia coli 70S Ribosomes are converted to 100S Ribosomes, and translational activity is lost. This conversion is caused by the binding of the ribosome modulation factor (RMF) to 70S Ribosomes. In order to elucidate the mechanisms by which 100S Ribosomes form and translational inactivation occurs, the shape of the 100S ribosome and the RMF ribosomal binding site were investigated by electron microscopy and protein-protein cross-linking, respectively. We show that (i) the 100S ribosome is formed by the dimerization of two 70S Ribosomes mediated by face-to-face contacts between their constituent 30S subunits, and (ii) RMF binds near the ribosomal proteins S13, L13, and L2. The positions of these proteins indicate that the RMF binding site is near the peptidyl transferase center or the P site (peptidyl-tRNA binding site). These observations are consistent with the translational inactivation of the ribosome by RMF binding. After the "Recycling" stage, Ribosomes can readily proceed to the "Initiation" stage during exponential growth, but during stationary phase, the majority of 70S Ribosomes are stored as 100S Ribosomes and are translationally inactive. We suggest that this conversion of 70S to 100S Ribosomes represents a newly identified stage of the ribosomal cycle in stationary phase cells, and we have termed it the "Hibernation" stage.
