Mechanical unfolding kinetics of the SRV-1 gag-pro mRNA pseudoknot: possible implications for -1 ribosomal frameshifting stimulation

NASA Astrophysics Data System (ADS)

Zhong, Zhensheng; Yang, Lixia; Zhang, Haiping; Shi, Jiahao; Vandana, J. Jeya; Lam, Do Thuy Uyen Ha; Olsthoorn, René C. L.; Lu, Lanyuan; Chen, Gang

2016-12-01

Minus-one ribosomal frameshifting is a translational recoding mechanism widely utilized by many RNA viruses to generate accurate ratios of structural and catalytic proteins. An RNA pseudoknot structure located in the overlapping region of the gag and pro genes of Simian Retrovirus type 1 (SRV-1) stimulates frameshifting. However, the experimental characterization of SRV-1 pseudoknot (un)folding dynamics and the effect of the base triple formation is lacking. Here, we report the results of our single-molecule nanomanipulation using optical tweezers and theoretical simulation by steered molecular dynamics. Our results directly reveal that the energetic coupling between loop 2 and stem 1 via minor-groove base triple formation enhances the mechanical stability. The terminal base pair in stem 1 (directly in contact with a translating ribosome at the slippery site) also affects the mechanical stability of the pseudoknot. The -1 frameshifting efficiency is positively correlated with the cooperative one-step unfolding force and inversely correlated with the one-step mechanical unfolding rate at zero force. A significantly improved correlation was observed between -1 frameshifting efficiency and unfolding rate at forces of 15-35 pN, consistent with the fact that the ribosome is a force-generating molecular motor with helicase activity. No correlation was observed between thermal stability and -1 frameshifting efficiency.

Global analysis of translation termination in E. coli.

PubMed

Baggett, Natalie E; Zhang, Yan; Gross, Carol A

2017-03-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins.

Two groups of phenylalanine biosynthetic operon leader peptides genes: a high level of apparently incidental frameshifting in decoding Escherichia coli pheL

PubMed Central

Gurvich, Olga L.; Näsvall, S. Joakim; Baranov, Pavel V.; Björk, Glenn R.; Atkins, John F.

2011-01-01

The bacterial pheL gene encodes the leader peptide for the phenylalanine biosynthetic operon. Translation of pheL mRNA controls transcription attenuation and, consequently, expression of the downstream pheA gene. Fifty-three unique pheL genes have been identified in sequenced genomes of the gamma subdivision. There are two groups of pheL genes, both of which are short and contain a run(s) of phenylalanine codons at an internal position. One group is somewhat diverse and features different termination and 5′-flanking codons. The other group, mostly restricted to Enterobacteria and including Escherichia coli pheL, has a conserved nucleotide sequence that ends with UUC_CCC_UGA. When these three codons in E. coli pheL mRNA are in the ribosomal E-, P- and A-sites, there is an unusually high level, 15%, of +1 ribosomal frameshifting due to features of the nascent peptide sequence that include the penultimate phenylalanine. This level increases to 60% with a natural, heterologous, nascent peptide stimulator. Nevertheless, studies with different tRNAPro mutants in Salmonella enterica suggest that frameshifting at the end of pheL does not influence expression of the downstream pheA. This finding of incidental, rather than utilized, frameshifting is cautionary for other studies of programmed frameshifting. PMID:21177642

A [Cu]rious Ribosomal Profiling Pattern Leads to the Discovery of Ribosomal Frameshifting in the Synthesis of a Copper Chaperone.

PubMed

Atkins, John F; Loughran, Gary; Baranov, Pavel V

2017-01-19

In many bacteria, separate genes encode a copper binding chaperone and a copper efflux pump, but in some the chaperone encoding gene has been elusive. In this issue of Molecular Cell, Meydan et al. (2017) report that ribosomes translating the ORF that encodes the copper pump frequently frameshift and terminate to produce the copper chaperone. Copyright © 2017 Elsevier Inc. All rights reserved.

High-Efficiency "-1" and "-2" Ribosomal Frameshiftings Revealed by Force Spectroscopy.

PubMed

Tsai, Te-Wei; Yang, Haopeng; Yin, Heng; Xu, Shoujun; Wang, Yuhong

2017-06-16

Ribosomal frameshifting is a rare but ubiquitous process that is being studied extensively. Meanwhile, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. We report unambiguous observation of highly efficient "-1" and "-2" frameshiftings on a GA 7 G slippery mRNA without the downstream secondary structure, using force-induced remnant magnetization spectroscopy combined with unique probing schemes. The result represents the first experimental evidence of multiple frameshifting steps. It is also one of the rare reports of the "-2" frameshifting. Our assay removed the ambiguity of transcriptional slippage involvement in other frameshifting assays. Two significant insights for the frameshifting mechanism were revealed. First, EF-G·GTP is indispensable to frameshifting. Although EFG·GDPCP has been shown to prompt translocation before, we found that it could not induce frameshifting. This implies that the GTP hydrolysis is responsible for the codon-anticodon re-pairing in frameshifting, which corroborates our previous mechanical force measurement of EF-G·GTP. Second, translation in all three reading frames of the slippery sequence can be induced by the corresponding in-frame aminoacyl tRNAs. Although A-site tRNA is known to affect the partition between "0" and "-1" frameshifting, it has not been reported that all three reading frames can be translated by their corresponding tRNAs. The in vitro results were confirmed by toe-printing assay and protein sequencing.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pallan, Pradeep S.; Marshall, William S.; Harp, Joel

To understand the role of structural elements of RNA pseudoknots in controlling the extent of -1-type ribosomal frameshifting, we determined the crystal structure of a high-efficiency frameshifting mutant of the pseudoknot from potato leaf roll virus (PLRV). Correlations of the structure with available in vitro frameshifting data for PLRV pseudoknot mutants implicate sequence and length of a stem-loop linker as modulators of frameshifting efficiency. Although the sequences and overall structures of the RNA pseudoknots from PLRV and beet western yellow virus (BWYV) are similar, nucleotide deletions in the linker and adjacent minor groove loop abolish frameshifting only with the latter.more » Conversely, mutant PLRV pseudoknots with up to four nucleotides deleted in this region exhibit nearly wild-type frameshifting efficiencies. The crystal structure helps rationalize the different tolerances for deletions in the PLRV and BWYV RNAs, and we have used it to build a three-dimensional model of the PRLV pseudoknot with a four-nucleotide deletion. The resulting structure defines a minimal RNA pseudoknot motif composed of 22 nucleotides capable of stimulating -1-type ribosomal frameshifts.« less

Global analysis of translation termination in E. coli

PubMed Central

Baggett, Natalie E.

2017-01-01

Terminating protein translation accurately and efficiently is critical for both protein fidelity and ribosome recycling for continued translation. The three bacterial release factors (RFs) play key roles: RF1 and 2 recognize stop codons and terminate translation; and RF3 promotes disassociation of bound release factors. Probing release factors mutations with reporter constructs containing programmed frameshifting sequences or premature stop codons had revealed a propensity for readthrough or frameshifting at these specific sites, but their effects on translation genome-wide have not been examined. We performed ribosome profiling on a set of isogenic strains with well-characterized release factor mutations to determine how they alter translation globally. Consistent with their known defects, strains with increasingly severe release factor defects exhibit increasingly severe accumulation of ribosomes over stop codons, indicative of an increased duration of the termination/release phase of translation. Release factor mutant strains also exhibit increased occupancy in the region following the stop codon at a significant number of genes. Our global analysis revealed that, as expected, translation termination is generally efficient and accurate, but that at a significant number of genes (≥ 50) the ribosome signature after the stop codon is suggestive of translation past the stop codon. Even native E. coli K-12 exhibits the ribosome signature suggestive of protein extension, especially at UGA codons, which rely exclusively on the reduced function RF2 variant of the K-12 strain for termination. Deletion of RF3 increases the severity of the defect. We unambiguously demonstrate readthrough and frameshifting protein extensions and their further accumulation in mutant strains for a few select cases. In addition to enhancing recoding, ribosome accumulation over stop codons disrupts attenuation control of biosynthetic operons, and may alter expression of some overlapping genes. Together, these functional alterations may either augment the protein repertoire or produce deleterious proteins. PMID:28301469

Minor groove RNA triplex in the crystal structure of a ribosomal frameshifting viral pseudoknot

NASA Technical Reports Server (NTRS)

Su, L.; Chen, L.; Egli, M.; Berger, J. M.; Rich, A.

1999-01-01

Many viruses regulate translation of polycistronic mRNA using a -1 ribosomal frameshift induced by an RNA pseudoknot. A pseudoknot has two stems that form a quasi-continuous helix and two connecting loops. A 1.6 A crystal structure of the beet western yellow virus (BWYV) pseudoknot reveals rotation and a bend at the junction of the two stems. A loop base is inserted in the major groove of one stem with quadruple-base interactions. The second loop forms a new minor-groove triplex motif with the other stem, involving 2'-OH and triple-base interactions, as well as sodium ion coordination. Overall, the number of hydrogen bonds stabilizing the tertiary interactions exceeds the number involved in Watson-Crick base pairs. This structure will aid mechanistic analyses of ribosomal frameshifting.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing-activated transfer of a mobile genetic element.

PubMed

Ramsay, Joshua P; Tester, Laura G L; Major, Anthony S; Sullivan, John T; Edgar, Christina D; Kleffmann, Torsten; Patterson-House, Jackson R; Hall, Drew A; Tate, Warren P; Hynes, Michael F; Ronson, Clive W

2015-03-31

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

PubMed Central

Ramsay, Joshua P.; Tester, Laura G. L.; Major, Anthony S.; Sullivan, John T.; Edgar, Christina D.; Kleffmann, Torsten; Patterson-House, Jackson R.; Hall, Drew A.; Tate, Warren P.; Hynes, Michael F.; Ronson, Clive W.

2015-01-01

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSymR7A is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNAphe from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSymR7A, suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSymR7A-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSymR7A excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSymR7A transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSymR7A transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels. PMID:25787256

Independent suppression of ribosomal +1 frameshifts by different tRNA anticodon loop modifications.

PubMed

Klassen, Roland; Bruch, Alexander; Schaffrath, Raffael

2017-09-02

Recently, a role for the anticodon wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U) has been revealed in the suppression of translational +1 frameshifts in Saccharomyces cerevisiae. Loss of either the mcm 5 U or s 2 U parts of the modification elevated +1 frameshift rates and results obtained with reporters involving a tRNA Lys UUU dependent frameshift site suggested these effects are caused by reduced ribosomal A-site binding of the hypomodified tRNA. Combined loss of mcm 5 U and s 2 U leads to increased ribosome pausing at tRNA Lys UUU dependent codons and synergistic growth defects but effects on +1 frameshift rates remained undefined to this end. We show in here that simultaneous removal of mcm 5 U and s 2 U results in synergistically increased +1 frameshift rates that are suppressible by extra copies of tRNA Lys UUU . Thus, two distinct chemical modifications of the same wobble base independently contribute to reading frame maintenance, loss of which may cause or contribute to observed growth defects. Since the thiolation pathway is sensitive to moderately elevated temperatures in yeast, we observe a heat-induced increase of +1 frameshift rates in wild type cells that depends on the sulfur transfer protein Urm1. Furthermore, we find that temperature-induced frameshifting is kept in check by the dehydration of N6-threonylcarbamoyladenosine (t 6 A) to its cyclic derivative (ct 6 A) at the anticodon adjacent position 37. Since loss of ct 6 A in elp3 or urm1 mutant cells is detrimental for temperature stress resistance we assume that conversion of t 6 A to ct 6 A serves to limit deleterious effects on translational fidelity caused by hypomodified states of wobble uridine bases.

Single-Molecule Mechanical (Un)folding of RNA Hairpins: Effects of Single A-U to A∙C Pair Substitutions and Single Proton Binding and Implications for mRNA Structure-Induced -1 Ribosomal Frameshifting.

PubMed

Yang, Lixia; Zhong, Zhensheng; Tong, Cailing; Jia, Huan; Liu, Yiran; Chen, Gang

2018-06-08

A wobble A∙C pair can be protonated at near physiological pH to form a more stable wobble A+∙C pair. Here, we constructed an RNA hairpin (rHP) and three mutants with one A-U base pair substituted with an A∙C mismatch on the top (near the loop, U22C), middle (U25C) and bottom (U29C) positions of the stem, respectively. Our results on single-molecule mechanical (un)folding using optical tweezers reveal the destabilization effect of A-U to A∙C pair substitution, and protonation-dependent enhancement of mechanical stability facilitated through an increased folding rate, or decreased unfolding rate, or both. Our data show that protonation may occur rapidly upon the formation of apparent mechanical folding transition state. Furthermore, we measured the bulk -1 ribosomal frameshifting efficiencies of the hairpins by a cell-free translation assay. For the mRNA hairpins studied, -1 frameshifting efficiency correlates with mechanical unfolding force at equilibrium and folding rate at around 15 pN. U29C has a frameshifting efficiency similar to that of rHP (~2%). Accordingly, the bottom 2-4 base pairs of U29C may not form under a stretching force at pH 7.3, which is consistent with the fact that the bottom base pairs of the hairpins may be disrupted by ribosome at the slippery site. U22C and U25C have a similar frameshifting efficiency (~1%), indicating that both unfolding and folding rates of an mRNA hairpin in a crowded environment may affect frameshifting. Our data indicate that mechanical (un)folding of RNA hairpins may mimic how mRNAs unfold and fold in the presence of translating ribosomes.

Efficient -2 frameshifting by mammalian ribosomes to synthesize an additional arterivirus protein.

PubMed

Fang, Ying; Treffers, Emmely E; Li, Yanhua; Tas, Ali; Sun, Zhi; van der Meer, Yvonne; de Ru, Arnoud H; van Veelen, Peter A; Atkins, John F; Snijder, Eric J; Firth, Andrew E

2012-10-23

Programmed -1 ribosomal frameshifting (-1 PRF) is a gene-expression mechanism used to express many viral and some cellular genes. In contrast, efficient natural utilization of -2 PRF has not been demonstrated previously in eukaryotic systems. Like all nidoviruses, members of the Arteriviridae (a family of positive-stranded RNA viruses) express their replicase polyproteins pp1a and pp1ab from two long ORFs (1a and 1b), where synthesis of pp1ab depends on -1 PRF. These polyproteins are posttranslationally cleaved into at least 13 functional nonstructural proteins. Here we report that porcine reproductive and respiratory syndrome virus (PRRSV), and apparently most other arteriviruses, use an additional PRF mechanism to access a conserved alternative ORF that overlaps the nsp2-encoding region of ORF1a in the +1 frame. We show here that this ORF is translated via -2 PRF at a conserved G_GUU_UUU sequence (underscores separate ORF1a codons) at an estimated efficiency of around 20%, yielding a transframe fusion (nsp2TF) with the N-terminal two thirds of nsp2. Expression of nsp2TF in PRRSV-infected cells was verified using specific Abs, and the site and direction of frameshifting were determined via mass spectrometric analysis of nsp2TF. Further, mutagenesis showed that the frameshift site and an unusual frameshift-stimulatory element (a conserved CCCANCUCC motif 11 nucleotides downstream) are required to direct efficient -2 PRF. Mutations preventing nsp2TF expression impair PRRSV replication and produce a small-plaque phenotype. Our findings demonstrate that -2 PRF is a functional gene-expression mechanism in eukaryotes and add another layer to the complexity of arterivirus genome expression.

A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA.

PubMed

Yordanova, Martina M; Wu, Cheng; Andreev, Dmitry E; Sachs, Matthew S; Atkins, John F

2015-07-17

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3' end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5' and 3' of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5' of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5' part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3' part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3' of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Extensive frameshift at all AGG and CCC codons in the mitochondrial cytochrome c oxidase subunit 1 gene of Perkinsus marinus (Alveolata; Dinoflagellata).

PubMed

Masuda, Isao; Matsuzaki, Motomichi; Kita, Kiyoshi

2010-10-01

Diverse mitochondrial (mt) genetic systems have evolved independently of the more uniform nuclear system and often employ modified genetic codes. The organization and genetic system of dinoflagellate mt genomes are particularly unusual and remain an evolutionary enigma. We determined the sequence of full-length cytochrome c oxidase subunit 1 (cox1) mRNA of the earliest diverging dinoflagellate Perkinsus and show that this gene resides in the mt genome. Apparently, this mRNA is not translated in a single reading frame with standard codon usage. Our examination of the nucleotide sequence and three-frame translation of the mRNA suggest that the reading frame must be shifted 10 times, at every AGG and CCC codon, to yield a consensus COX1 protein. We suggest two possible mechanisms for these translational frameshifts: a ribosomal frameshift in which stalled ribosomes skip the first bases of these codons or specialized tRNAs recognizing non-triplet codons, AGGY and CCCCU. Regardless of the mechanism, active and efficient machinery would be required to tolerate the frameshifts predicted in Perkinsus mitochondria. To our knowledge, this is the first evidence of translational frameshifts in protist mitochondria and, by far, is the most extensive case in mitochondria.

Orsay virus utilizes ribosomal frameshifting to express a novel protein that is incorporated into virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Hongbing; Franz, Carl J.; Wu, Guang

2014-02-15

Orsay virus is the first identified virus that is capable of naturally infecting Caenorhabditis elegans. Although it is most closely related to nodaviruses, Orsay virus differs from nodaviruses in its genome organization. In particular, the Orsay virus RNA2 segment encodes a putative novel protein of unknown function, termed delta, which is absent from all known nodaviruses. Here we present evidence that Orsay virus utilizes a ribosomal frameshifting strategy to express a novel fusion protein from the viral capsid (alpha) and delta ORFs. Moreover, the fusion protein was detected in purified virus fractions, demonstrating that it is most likely incorporated intomore » Orsay virions. Furthermore, N-terminal sequencing of both the fusion protein and the capsid protein demonstrated that these proteins must be translated from a non-canonical initiation site. While the function of the alpha–delta fusion remains cryptic, these studies provide novel insights into the fundamental properties of this new clade of viruses. - Highlights: • Orsay virus encodes a novel fusion protein by a ribosomal frameshifting mechanism. • Orsay capsid and fusion protein is translated from a non-canonical initiation site. • The fusion protein is likely incorporated into Orsay virions.« less

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. Copyright © 2016 Elsevier Inc. All rights reserved.

CCC CGA is a weak translational recoding site in Escherichia coli.

PubMed

Shu, Ping; Dai, Huacheng; Mandecki, Wlodek; Goldman, Emanuel

2004-12-08

Previously published experiments had indicated unexpected expression of a control vector in which a beta-galactosidase reporter was in the +1 reading frame relative to the translation start. This control vector contained the codon pair CCC CGA in the zero reading frame, raising the possibility that ribosomes rephased on this sequence, with peptidyl-tRNA(Pro) pairing with CCC in the +1 frame. This putative rephasing might also be exacerbated by the rare CGA Arg codon in the second position due to increased vacancy of the ribosomal A-site. To test this hypothesis, a series of site-directed mutants was constructed, including mutations in both the first and second codons of this codon pair. The results show that interrupting the continuous run of C residues with synonymous codon changes essentially abolishes the frameshift. Further, changing the rare Arg codon to a common Arg codon also reduces the frequency of the frameshift. These results provide strong support for the hypothesis that CCC CGA in the zero frame is indeed a weak translational frameshift site in Escherichia coli, with a 1-2% efficiency. Because the vector sequence also contains another CCC triplet in the +1 reading frame starting within the next codon after the CGA, our data also support possible contribution to expression of a +7 nucleotide ribosome hop into the same +1 reading frame. We also confirm here a previous report that CCC UGA is a translational frameshift site, in these experiments, with about 5% efficiency.

Replacement of Murine Leukemia Virus Readthrough Mechanism by Human Immunodeficiency Virus Frameshift Allows Synthesis of Viral Proteins and Virus Replication

PubMed Central

Brunelle, Marie-Noëlle; Brakier-Gingras, Léa; Lemay, Guy

2003-01-01

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model. PMID:12584361

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

N -Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

DOE PAGES

Hilimire, Thomas A.; Bennett, Ryan P.; Stewart, Ryan A.; ...

2015-10-23

Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stemmore » loop with low nanomolar afinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.« less

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

PubMed

Cornish, Peter V; Hennig, Mirko; Giedroc, David P

2005-09-06

The molecular determinants of stimulation of -1 programmed ribosomal frameshifting (-1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes -1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3' nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2'-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1-2'-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2-S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of -1 PRF (15 +/- 2%) relative to the BWYV pseudoknot (6 +/- 1%), a difference traced largely to the identity of the 3' nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by approximately 1.5 kcal x mol(-1) (pH 7.0, 37 degrees C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs.

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

PubMed Central

Cornish, Peter V.; Hennig, Mirko; Giedroc, David P.

2005-01-01

The molecular determinants of stimulation of –1 programmed ribosomal frameshifting (–1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes –1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3′ nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2′-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1–2′-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2–S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of –1 PRF (15 ± 2%) relative to the BWYV pseudoknot (6 ± 1%), a difference traced largely to the identity of the 3′ nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by ≈1.5 kcal·mol–1 (pH 7.0, 37°C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs. PMID:16123125

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites.

PubMed

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-04-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass ( Zostera marina ) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae . They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

Identification of Two Novel Amalgaviruses in the Common Eelgrass (Zostera marina) and in Silico Analysis of the Amalgavirus +1 Programmed Ribosomal Frameshifting Sites

PubMed Central

Park, Dongbin; Goh, Chul Jun; Kim, Hyein; Hahn, Yoonsoo

2018-01-01

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses. PMID:29628822

Evidence for an RNA pseudoknot loop-helix interaction essential for efficient -1 ribosomal frameshifting.

PubMed

Liphardt, J; Napthine, S; Kontos, H; Brierley, I

1999-05-07

RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2. Copyright 1999 Academic Press.

Expanded ATXN3 frameshifting events are toxic in Drosophila and mammalian neuron models.

PubMed

Stochmanski, Shawn J; Therrien, Martine; Laganière, Janet; Rochefort, Daniel; Laurent, Sandra; Karemera, Liliane; Gaudet, Rebecca; Vyboh, Kishanda; Van Meyel, Don J; Di Cristo, Graziella; Dion, Patrick A; Gaspar, Claudia; Rouleau, Guy A

2012-05-15

Spinocerebellar ataxia type 3 is caused by the expansion of the coding CAG repeat in the ATXN3 gene. Interestingly, a -1 bp frameshift occurring within an (exp)CAG repeat would henceforth lead to translation from a GCA frame, generating polyalanine stretches instead of polyglutamine. Our results show that transgenic expression of (exp)CAG ATXN3 led to -1 frameshifting events, which have deleterious effects in Drosophila and mammalian neurons. Conversely, transgenic expression of polyglutamine-encoding (exp)CAA ATXN3 was not toxic. Furthermore, (exp)CAG ATXN3 mRNA does not contribute per se to the toxicity observed in our models. Our observations indicate that expanded polyglutamine tracts in Drosophila and mouse neurons are insufficient for the development of a phenotype. Hence, we propose that -1 ribosomal frameshifting contributes to the toxicity associated with (exp)CAG repeats.

Structural insights into translational recoding by frameshift suppressor tRNASufJ

PubMed Central

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; Miles, Stacey J.

2014-01-01

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5′ or 3′ direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNASufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNASufJ contains an insertion 5′ to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASLSufJ or tRNASufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASLSufJ and ASLThr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASLSufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNASufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting. PMID:25352689

The ribosome uses two active mechanisms to unwind messenger RNA during translation.

PubMed

Qu, Xiaohui; Wen, Jin-Der; Lancaster, Laura; Noller, Harry F; Bustamante, Carlos; Tinoco, Ignacio

2011-07-06

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs. ©2011 Macmillan Publishers Limited. All rights reserved

Adjustable under-expression of yeast mating pathway proteins in Saccharomyces cerevisiae using a programmed ribosomal frameshift.

PubMed

Choi, Min-Yeon; Park, Sang-Hyun

2016-06-01

Experimental research in molecular biology frequently relies on the promotion or suppression of gene expression, an important tool in the study of its functions. Although yeast is among the most studied model systems with the ease of maintenance and manipulation, current experimental methods are mostly limited to gene deletion, suppression or overexpression of genes. Therefore, the ability to reduce protein expressions and then observing the effects would promote a better understanding of the exact functions and their interactions. Reducing protein expression is mainly limited by the difficulties associated with controlling the reduction level, and in some cases, the initial endogenous abundance is too low. For the under-expression to be useful as an experimental tool, repeatability and stability of reduced expression is important. We found that cis-elements in programmed -1 ribosomal frameshifting (-1RFS) of beet western yellow virus (BWYV) could be utilized to reduced protein expression in Saccharomyces cerevisiae. The two main advantages of using -1RFS are adjustable reduction rates and ease of use. To demonstrate the utility of this under-expression system, examples of reduced protein abundance were shown using yeast mating pathway components. The abundance of MAP kinase Fus3 was reduced to approximately 28-75 % of the wild-type value. Other MAP kinase mating pathway components, including Ste5, Ste11, and Ste7, were also under-expressed to verify that the -1RFS system works with different proteins. Furthermore, reduced Fus3 abundance altered the overall signal transduction outcome of the mating pathway, demonstrating the potential for further studies of signal transduction adjustment via under-expression.

A +1 ribosomal frameshifting motif prevalent among plant amalgaviruses.

PubMed

Nibert, Max L; Pyle, Jesse D; Firth, Andrew E

2016-11-01

Sequence accessions attributable to novel plant amalgaviruses have been found in the Transcriptome Shotgun Assembly database. Sixteen accessions, derived from 12 different plant species, appear to encompass the complete protein-coding regions of the proposed amalgaviruses, which would substantially expand the size of genus Amalgavirus from 4 current species. Other findings include evidence for UUU_CGN as a +1 ribosomal frameshifting motif prevalent among plant amalgaviruses; for a variant version of this motif found thus far in only two amalgaviruses from solanaceous plants; for a region of α-helical coiled coil propensity conserved in a central region of the ORF1 translation product of plant amalgaviruses; and for conserved sequences in a C-terminal region of the ORF2 translation product (RNA-dependent RNA polymerase) of plant amalgaviruses, seemingly beyond the region of conserved polymerase motifs. These results additionally illustrate the value of mining the TSA database and others for novel viral sequences for comparative analyses. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Structural insights into translational recoding by frameshift suppressor tRNA SufJ

DOE PAGES

Fagan, Crystal E.; Maehigashi, Tatsuya; Dunkle, Jack A.; ...

2014-10-28

The three-nucleotide mRNA reading frame is tightly regulated during translation to ensure accurate protein expression. Translation errors that lead to aberrant protein production can result from the uncoupled movement of the tRNA in either the 5' or 3' direction on mRNA. Here, we report the biochemical and structural characterization of +1 frameshift suppressor tRNA SufJ, a tRNA known to decode four, instead of three, nucleotides. Frameshift suppressor tRNA SufJ contains an insertion 5' to its anticodon, expanding the anticodon loop from seven to eight nucleotides. Our results indicate that the expansion of the anticodon loop of either ASL SufJ ormore » tRNA SufJ does not affect its affinity for the A site of the ribosome. Structural analyses of both ASL SufJ and ASL Thr bound to the Thermus thermophilus 70S ribosome demonstrate both ASLs decode in the zero frame. Although the anticodon loop residues 34–37 are superimposable with canonical seven-nucleotide ASLs, the single C31.5 insertion between nucleotides 31 and 32 in ASL SufJ imposes a conformational change of the anticodon stem, that repositions and tilts the ASL toward the back of the A site. Further modeling analyses reveal that this tilting would cause a distortion in full-length A-site tRNA SufJ during tRNA selection and possibly impede gripping of the anticodon stem by 16S rRNA nucleotides in the P site. Together, these data implicate tRNA distortion as a major driver of noncanonical translation events such as frameshifting.« less

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals.

PubMed

El Houmami, Nawal; Seligmann, Hervé

2017-01-01

We present an evolutionary hypothesis assuming that signals marking nucleotide synthesis (DNA replication and RNA transcription) evolved from multi- to unidimensional structures, and were carried over from transcription to translation. This evolutionary scenario presumes that signals combining secondary and primary nucleotide structures are evolutionary transitions. Mitochondrial replication initiation fits this scenario. Some observations reported in the literature corroborate that several signals for nucleotide synthesis function in translation, and vice versa. (a) Polymerase-induced frameshift mutations occur preferentially at translational termination signals (nucleotide deletion is interpreted as termination of nucleotide polymerization, paralleling the role of stop codons in translation). (b) Stem-loop hairpin presence/absence modulates codon-amino acid assignments, showing that translational signals sometimes combine primary and secondary nucleotide structures (here codon and stem-loop). (c) Homopolymer nucleotide triplets (AAA, CCC, GGG, TTT) cause transcriptional and ribosomal frameshifts. Here we find in recently described human mitochondrial RNAs that systematically lack mono-, dinucleotides after each trinucleotide (delRNAs) that delRNA triplets include 2x more homopolymers than mitogenome regions not covered by delRNA. Further analyses of delRNAs show that the natural circular code X (a little-known group of 20 translational signals enabling ribosomal frame retrieval consisting of 20 codons {AAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC} universally overrepresented in coding versus other frames of gene sequences), regulates frameshift in transcription and translation. This dual transcription and translation role confirms for X the hypothesis that translational signals were carried over from transcriptional signals.

A conserved predicted pseudoknot in the NS2A-encoding sequence of West Nile and Japanese encephalitis flaviviruses suggests NS1' may derive from ribosomal frameshifting

PubMed Central

Firth, Andrew E; Atkins, John F

2009-01-01

Japanese encephalitis, West Nile, Usutu and Murray Valley encephalitis viruses form a tight subgroup within the larger Flavivirus genus. These viruses utilize a single-polyprotein expression strategy, resulting in ~10 mature proteins. Plotting the conservation at synonymous sites along the polyprotein coding sequence reveals strong conservation peaks at the very 5' end of the coding sequence, and also at the 5' end of the sequence encoding the NS2A protein. Such peaks are generally indicative of functionally important non-coding sequence elements. The second peak corresponds to a predicted stable pseudoknot structure whose biological importance is supported by compensatory mutations that preserve the structure. The pseudoknot is preceded by a conserved slippery heptanucleotide (Y CCU UUU), thus forming a classical stimulatory motif for -1 ribosomal frameshifting. We hypothesize, therefore, that the functional importance of the pseudoknot is to stimulate a portion of ribosomes to shift -1 nt into a short (45 codon), conserved, overlapping open reading frame, termed foo. Since cleavage at the NS1-NS2A boundary is known to require synthesis of NS2A in cis, the resulting transframe fusion protein is predicted to be NS1-NS2AN-term-FOO. We hypothesize that this may explain the origin of the previously identified NS1 'extension' protein in JEV-group flaviviruses, known as NS1'. PMID:19196463

endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1.

PubMed Central

Cavicchioli, R; East, P D; Watson, K

1991-01-01

The complete nucleotide sequence of endAFS, an endoglucanase gene isolated from the ruminal anaerobe Fibrobacter succinogenes AR1, was determined. endAFS encodes two overlapping open reading frames (ORF1 and ORF2), and it was proposed that a -1 ribosomal frameshift was required to allow contiguous synthesis of a 453-amino-acid endoglucanase. A proline- and threonine-rich region at the C terminus of ORF1 and rare codons for arginine and threonine were coincident with the proposed frameshift site. ENDAFS is proposed to be a member of subgroup 1 of family E endoglucanases, of which endoglucanases from Thermomonospora fusca and Persea americana (avocado) are also members. Endoglucanases from Clostridium thermocellum and Pseudomonas fluorescens form subgroup 2. Images PMID:1708767

Genetic characterization of frameshift suppressors with new decoding properties.

PubMed Central

Hughes, D; Thompson, S; O'Connor, M; Tuohy, T; Nichols, B P; Atkins, J F

1989-01-01

Suppressor mutants that cause ribosomes to shift reading frame at specific and new sequences are described. Suppressors for trpE91, the only known suppressible -1 frameshift mutant, have been isolated in Escherichia coli and in Salmonella typhimurium. E. coli hopR acts on trpE91 within the 9-base-pair sequence GGA GUG UGA, is dominant, and is located at min 52 on the chromosome. Its Salmonella homolog maps at an equivalent position and arises as a rarer class in that organism as compared with E. coli. The Salmonella suppressor, hopE, believed to be in a duplicate copy of the same gene, maps at min 17. The +1 suppressor, sufT, acts at the nonmonotonous sequence CCGU, is dominant, and maps at min 59 on the Salmonella chromosome. PMID:2644219

Frameshift mutations of TAF1C gene, a core component for transcription by RNA polymerase I, and its regional heterogeneity in gastric and colorectal cancers.

PubMed

Oh, Hye Rim; An, Chang Hyeok; Yoo, Nam Jin; Lee, Sug Hyung

2015-02-01

Initiation of transcription for ribosomal RNA (rRNA) by RNA polymerase I requires TATA-binding protein (TBP) and TBP-associated factors (TAF1A, TAF1B and TAF1C). p53 tumour suppressor inhibits rRNA transcription by blocking TAF1C-UBF interaction, but alterations of TAF1C itself in tumorigenesis remain unknown. The aim of this study was to explore whether TAF1C gene was mutated in gastric (GC) and colorectal cancers (CRC).In a public database, we found that TAF1C gene had a mononucleotide repeat (C8) in the coding sequences that might be a mutation target in the cancers with microsatellite instability (MSI). We analysed 79 GC and 124 CRC by single-strand conformation polymorphism and DNA sequencing analyses. In this study, we found TAF1C frameshift mutations (8.8% of GC and 10.1% of CRC with MSI-H), which were not found in stable MSI/low MSI (MSS/MSI-L) (0/90). In addition, we analysed intratumoural heterogeneity (ITH) of TAF1C frameshift mutations in 16 CRC and found that three CRC (18.8%) harboured regional ITH of the TAF1C frameshift mutations. Our results indicate that TAF1C gene harboured not only somatic frameshift mutations but also the mutational ITH, which together might play a role in tumourigenesis of GC and CRC. Our data also suggest that multi-regional mutation analysis is needed for a better evaluation of the mutation status in CRC.

Analyses of frameshifting at UUU-pyrimidine sites.

PubMed

Schwartz, R; Curran, J F

1997-05-15

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage.

Analyses of frameshifting at UUU-pyrimidine sites.

PubMed Central

Schwartz, R; Curran, J F

1997-01-01

Others have recently shown that the UUU phenylalanine codon is highly frameshift-prone in the 3'(rightward) direction at pyrimidine 3'contexts. Here, several approaches are used to analyze frameshifting at such sites. The four permutations of the UUU/C (phenylalanine) and CGG/U (arginine) codon pairs were examined because they vary greatly in their expected frameshifting tendencies. Furthermore, these synonymous sites allow direct tests of the idea that codon usage can control frameshifting. Frameshifting was measured for these dicodons embedded within each of two broader contexts: the Escherichia coli prfB (RF2 gene) programmed frameshift site and a 'normal' message site. The principal difference between these contexts is that the programmed frameshift contains a purine-rich sequence upstream of the slippery site that can base pair with the 3'end of 16 S rRNA (the anti-Shine-Dalgarno) to enhance frameshifting. In both contexts frameshift frequencies are highest if the slippery tRNAPhe is capable of stable base pairing in the shifted reading frame. This requirement is less stringent in the RF2 context, as if the Shine-Dalgarno interaction can help stabilize a quasi-stable rephased tRNA:message complex. It was previously shown that frameshifting in RF2 occurs more frequently if the codon 3'to the slippery site is read by a rare tRNA. Consistent with that earlier work, in the RF2 context frameshifting occurs substantially more frequently if the arginine codon is CGG, which is read by a rare tRNA. In contrast, in the 'normal' context frameshifting is only slightly greater at CGG than at CGU. It is suggested that the Shine-Dalgarno-like interaction elevates frameshifting specifically during the pause prior to translation of the second codon, which makes frameshifting exquisitely sensitive to the rate of translation of that codon. In both contexts frameshifting increases in a mutant strain that fails to modify tRNA base A37, which is 3'of the anticodon. Thus, those base modifications may limit frameshifting at UUU codons. Finally, statistical analyses show that UUU Ynn dicodons are extremely rare in E.coli genes that have highly biased codon usage. PMID:9115369

Novel divergent nidovirus in a python with pneumonia.

PubMed

Bodewes, Rogier; Lempp, Charlotte; Schürch, Anita C; Habierski, Andre; Hahn, Kerstin; Lamers, Mart; von Dörnberg, Katja; Wohlsein, Peter; Drexler, Jan Felix; Haagmans, Bart L; Smits, Saskia L; Baumgärtner, Wolfgang; Osterhaus, Albert D M E

2014-11-01

The order Nidovirales contains large, enveloped viruses with a non-segmented positive-stranded RNA genome. Nidoviruses have been detected in man and various animal species, but, to date, there have been no reports of nidovirus in reptiles. In the present study, we describe the detection, characterization, phylogenetic analyses and disease association of a novel divergent nidovirus in the lung of an Indian python (Python molurus) with necrotizing pneumonia. Characterization of the partial genome (>33 000 nt) of this virus revealed several genetic features that are distinct from other nidoviruses, including a very large polyprotein 1a, a putative ribosomal frameshift signal that was identical to the frameshift signal of astroviruses and retroviruses and an accessory ORF that showed some similarity with the haemagglutinin-neuraminidase of paramyxoviruses. Analysis of genome organization and phylogenetic analysis of polyprotein 1ab suggests that this virus belongs to the subfamily Torovirinae. Results of this study provide novel insights into the genetic diversity within the order Nidovirales. © 2014 The Authors.

High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

PubMed Central

Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

2016-01-01

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. PMID:26919232

New Insights into Flavivirus Evolution, Taxonomy and Biogeographic History, Extended by Analysis of Canonical and Alternative Coding Sequences

PubMed Central

Moureau, Gregory; Cook, Shelley; Lemey, Philippe; Nougairede, Antoine; Forrester, Naomi L.; Khasnatinov, Maxim; Charrel, Remi N.; Firth, Andrew E.; Gould, Ernest A.; de Lamballerie, Xavier

2015-01-01

To generate the most diverse phylogenetic dataset for the flaviviruses to date, we determined the genomic sequences and phylogenetic relationships of 14 flaviviruses, of which 10 are primarily associated with Culex spp. mosquitoes. We analyze these data, in conjunction with a comprehensive collection of flavivirus genomes, to characterize flavivirus evolutionary and biogeographic history in unprecedented detail and breadth. Based on the presumed introduction of yellow fever virus into the Americas via the transatlantic slave trade, we extrapolated a timescale for a relevant subset of flaviviruses whose evolutionary history, shows that different Culex-spp. associated flaviviruses have been introduced from the Old World to the New World on at least five separate occasions, with 2 different sets of factors likely to have contributed to the dispersal of the different viruses. We also discuss the significance of programmed ribosomal frameshifting in a central region of the polyprotein open reading frame in some mosquito-associated flaviviruses. PMID:25719412

An RNA Element That Facilitates Programmed Ribosomal Readthrough in Turnip Crinkle Virus Adopts Multiple Conformations

PubMed Central

Kuhlmann, Micki M.; Chattopadhyay, Maitreyi; Stupina, Vera A.; Gao, Feng

2016-01-01

ABSTRACT Ribosome recoding is used by RNA viruses for translational readthrough or frameshifting past termination codons for the synthesis of extension products. Recoding sites, along with downstream recoding stimulatory elements (RSEs), have long been studied in reporter constructs, because these fragments alone mediate customary levels of recoding and are thus assumed to contain complete instructions for establishment of the proper ratio of termination to recoding. RSEs from the Tombusviridae and Luteoviridae are thought to be exceptions, since they contain a long-distance RNA-RNA connection with the 3′ end. This interaction has been suggested to substitute for pseudoknots, thought to be missing in tombusvirid RSEs. We provide evidence that the phylogenetically conserved RSE of the carmovirus Turnip crinkle virus (TCV) adopts an alternative, smaller structure that extends an upstream conserved hairpin and that this alternative structure is the predominant form of the RSE within nascent viral RNA in plant cells and when RNA is synthesized in vitro. The TCV RSE also contains an internal pseudoknot along with the long-distance interaction, and the pseudoknot is not compatible with the phylogenetically conserved structure. Conserved residues just past the recoding site are important for recoding, and these residues are also conserved in the RSEs of gammaretroviruses. Our data demonstrate the dynamic nature of the TCV RSE and suggest that studies using reporter constructs may not be effectively recapitulating RSE-mediated recoding within viral genomes. IMPORTANCE Ribosome recoding is used by RNA viruses to enable ribosomes to extend translation past termination codons for the synthesis of longer products. Recoding sites and a downstream recoding stimulatory element (RSE) mediate expected levels of recoding when excised and placed in reporter constructs and thus are assumed to contain complete instructions for the establishment of the proper ratio of termination to recoding. We provide evidence that most of the TCV RSE adopts an alternative structure that extends an upstream conserved hairpin and that this alternative structure, and not the phylogenetically conserved structure, is the predominant form of the RSE in RNA synthesized in vitro and in plant cells. The TCV RSE also contains an internal pseudoknot that is not compatible with the phylogenetically conserved structure and an RNA bridge to the 3′ end. These data suggest that the TCV RSE is structurally dynamic and that multiple conformations are likely required to regulate ribosomal readthrough. PMID:27440887

Complete nucleotide sequence and annotation of the temperate corynephage ϕ16 genome.

PubMed

Lobanova, Juliya S; Gak, Evgueni R; Andreeva, Irina G; Rybak, Konstantin V; Krylov, Alexander A; Mashko, Sergey V

2017-08-01

The complete genome of ϕ16, a temperate corynephage from Corynebacterium glutamicum ATCC 21792, was sequenced and annotated (GenBank: KY250482). The electron microscopy study of ϕ16 virion confirmed that it belongs to the family Siphoviridae. The ϕ16 genome consists of a linear double-stranded DNA molecule of 58,200 bp (G+C = 52.2%) with protruding cohesive 3'-ends of 14 nt. Four major structural proteins were separated by SDS-PAGE and identified by peptide mass fingerprinting technique. Using bioinformatics analysis, 101 putative ORFs and 5 tRNA genes were predicted. Only 27 putative gene products could be assigned to known biological functions. The ϕ16 genome was divided into functional modules. Seven putative promoters and eight putative unidirectional intrinsic terminators were predicted. One site of putative «-1» programmed ribosomal frameshifting was proposed in the phage tail assembly genome region. C. glutamicum genetic tools could be broadened by exploiting the known integrase gene (gp33) and the newly identified excisionase gene (gp47), participating in site-specific recombination between ϕ16-attP/attB.

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia.

PubMed

Wang, RuNan; Yoshida, Kenichi; Toki, Tsutomu; Sawada, Takafumi; Uechi, Tamayo; Okuno, Yusuke; Sato-Otsubo, Aiko; Kudo, Kazuko; Kamimaki, Isamu; Kanezaki, Rika; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Sato, Tomohiko; Iribe, Yuji; Ohga, Shouichi; Kuramitsu, Madoka; Hamaguchi, Isao; Ohara, Akira; Hara, Junichi; Goi, Kumiko; Matsubara, Kousaku; Koike, Kenichi; Ishiguro, Akira; Okamoto, Yasuhiro; Watanabe, Kenichiro; Kanno, Hitoshi; Kojima, Seiji; Miyano, Satoru; Kenmochi, Naoya; Ogawa, Seishi; Ito, Etsuro

2015-03-01

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia. © 2014 John Wiley & Sons Ltd.

Gene Model Annotations for Drosophila melanogaster: The Rule-Benders

PubMed Central

Crosby, Madeline A.; Gramates, L. Sian; dos Santos, Gilberto; Matthews, Beverley B.; St. Pierre, Susan E.; Zhou, Pinglei; Schroeder, Andrew J.; Falls, Kathleen; Emmert, David B.; Russo, Susan M.; Gelbart, William M.

2015-01-01

In the context of the FlyBase annotated gene models in Drosophila melanogaster, we describe the many exceptional cases we have curated from the literature or identified in the course of FlyBase analysis. These range from atypical but common examples such as dicistronic and polycistronic transcripts, noncanonical splices, trans-spliced transcripts, noncanonical translation starts, and stop-codon readthroughs, to single exceptional cases such as ribosomal frameshifting and HAC1-type intron processing. In FlyBase, exceptional genes and transcripts are flagged with Sequence Ontology terms and/or standardized comments. Because some of the rule-benders create problems for handlers of high-throughput data, we discuss plans for flagging these cases in bulk data downloads. PMID:26109356

The mitochondrial genome of the deep-sea glass sponge Lophophysema eversa (Porifera, Hexacinellida, Hyalonematidae).

PubMed

Zhang, Yanjie; Sun, Jin; Li, Xinzheng; Qiu, Jian-Wen

2016-01-01

We reported a nearly complete mitochondrial genome (mitogenome) from the glass sponge Lophophysema eversa, the second mitogenome in the order Amphidiscosida and the ninth in the class Hexactinellida. It is 20,651 base pairs in length and contains 39 genes including 13 protein-coding genes, 2 ribosomal RNA subunit genes and 24 tRNA genes. The gene content and order of L. eversa are identical to those of Tabachnickia sp., the other species with a sequenced mitogenome in Amphidiscosida, except with two additional tRNAs and three tRNA translocations. The cob gene has a +1 translational frameshift. These results will contribute to a better understanding of the phylogeny of glass sponges.

Structural insights into translational fidelity.

PubMed

Ogle, James M; Ramakrishnan, V

2005-01-01

The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting.

Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis

PubMed Central

Shoji, Shinichiro; Janssen, Brian D.; Hayes, Christopher S.; Fredrick, Kurt

2009-01-01

LepA is a translational GTPase highly conserved in bacterial lineages. While it has been shown that LepA can catalyze reverse ribosomal translocation in vitro, the role of LepA in the cell remains unclear. Here, we show that deletion of the lepA gene (ΔlepA) in E. coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. ΔlepA does not increase miscoding or frameshifting errors under normal or stress conditions, indicating that LepA does not contribute to the fidelity of translation. Overexpression of LepA interferes with tmRNA-mediated peptide tagging and A-site mRNA cleavage, suggesting that LepA is a bona fide translation factor that can act on stalled ribosomes with a vacant A site in vivo. Together these results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation. PMID:19925844

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

PubMed

Valles, Steven M; Bell, Susanne; Firth, Andrew E

2014-01-01

Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

PubMed

Malagnac, Fabienne; Fabret, Céline; Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

2013-01-01

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.

PubMed

Mora, Liliana; Heurgué-Hamard, Valérie; de Zamaroczy, Miklos; Kervestin, Stephanie; Buckingham, Richard H

2007-12-07

Bacterial release factors RF1 and RF2 are methylated on the Gln residue of a universally conserved tripeptide motif GGQ, which interacts with the peptidyl transferase center of the large ribosomal subunit, triggering hydrolysis of the ester bond in peptidyl-tRNA and releasing the newly synthesized polypeptide from the ribosome. In vitro experiments have shown that the activity of RF2 is stimulated by Gln methylation. The viability of Escherichia coli K12 strains depends on the integrity of the release factor methyltransferase PrmC, because K12 strains are partially deficient in RF2 activity due to the presence of a Thr residue at position 246 instead of Ala. Here, we study in vivo RF1 and RF2 activity at termination codons in competition with programmed frameshifting and the effect of the Ala-246 --> Thr mutation. PrmC inactivation reduces the specific termination activity of RF1 and RF2(Ala-246) by approximately 3- to 4-fold. The mutation Ala-246 --> Thr in RF2 reduces the termination activity in cells approximately 5-fold. After correction for the decrease in level of RF2 due to the autocontrol of RF2 synthesis, the mutation Ala-246 --> Thr reduced RF2 termination activity by approximately 10-fold at UGA codons and UAA codons. PrmC inactivation had no effect on cell growth in rich media but reduced growth considerably on poor carbon sources. This suggests that the expression of some genes needed for optimal growth under such conditions can become growth limiting as a result of inefficient translation termination.

Using secondary structure to identify ribosomal numts: cautionary examples from the human genome.

PubMed

Olson, Link E; Yoder, Anne D

2002-01-01

The identification of inadvertently sequenced mitochondrial pseudogenes (numts) is critical to any study employing mitochondrial DNA sequence data. Failure to discriminate numts correctly can confound phylogenetic reconstruction and studies of molecular evolution. This is especially problematic for ribosomal mtDNA genes. Unlike protein-coding loci, whose pseudogenes tend to accumulate diagnostic frameshift or premature stop mutations, functional ribosomal genes are not constrained to maintain a reading frame and can accumulate insertion-deletion events of varying length, particularly in nonpairing regions. Several authors have advocated using structural features of the transcribed rRNA molecule to differentiate functional mitochondrial rRNA genes from their nuclear paralogs. We explored this approach using the mitochondrial 12S rRNA gene and three known 12S numts from the human genome in the context of anthropoid phylogeny and the inferred secondary structure of primate 12S rRNA. Contrary to expectation, each of the three human numts exhibits striking concordance with secondary structure models, with little, if any, indication of their pseudogene status, and would likely escape detection based on structural criteria alone. Furthermore, we show that the unwitting inclusion of a particularly ancient (18-25 Myr old) and surprisingly cryptic human numt in a phylogenetic analysis would yield a well-supported but dramatically incorrect conclusion regarding anthropoid relationships. Though we endorse the use of secondary structure models for inferring positional homology wholeheartedly, we caution against reliance on structural criteria for the discrimination of rRNA numts, given the potential fallibility of this approach.

Rab-GDI Complex Dissociation Factor Expressed through Translational Frameshifting in Filamentous Ascomycetes

PubMed Central

Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

2013-01-01

In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild. PMID:24069231

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

PubMed

Gong, Xiao-Qian; Sun, Ying-Feng; Ruan, Bao-Yang; Liu, Xiao-Min; Wang, Qi; Yang, Hai-Ming; Wang, Shuai-Yong; Zhang, Peng; Wang, Xiu-Hui; Shan, Tong-Ling; Tong, Wu; Zhou, Yan-Jun; Li, Guo-Xin; Zheng, Hao; Tong, Guang-Zhi; Yu, Hai

2017-06-01

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

PubMed Central

Ahern, Chris A; Vallejo, Paola; Mortenson, Lindsay; Coronado, Roberto

2001-01-01

Background The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. Results Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S) expressed the N-terminal half of α1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. Discussion A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel. PMID:11806762

FrameD: A flexible program for quality check and gene prediction in prokaryotic genomes and noisy matured eukaryotic sequences.

PubMed

Schiex, Thomas; Gouzy, Jérôme; Moisan, Annick; de Oliveira, Yannick

2003-07-01

We describe FrameD, a program that predicts coding regions in prokaryotic and matured eukaryotic sequences. Initially targeted at gene prediction in bacterial GC rich genomes, the gene model used in FrameD also allows to predict genes in the presence of frameshifts and partially undetermined sequences which makes it also very suitable for gene prediction and frameshift correction in unfinished sequences such as EST and EST cluster sequences. Like recent eukaryotic gene prediction programs, FrameD also includes the ability to take into account protein similarity information both in its prediction and its graphical output. Its performances are evaluated on different bacterial genomes. The web site (http://genopole.toulouse.inra.fr/bioinfo/FrameD/FD) allows direct prediction, sequence correction and translation and the ability to learn new models for new organisms.

The importance of ribosome production, and the 5S RNP-MDM2 pathway, in health and disease.

PubMed

Pelava, Andria; Schneider, Claudia; Watkins, Nicholas J

2016-08-15

Ribosomes are abundant, large RNA-protein complexes that are the source of all protein synthesis in the cell. The production of ribosomes is an extremely energetically expensive cellular process that has long been linked to human health and disease. More recently, it has been shown that ribosome biogenesis is intimately linked to multiple cellular signalling pathways and that defects in ribosome production can lead to a wide variety of human diseases. Furthermore, changes in ribosome production in response to nutrient levels in the diet lead to metabolic re-programming of the liver. Reduced or abnormal ribosome production in response to cellular stress or mutations in genes encoding factors critical for ribosome biogenesis causes the activation of the tumour suppressor p53, which leads to re-programming of cellular transcription. The ribosomal assembly intermediate 5S RNP (ribonucleoprotein particle), containing RPL5, RPL11 and the 5S rRNA, accumulates when ribosome biogenesis is blocked. The excess 5S RNP binds to murine double minute 2 (MDM2), the main p53-suppressor in the cell, inhibiting its function and leading to p53 activation. Here, we discuss the involvement of ribosome biogenesis in the homoeostasis of p53 in the cell and in human health and disease. © 2016 The Author(s).

Modified nucleoside dependent Watson-Crick and wobble codon binding by tRNALysUUU species.

PubMed

Yarian, C; Marszalek, M; Sochacka, E; Malkiewicz, A; Guenther, R; Miskiewicz, A; Agris, P F

2000-11-07

Nucleoside modifications are important to the structure of all tRNAs and are critical to the function of some tRNA species. The transcript of human tRNA(Lys3)(UUU) with a UUU anticodon, and the corresponding anticodon stem and loop domain (ASL(Lys3)(UUU)), are unable to bind to poly-A programmed ribosomes. To determine if specific anticodon domain modified nucleosides of tRNA(Lys) species would restore ribosomal binding and also affect thermal stability, we chemically synthesized ASL(Lys) heptadecamers and site-specifically incorporated the anticodon domain modified nucleosides pseudouridine (Psi(39)), 5-methylaminomethyluridine (mnm(5)U(34)) and N6-threonylcarbamoyl-adenosine (t(6)A(37)). Incorporation of t(6)A(37) and mnm(5)U(34) contributed structure to the anticodon loop, apparent by increases in DeltaS, and significantly enhanced the ability of ASL(Lys3)(UUU) to bind poly-A programmed ribosomes. Neither ASL(Lys3)(UUU)-t(6)A(37) nor ASL(Lys3)(UUU)-mnm(5)U(34) bound AAG programmed ribosomes. Only the presence of both t(6)A(37) and mnm(5)U(34) enabled ASL(Lys3)(UUU) to bind AAG programmed ribosomes, as well as increased its affinity for poly-A programmed ribosomes to the level of native Escherichia coli tRNA(Lys). The completely unmodified anticodon stem and loop of human tRNA(Lys1,2)(CUU) with a wobble position-34 C bound AAG, but did not wobble to AAA, even when the ASL was modified with t(6)A(37). The data suggest that tRNA(Lys)(UUU) species require anticodon domain modifications in the loop to impart an ordered structure to the anticodon for ribosomal binding to AAA and require a combination of modified nucleosides to bind AAG.

Cis- and trans-regulation of luteovirus gene expression by the 3’ end of the viral genome

PubMed Central

Miller, W. Allen; Jackson, Jacquelyn; Feng, Ying

2016-01-01

Translation of the 5.7 kb luteovirus genome is controlled by the 3’ untranslated region (UTR). Base pairing between regions of the 3’ UTR and sequences kilobases upstream is required for cap-independent translation and ribosomal frameshifting needed to synthesize the viral replicase. Luteoviruses produce subgenomic RNAs, which can serve as mRNA, but one sgRNA also regulates translation initiation in trans. As on all viruses, the 3’ and 5’ ends contain structures that are presumed to facilitate RNA synthesis. This review describes the structures and interactions of Barley yellow dwarf virus RNA that facilitate the complex interplay between the above events and result in a successful virus infection. We also present surprising results on the apparent lack of need for some subgenomic RNAs for the virus to infect cells or whole plants. In summary, the UTRs of luteoviruses are highly complex entities that control and fine-tune many key events of the virus replication cycle. PMID:25858272

Identification of a novel plant amalgavirus (Amalgavirus, Amalgaviridae) genome sequence in Cistus incanus.

PubMed

Goh, C J; Park, D; Lee, J S; Sebastiani, F; Hahn, Y

2018-01-01

Amalgaviridae is a family of double-stranded, monosegmented RNA viruses that are associated with plants, fungi, microsporidians, and animals. A sequence contig derived from the transcriptome of a eudicot, Cistus incanus (the family Cistaceae; commonly known as hoary rockrose), was identified as the genome sequence of a novel plant RNA virus and named Cistus incanus RNA virus 1 (CiRV1). Sequence comparison and phylogenetic analysis indicated that CiRV1 is a novel species of the genus Amalgavirus in the family Amalgaviridae. The CiRV1 genome contig has two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. An ORF1+2 fusion protein, which functions in viral RNA replication, is produced by a +1 programmed ribosomal frameshifting (PRF) mechanism. A +1 PRF motif UUU_CGU, which matches the conserved amalgavirus +1 PRF consensus sequence UUU_CGN, was found at the boundary of CiRV1 ORF1 and ORF2. Comparison of 25 amalgavirus ORF1+2 fusion proteins revealed that only three different positions within a 13-amino acid segment were recurrently used at the boundary, possibly being selected so as not to interfere with correct folding and function of the fusion protein. CiRV1 is the first virus found to be associated with the Cistus species and may be useful for studying amalgaviruses.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

PubMed

Penno, Christophe; Sharma, Virag; Coakley, Arthur; O'Connell Motherway, Mary; van Sinderen, Douwe; Lubkowska, Lucyna; Kireeva, Maria L; Kashlev, Mikhail; Baranov, Pavel V; Atkins, John F

2015-04-21

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

A GTPase reaction accompanying the rejection of Leu-tRNA2 by UUU-programmed ribosomes. Proofreading of the codon-anticodon interaction by ribosomes.

PubMed

Thompson, R C; Dix, D B; Gerson, R B; Karim, A M

1981-01-10

The characteristics of a GTPase reaction between poly(U)-programmed ribosomes, EFTu . GTP, and the near-cognate aminoacyl (aa)-tRNA, Leu-tRNA Leu 2, have been studied to assess the role of this reaction in proofreading of the codon-anticodon interaction. The reaction resembles the GTPase reaction with cognate aa-tRNAs and EFTu . GTP in its substrate requirements, in its involving EFTu . GTP . aa-tRNA ternary complexes, and in its requiring a free ribosomal A-site. The noncognate reaction differs from the cognate one in that aa-tRNA becomes stably bound to the ribosomes only 5% of the time; it therefore seems best characterized as an abortive enzymatic binding reaction. The rate of reaction is a significant fraction (4%) of that of the cognate aa-tRNA, indicating that recognition of ternary complexes by ribosomes involves a level of error greater than that of translation as a whole. The rejection of the noncognate aa-tRNA following GTP hydrolysis is therefore a vital step in the translation process and fulfills the criteria set for a proofreading reaction. Leu-tRNA Leu 2 which escapes rejection through proofreading, forms a stable complex with the ribosomal A-site, so it appears that the Leu-tRNA2 which was rejected never reached the A-site and that proofreading precedes full A-site binding.

Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

PubMed Central

Nakagawa, K.; Lokugamage, K.G.; Makino, S.

2017-01-01

Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

Pseudo-polyprotein translated from the full-length ORF1 of capillovirus is important for pathogenicity, but a truncated ORF1 protein without variable and CP regions is sufficient for replication.

PubMed

Hirata, Hisae; Yamaji, Yasuyuki; Komatsu, Ken; Kagiwada, Satoshi; Oshima, Kenro; Okano, Yukari; Takahashi, Shuichiro; Ugaki, Masashi; Namba, Shigetou

2010-09-01

The first open-reading frame (ORF) of the genus Capillovirus encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP), while other viruses in the family Flexiviridae have separate ORFs encoding these proteins. To investigate the role of the full-length ORF1 polyprotein of capillovirus, we generated truncation mutants of ORF1 of apple stem grooving virus by inserting a termination codon into the variable region located between the putative Rep- and CP-coding regions. These mutants were capable of systemic infection, although their pathogenicity was attenuated. In vitro translation of ORF1 produced both the full-length polyprotein and the smaller Rep protein. The results of in vivo reporter assays suggested that the mechanism of this early termination is a ribosomal -1 frame-shift occurring downstream from the conserved Rep domains. The mechanism of capillovirus gene expression and the very close evolutionary relationship between the genera Capillovirus and Trichovirus are discussed. Copyright (c) 2010. Published by Elsevier B.V.

Animal Mitochondrial DNA as We Do Not Know It: mt-Genome Organization and Evolution in Nonbilaterian Lineages

PubMed Central

Pett, Walker

2016-01-01

Abstract Animal mitochondrial DNA (mtDNA) is commonly described as a small, circular molecule that is conserved in size, gene content, and organization. Data collected in the last decade have challenged this view by revealing considerable diversity in animal mitochondrial genome organization. Much of this diversity has been found in nonbilaterian animals (phyla Cnidaria, Ctenophora, Placozoa, and Porifera), which, from a phylogenetic perspective, form the main branches of the animal tree along with Bilateria. Within these groups, mt-genomes are characterized by varying numbers of both linear and circular chromosomes, extra genes (e.g. atp9, polB, tatC), large variation in the number of encoded mitochondrial transfer RNAs (tRNAs) (0–25), at least seven different genetic codes, presence/absence of introns, tRNA and mRNA editing, fragmented ribosomal RNA genes, translational frameshifting, highly variable substitution rates, and a large range of genome sizes. This newly discovered diversity allows a better understanding of the evolutionary plasticity and conservation of animal mtDNA and provides insights into the molecular and evolutionary mechanisms shaping mitochondrial genomes. PMID:27557826

Evolution of genome size and complexity in the rhabdoviridae.

PubMed

Walker, Peter J; Firth, Cadhla; Widen, Steven G; Blasdell, Kim R; Guzman, Hilda; Wood, Thomas G; Paradkar, Prasad N; Holmes, Edward C; Tesh, Robert B; Vasilakis, Nikos

2015-02-01

RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3' to 5' direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae.

Evolution of Genome Size and Complexity in the Rhabdoviridae

PubMed Central

Walker, Peter J.; Firth, Cadhla; Widen, Steven G.; Blasdell, Kim R.; Guzman, Hilda; Wood, Thomas G.; Paradkar, Prasad N.; Holmes, Edward C.; Tesh, Robert B.; Vasilakis, Nikos

2015-01-01

RNA viruses exhibit substantial structural, ecological and genomic diversity. However, genome size in RNA viruses is likely limited by a high mutation rate, resulting in the evolution of various mechanisms to increase complexity while minimising genome expansion. Here we conduct a large-scale analysis of the genome sequences of 99 animal rhabdoviruses, including 45 genomes which we determined de novo, to identify patterns of genome expansion and the evolution of genome complexity. All but seven of the rhabdoviruses clustered into 17 well-supported monophyletic groups, of which eight corresponded to established genera, seven were assigned as new genera, and two were taxonomically ambiguous. We show that the acquisition and loss of new genes appears to have been a central theme of rhabdovirus evolution, and has been associated with the appearance of alternative, overlapping and consecutive ORFs within the major structural protein genes, and the insertion and loss of additional ORFs in each gene junction in a clade-specific manner. Changes in the lengths of gene junctions accounted for as much as 48.5% of the variation in genome size from the smallest to the largest genome, and the frequency with which new ORFs were observed increased in the 3’ to 5’ direction along the genome. We also identify several new families of accessory genes encoded in these regions, and show that non-canonical expression strategies involving TURBS-like termination-reinitiation, ribosomal frame-shifts and leaky ribosomal scanning appear to be common. We conclude that rhabdoviruses have an unusual capacity for genomic plasticity that may be linked to their discontinuous transcription strategy from the negative-sense single-stranded RNA genome, and propose a model that accounts for the regular occurrence of genome expansion and contraction throughout the evolution of the Rhabdoviridae. PMID:25679389

Mechanisms employed by retroviruses to exploit host factors for translational control of a complicated proteome

PubMed Central

Bolinger, Cheryl; Boris-Lawrie, Kathleen

2009-01-01

Retroviruses have evolved multiple strategies to direct the synthesis of a complex proteome from a single primary transcript. Their mechanisms are modulated by a breadth of virus-host interactions, which are of significant fundamental interest because they ultimately affect the efficiency of virus replication and disease pathogenesis. Motifs located within the untranslated region (UTR) of the retroviral RNA have established roles in transcriptional trans-activation, RNA packaging, and genome reverse transcription; and a growing literature has revealed a necessary role of the UTR in modulating the efficiency of viral protein synthesis. Examples include a 5' UTR post-transcriptional control element (PCE), present in at least eight retroviruses, that interacts with cellular RNA helicase A to facilitate cap-dependent polyribosome association; and 3' UTR constitutive transport element (CTE) of Mason-Pfizer monkey virus that interacts with Tap/NXF1 and SR protein 9G8 to facilitate RNA export and translational utilization. By contrast, nuclear protein hnRNP E1 negatively modulates HIV-1 Gag, Env, and Rev protein synthesis. Alternative initiation strategies by ribosomal frameshifting and leaky scanning enable polycistronic translation of the cap-dependent viral transcript. Other studies posit cap-independent translation initiation by internal ribosome entry at structural features of the 5' UTR of selected retroviruses. The retroviral armamentarium also commands mechanisms to counter cellular post-transcriptional innate defenses, including protein kinase R, 2',5'-oligoadenylate synthetase and the small RNA pathway. This review will discuss recent and historically-recognized insights into retrovirus translational control. The expanding knowledge of retroviral post-transcriptional control is vital to understanding the biology of the retroviral proteome. In a broad perspective, each new insight offers a prospective target for antiviral therapy and strategic improvement of gene transfer vectors. PMID:19166625

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

Ribosome Levels Selectively Regulate Translation and Lineage Commitment in Human Hematopoiesis.

PubMed

Khajuria, Rajiv K; Munschauer, Mathias; Ulirsch, Jacob C; Fiorini, Claudia; Ludwig, Leif S; McFarland, Sean K; Abdulhay, Nour J; Specht, Harrison; Keshishian, Hasmik; Mani, D R; Jovanovic, Marko; Ellis, Steven R; Fulco, Charles P; Engreitz, Jesse M; Schütz, Sabina; Lian, John; Gripp, Karen W; Weinberg, Olga K; Pinkus, Geraldine S; Gehrke, Lee; Regev, Aviv; Lander, Eric S; Gazda, Hanna T; Lee, Winston Y; Panse, Vikram G; Carr, Steven A; Sankaran, Vijay G

2018-03-22

Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease. We show that in DBA, the pool of available ribosomes is limited, while ribosome composition remains constant. Surprisingly, this global reduction in ribosome levels more profoundly alters translation of a select subset of transcripts. We show how the reduced translation of select transcripts in HSPCs can impair erythroid lineage commitment, illuminating a regulatory role for ribosome levels in cellular differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

Frameshifted prion proteins as pathological agents: quantitative considerations.

PubMed

Wills, Peter R

2013-05-21

A quantitatively consistent explanation for the titres of infectivity found in a variety of prion-containing preparations is provided on the basis that the ætiological agents of transmissible spongiform encephalopathy comprise a very small population fraction of prion protein (PrP) variants, which contain frameshifted elements in their N-terminal octapeptide-repeat regions. A mechanism for the replication of frameshifted prions is described and calculations are performed to obtain estimates of the concentration of these PrP variants in normal and infected brain, as well as their enrichment in products of protein misfolding cyclic amplification. These calculations resolve the lack of proper quantitative correlation between measures of infectivity and the presence of conformationally-altered, protease-resistant variants of PrP. Experiments, which could confirm or eventually exclude the role of frameshifted variants in the ætiology of prion disease, are suggested. Copyright © 2013 Elsevier Ltd. All rights reserved.

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. V. Isolation and Genetic Properties of Nongroup-Specific Suppressors

PubMed Central

Culbertson, Michael R.; Gaber, Richard F.; Cummins, Claudia M.

1982-01-01

Two classes of frameshift suppressors distributed at 22 different loci were identified in previous studies in the yeast Saccharomyces cerevisiae. These suppressors exhibited allele-specific suppression of +1 G:C insertion mutations in either glycine or proline codons, designated as group II and group III frameshift mutations, respectively. Genes corresponding to representative suppressors of each group have been shown to encode altered glycine or proline tRNAs containing four base anticodons.—This communication reports the existence of a third class of frameshift suppressor that exhibits a wider range in specificity of suppression. The suppressors map at three loci, suf12, suf13, and suf14, which are located on chromosomes IV, XV, and XIV, respectively. The phenotypes of these suppressors suggest that suppression may be mediated by genes other than those encoding the primary structure of glycine or proline tRNAs. PMID:6757053

Frameshift Suppression in SACCHAROMYCES CEREVISIAE VI. Complete Genetic Map of Twenty-Five Suppressor Genes

PubMed Central

Gaber, Richard F.; Mathison, Lorilee; Edelman, Irv; Culbertson, Michael R.

1983-01-01

Five previously unmapped frameshift suppressor genes have been located on the yeast genetic map. In addition, we have further characterized the map positions of two suppressors whose approximate locations were determined in an earlier study. These results represent the completion of genetic mapping studies on all 25 of the known frameshift suppressor genes in yeast.—The approximate location of each suppressor gene was initially determined through the use of a set of mapping strains containing 61 signal markers distributed throughout the yeast genome. Standard meiotic linkage was assayed in crosses between strains carrying the suppressors and the mapping strains. Subsequent to these approximate linkage determinations, each suppressor gene was more precisely located in multi-point crosses. The implications of these mapping results for the genomic distribution of frameshift suppressor genes, which include both glycine and proline tRNA genes, are discussed. PMID:17246112

Frameshifting in the expression of the Escherichia coli trpR gene is modulated by translation initiation.

PubMed Central

Benhar, I; Miller, C; Engelberg-Kulka, H

1993-01-01

The Escherichia coli trpR gene encodes the 108-amino-acid-long Trp repressor. We have shown previously that a +1 frameshifting event occurs during the expression of trpR, resulting in the synthesis of an additional (+1 frame) polypeptide. Using trpR-lac'Z fusions, we have recently found that the transition from the 0 to the +1 frame occurs via the bypassing of a 55-nucleotide-long segment of the trpR+1-lac'Z mRNA (I. Benhar, and H. Engelberg-Kulka, Cell 72:121-130, 1993). Here we show that the frequency of trpR frameshifting (or bypassing) can be regulated both in vivo and in vitro. This frequency is inversely proportional to the rate of initiation of translation of the trpR gene. Hence, modulating the level of translation initiation affects the frequency of frameshifting. Images PMID:8491735

pKa shifting in double-stranded RNA is highly dependent upon nearest neighbors and bulge positioning.

PubMed

Wilcox, Jennifer L; Bevilacqua, Philip C

2013-10-22

Shifting of pKa's in RNA is important for many biological processes; however, the driving forces responsible for shifting are not well understood. Herein, we determine how structural environments surrounding protonated bases affect pKa shifting in double-stranded RNA (dsRNA). Using (31)P NMR, we determined the pKa of the adenine in an A(+)·C base pair in various sequence and structural environments. We found a significant dependence of pKa on the base pairing strength of nearest neighbors and the location of a nearby bulge. Increasing nearest neighbor base pairing strength shifted the pKa of the adenine in an A(+)·C base pair higher by an additional 1.6 pKa units, from 6.5 to 8.1, which is well above neutrality. The addition of a bulge two base pairs away from a protonated A(+)·C base pair shifted the pKa by only ~0.5 units less than a perfectly base paired hairpin; however, positioning the bulge just one base pair away from the A(+)·C base pair prohibited formation of the protonated base pair as well as several flanking base pairs. Comparison of data collected at 25 °C and 100 mM KCl to biological temperature and Mg(2+) concentration revealed only slight pKa changes, suggesting that similar sequence contexts in biological systems have the potential to be protonated at biological pH. We present a general model to aid in the determination of the roles protonated bases may play in various dsRNA-mediated processes including ADAR editing, miRNA processing, programmed ribosomal frameshifting, and general acid-base catalysis in ribozymes.

Last 20 aa of the West Nile virus NS1' protein are responsible for its retention in cells and the formation of unique heat-stable dimers.

PubMed

Young, Lucy B; Melian, Ezequiel Balmori; Setoh, Yin Xiang; Young, Paul R; Khromykh, Alexander A

2015-05-01

West Nile virus (WNV), a mosquito-borne flavivirus, is the major cause of arboviral encephalitis in the USA. As with other members of the Japanese encephalitis virus serogroup, WNV produces an additional non-structural protein, NS1', a C-terminal extended product of NS1 generated as the result of a -1 programmed ribosomal frameshift (PRF). We have previously shown that mutations abolishing the PRF, and consequently NS1', resulted in reduced neuroinvasiveness. However, whether this was caused by the PRF event itself or by the lack of a PRF product, NS1', or a combination of both, remains undetermined. Here, we showed that WNV NS1' formed a unique subpopulation of heat- and low-pH-stable dimers. C-terminal truncations and mutational analysis employing an NS1'-expressing plasmid showed that stability of NS1' dimers was linked to the penultimate 10 aa. To examine the role of NS1' heat-stable dimers in virus replication and pathogenicity, a stop codon mutation was introduced into NS1' to create a WNV producing a truncated version of NS1' lacking the last 20 aa, but not affecting the PRF. NS1' protein produced by this mutant virus was secreted more efficiently than WT NS1', indicating that the sequence of the last 20 aa of NS1' was responsible for its cellular retention. Further analysis of this mutant showed growth kinetics in cells and virulence in weanling mice after peripheral infection similar to the WT WNVKUN, suggesting that full-length NS1' was not essential for virus replication in vitro and for virulence in mice. © 2015 The Authors.

Quinupristin-Dalfopristin Resistance in Streptococcus pneumoniae: Novel L22 Ribosomal Protein Mutation in Two Clinical Isolates from the SENTRY Antimicrobial Surveillance Program

PubMed Central

Jones, Ronald N.; Farrell, David J.; Morrissey, Ian

2003-01-01

Resistance to quinupristin-dalfopristin (Q/D) among gram-positive cocci has been very uncommon. Two clinical isolates among 8,837 (0.02%) Streptococcus pneumoniae isolates were discovered in 2001 to 2002 with Q/D MICs of 4 μg/ml. Each had a 5-amino-acid tandem duplication (RTAHI) in the L22 ribosomal protein gene (rplV) preventing synergistic ribosomal binding of the streptogramin combination. Similar gene duplication has been reported in Q/D-resistant Staphylococcus aureus. PMID:12878545

Molecular characterization of a new monopartite dsRNA mycovirus from mycorrhizal Thelephora terrestris (Ehrh.) and its detection in soil oribatid mites (Acari: Oribatida)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petrzik, Karel, E-mail: petrzik@umbr.cas.cz; Sarkisova, Tatiana; Starý, Josef

2016-02-15

A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris (Ehrh.) and sequenced. This virus, named Thelephora terrestris virus 1 (TtV1), contains two reading frames in different frames but with the possibility that ORF2 could be translated as a fusion polyprotein after ribosomal -1 frameshifting. Picornavirus 2A-like motif, nudix hydrolase, phytoreovirus S7, and RdRp domains were found in a unique arrangement on the polyprotein. A new genus named Phlegivirus and containing TtV1, PgLV1, RfV1 and LeV is therefore proposed. Twenty species of oribatid mites were identified in soil material in the vicinity of T. terrestris. TtV1 was detectedmore » in large amounts in Steganacarus (Tropacarus) carinatus (C.L. Koch, 1841) and in much smaller amounts in Nothrus silvestris (Nicolet). This is the first description of mycovirus presence in oribatid mites. - Highlights: • A novel dsRNA virus was identified in the mycorrhizal fungus Thelephora terrestris. • A new virus genus Phlegivirus is proposed. • The mycovirus was firstly detected in oribatid mites.« less

Remaining Mysteries of Molecular Biology: The Role of Polyamines in the Cell.

PubMed

Miller-Fleming, Leonor; Olin-Sandoval, Viridiana; Campbell, Kate; Ralser, Markus

2015-10-23

The polyamines (PAs) spermidine, spermine, putrescine and cadaverine are an essential class of metabolites found throughout all kingdoms of life. In this comprehensive review, we discuss their metabolism, their various intracellular functions and their unusual and conserved regulatory features. These include the regulation of translation via upstream open reading frames, the over-reading of stop codons via ribosomal frameshifting, the existence of an antizyme and an antizyme inhibitor, ubiquitin-independent proteasomal degradation, a complex bi-directional membrane transport system and a unique posttranslational modification-hypusination-that is believed to occur on a single protein only (eIF-5A). Many of these features are broadly conserved indicating that PA metabolism is both concentration critical and evolutionary ancient. When PA metabolism is disrupted, a plethora of cellular processes are affected, including transcription, translation, gene expression regulation, autophagy and stress resistance. As a result, the role of PAs has been associated with cell growth, aging, memory performance, neurodegenerative diseases, metabolic disorders and cancer. Despite comprehensive studies addressing PAs, a unifying concept to interpret their molecular role is missing. The precise biochemical function of polyamines is thus one of the remaining mysteries of molecular cell biology. Copyright © 2015. Published by Elsevier Ltd.

A novel monopartite dsRNA virus isolated from the phytopathogenic fungus Ustilaginoidea virens and ancestrally related to a mitochondria-associated dsRNA in the green alga Bryopsis.

PubMed

Zhang, Tingting; Jiang, Yinhui; Dong, Wubei

2014-08-01

In this study, we describe a novel mycovirus isolated from Ustilaginoidea virens, which was designated Ustilaginoidea virens nonsegmented virus 1 (UvNV-1). The sequence analysis revealed that UvNV-1 has two open reading frames (ORFs). ORF1 encodes an unknown protein, which is similar to the hypothetical protein BN7_5177 of Wickerhamomyces ciferrii. ORF2 encodes a putative RNA-dependent RNA polymerase (RdRp), which is most closely related to Bryopsis mitochondria-associated dsRNA (BDRM) and is likely expressed by a +1 ribosomal frameshift within the sequence CCC_UUU_CGA. The phylogenetic analysis of the RdRp of UvNV-1 showed that UvNV-1 represents a new virus taxon of mycoviruses with a partitivirus-like lineage that is classified into the family of picorna-like viruses. Based on northern hybridization, UvNV-1 was found to be common to U. virens from different geographic locations in China. The biological comparison of virus-free and infected fungal strains revealed that UvNV-1 is likely to be cryptic to its host. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular dynamics of the frame-shifting pseudoknot from beet western yellows virus: the role of non-Watson-Crick base-pairing, ordered hydration, cation binding and base mutations on stability and unfolding.

PubMed

Csaszar, K; Spacková, N; Stefl, R; Sponer, J; Leontis, N B

2001-11-09

Molecular dynamics simulations of the frame-shifting pseudoknot from beet western yellows virus (BWYV, NDB file UR0004) were performed with explicit inclusion of solvent and counterions. In all, 33 ns of simulation were carried out, including 10 ns of the native structure with protonation of the crucial cytosine residue, C8(N3+). The native structure exhibited stable trajectories retaining all Watson-Crick and tertiary base-pairs, except for fluctuations or transient disruptions at specific sites. The most significant fluctuations involved the change or disruption of hydrogen-bonding between C8(N3+) and bases G12, A25, and C26, as well as disruption of the water bridges linking C8(N3+) with A25 and C26. To increase sampling of rare events, the native simulation was continued at 400 K. A partial, irreversible unfolding of the molecule was initiated by slippage of C8(N3+) relative to G12 and continued by sudden concerted changes in hydrogen-bonding involving A23, A24, and A25. These events were followed by a gradual loss of stacking interactions in loop 2. Of the Watson-Crick base-pairs, only the 5'-terminal pair of stem 1 dissociated at 400 K, while the trans sugar-edge/sugar-edge A20.G4 interaction remained surprisingly stable. Four additional room-temperature simulations were carried out to obtain insights into the structural and dynamic effects of selected mutations. In two of these, C8 was left unprotonated. Considerable local rearrangements occurred that were not observed in the crystal structure, thus confirming N3-protonation of C8 in the native molecule. We also investigated the effect of mutating C8(N3+) to U8, to correlate with experimental and phylogenetic studies, and of changing the G4 x C17 base-pair to A4 x U17 to weaken the trans sugar-edge interaction between positions 4 and 20 and to test models of unfolding. The simulations indicate that the C8 x G12 x C26 base-triple at the junction is the most labile region of the frame-shifting pseudoknot. They provide insights into the roles of the other non-Watson-Crick base-pairs in the early stages of unfolding of the pseudoknot, which must occur to allow readthrough of the message by the ribosome. The simulations revealed several critical, highly ordered hydration sites with close to 100 % occupancies and residency times of individual water molecules of up to 5 ns. Sodium cation coordination sites with occupancies above 50 % were also observed. Copyright 2001 Academic Press.

Mutations in a Highly Conserved Motif of nsp1β Protein Attenuate the Innate Immune Suppression Function of Porcine Reproductive and Respiratory Syndrome Virus

PubMed Central

Li, Yanhua; Shyu, Duan-Liang; Shang, Pengcheng; Bai, Jianfa; Ouyang, Kang; Dhakal, Santosh; Hiremath, Jagadish; Binjawadagi, Basavaraj

2016-01-01

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) nonstructural protein 1β (nsp1β) is a multifunctional viral protein, which is involved in suppressing the host innate immune response and activating a unique −2/−1 programmed ribosomal frameshifting (PRF) signal for the expression of frameshifting products. In this study, site-directed mutagenesis analysis showed that the R128A or R129A mutation introduced into a highly conserved motif (123GKYLQRRLQ131) reduced the ability of nsp1β to suppress interferon beta (IFN-β) activation and also impaired nsp1β's function as a PRF transactivator. Three recombinant viruses, vR128A, vR129A, and vRR129AA, carrying single or double mutations in the GKYLQRRLQ motif were characterized. In comparison to the wild-type (WT) virus, vR128A and vR129A showed slightly reduced growth abilities, while the vRR129AA mutant had a significantly reduced growth ability in infected cells. Consistent with the attenuated growth phenotype in vitro, pigs infected with nsp1β mutants had lower levels of viremia than did WT virus-infected pigs. Compared to the WT virus in infected cells, all three mutated viruses stimulated high levels of IFN-α expression and exhibited a reduced ability to suppress the mRNA expression of selected interferon-stimulated genes (ISGs). In pigs infected with nsp1β mutants, IFN-α production was increased in the lungs at early time points postinfection, which was correlated with increased innate NK cell function. Furthermore, the augmented innate response was consistent with the increased production of IFN-γ in pigs infected with mutated viruses. These data demonstrate that residues R128 and R129 are critical for nsp1β function and that modifying these key residues in the GKYLQRRLQ motif attenuates virus growth ability and improves the innate and adaptive immune responses in infected animals. IMPORTANCE PRRSV infection induces poor antiviral innate IFN and cytokine responses, which results in weak adaptive immunity. One of the strategies in next-generation vaccine construction is to manipulate viral proteins/genetic elements involved in antagonizing the host immune response. PRRSV nsp1β was identified to be a strong innate immune antagonist. In this study, two basic amino acids, R128 and R129, in a highly conserved GKYLQRRLQ motif were determined to be critical for nsp1β function. Mutations introduced into these two residues attenuated virus growth and improved the innate and adaptive immune responses of infected animals. Technologies developed in this study could be broadly applied to current commercial PRRSV modified live-virus (MLV) vaccines and other candidate vaccines. PMID:26792733

Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange.

PubMed Central

Farber, N; Cantor, C R

1980-01-01

The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment. This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)-programed tight-couple 70S ribosomes of Escherichia coli. The tritium incorporation into specific sites of the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments. Ribosome binding leads to marked suppression in the exchange rate of a number of fragments. This delineates extensive regions of tRNA-ribosome contact. No change in exchange rates is seen for fragments from the corner of the molecule, indicating that this region of bound tRNA is readily accessible to the solvent. Ribosome binding results in an enhanced exchange rate at the T loop. This appears to be the result of a conformational change that is most likely an unfolding of the T and D loops. Additional tritium exchange reactions suggest this conformational change is induced by ribosomes and not by messenger. PMID:7001473

Frameshifting in the p6 cDNA phage display system.

PubMed

Govarts, Cindy; Somers, Klaartje; Stinissen, Piet; Somers, Veerle

2010-12-20

Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.

Mutations in Elongation Factor Ef-1α Affect the Frequency of Frameshifting and Amino Acid Misincorporation in Saccharomyces Cerevisiae

PubMed Central

Sandbaken, M. G.; Culbertson, M. R.

1988-01-01

A mutational analysis of the eukaryotic elongation factor EF-1α indicates that this protein functions to limit the frequency of errors during genetic code translation. We found that both amino acid misincorporation and reading frame errors are controlled by EF-1α. In order to examine the function of this protein, the TEF2 gene, which encodes EF-1α in Saccharomyces cerevisiae, was mutagenized in vitro with hydroxylamine. Sixteen independent TEF2 alleles were isolated by their ability to suppress frameshift mutations. DNA sequence analysis identified eight different sites in the EF-1α protein that elevate the frequency of mistranslation when mutated. These sites are located in two different regions of the protein. Amino acid substitutions located in or near the GTP-binding and hydrolysis domain of the protein cause suppression of frameshift and nonsense mutations. These mutations may effect mistranslation by altering the binding or hydrolysis of GTP. Amino acid substitutions located adjacent to a putative aminoacyl-tRNA binding region also suppress frameshift and nonsense mutations. These mutations may alter the binding of aminoacyl-tRNA by EF-1α. The identification of frameshift and nonsense suppressor mutations in EF-1α indicates a role for this protein in limiting amino acid misincorporation and reading frame errors. We suggest that these types of errors are controlled by a common mechanism or closely related mechanisms. PMID:3066688

Frameshift Suppression in SACCHAROMYCES CEREVISIAE. IV. New Suppressors among Spontaneous Co-Revertants of the Group II HIS4-206 and LEU2-3 Frameshift Mutations

PubMed Central

Gaber, Richard F.; Culbertson, Michael R.

1982-01-01

ICR-induced frameshift mutations at the his4 locus in Saccharomyces cerevisiae have been classified into several groups on the basis of their reversion and suppression properties. One group of externally suppressible his4 mutations, designated Group II, have been shown to contain +1 G:C insertions in glycine codons and are suppressed by any one of five suppressor mutations described previously (SUF1, SUF3, SUF4, SUF5, and SUF6). The suppressor genes are believed to encode glycine tRNAs containing four base anticodons.—An analysis of spontaneous co-revertants of the Group II frameshift mutations his4-206 and leu2-3 has revealed the existence of eleven new Group II-specific suppressor genes (SUF15 through SUF25). The locations of the new suppressor loci on the yeast genetic map have been determined.—By comparing the ability or inability of Group II-specific suppressors mapping at 16 different loci to suppress different Group II his4 mutations, two subclasses of suppressors have been defined. One subclass suppresses his4-38 and his4-519, which contain the altered four base mRNA codons 5'-GGGU-3' and 5'-GGGG-3', respectively. The other subclass suppresses his4-38, but fails to suppress his4-519. The mechanism of tRNA-mediated frameshift suppression and the molecular basis for this division of the suppressors into two subclasses is discussed. PMID:6757051

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

PubMed

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

PubMed Central

DeMarini, D M; Shelton, M L; Abu-Shakra, A; Szakmary, A; Levine, J G

1998-01-01

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins. PMID:9584083

Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor.

PubMed

Belsham, Graham J; Polacek, Charlotta; Breum, Solvej Ø; Larsen, Lars E; Bøtner, Anette

2010-01-16

Myxoma virus is a member of the Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy. An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature. It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis.

Detection of myxoma viruses encoding a defective M135R gene from clinical cases of myxomatosis; possible implications for the role of the M135R protein as a virulence factor

PubMed Central

2010-01-01

Background Myxoma virus is a member of the Poxviridae and causes disease in European rabbits. Laboratory confirmation of the clinical disease, which occurs in the autumn of most years in Denmark, has been achieved previously using antigen ELISA and electron microscopy. Results An unusually large number of clinically suspected cases of myxomatosis were observed in Denmark during 2007. Myxoma virus DNA was detected, using a new real time PCR assay which targets the M029L gene, in over 70% of the clinical samples submitted for laboratory confirmation. Unexpectedly, further analysis revealed that a high proportion of these viral DNA preparations contained a frame-shift mutation within the M135R gene that has previously been identified as a virulence factor. This frame-shift mutation results in expression of a greatly truncated product. The same frame-shift mutation has also been found recently within an avirulent strain of myxoma virus (6918). However, three other frame-shift mutations found in this strain (in the genes M009L, M036L and M148R) were not shared with the Danish viruses but a single nucleotide deletion in the M138R/M139R intergenic region was a common feature. Conclusions It appears that expression of the full-length myxoma virus M135R protein is not required for virulence in rabbits. Hence, the frame-shift mutation in the M135R gene in the nonpathogenic 6918 virus strain is not sufficient to explain the attenuation of this myxoma virus but one/some of the other frame-shift mutations alone or in conjunction with one/some of the thirty two amino acid substitutions must also contribute. The real time PCR assay for myxoma virus is a useful diagnostic tool for laboratory confirmation of suspected cases of myxomatosis. PMID:20078890

Biopolymers Containing Unnatural Amino Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

Biopolymers Containing Unnatural Building Blocks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schultz, Peter G.

2013-06-30

Although the main chain structure of polymers has a profound effect on their materials properties, the side groups can also have dramatic effects on their properties including conductivity, liquid crystallinity, hydrophobicity, elasticity and biodegradability. Unfortunately control over the side chain structure of polymers remains a challenge – it is difficult to control the sequence of chain elongation when mixtures of monomers are polymerized, and postpolymerization side chain modification is made difficult by polymer effects on side chain reactivity. In contrast, the mRNA templated synthesis of polypeptides on the ribosome affords absolute control over the primary sequence of the twenty aminomore » acid monomers. Moreover, the length of the biopolymer is precisely controlled as are sites of crosslinking. However, whereas synthetic polymers can be synthesized from monomers with a wide range of chemically defined structures, ribosomal biosynthesis is largely limited to the 20 canonical amino acids. For many applications in material sciences, additional building blocks would be desirable, for example, amino acids containing metallocene, photoactive, and halogenated side chains. To overcome this natural constraint we have developed a method that allows unnatural amino acids, beyond the common twenty, to be genetically encoded in response to nonsense or frameshift codons in bacteria, yeast and mammalian cells with high fidelity and good yields. Here we have developed methods that allow identical or distinct noncanonical amino acids to be incorporated at multiple sites in a polypeptide chain, potentially leading to a new class of templated biopolymers. We have also developed improved methods for genetically encoding unnatural amino acids. In addition, we have genetically encoded new amino acids with novel physical and chemical properties that allow selective modification of proteins with synthetic agents. Finally, we have evolved new metal-ion binding sites in proteins using a novel metal-ion binding amino acid, which may facilitate our ability to generate new protein based sensors and catalysts.« less

A new version of the RDP (Ribosomal Database Project)

NASA Technical Reports Server (NTRS)

Maidak, B. L.; Cole, J. R.; Parker, C. T. Jr; Garrity, G. M.; Larsen, N.; Li, B.; Lilburn, T. G.; McCaughey, M. J.; Olsen, G. J.; Overbeek, R.;

RBSDesigner: software for designing synthetic ribosome binding sites that yields a desired level of protein expression.

PubMed

Na, Dokyun; Lee, Doheon

2010-10-15

RBSDesigner predicts the translation efficiency of existing mRNA sequences and designs synthetic ribosome binding sites (RBSs) for a given coding sequence (CDS) to yield a desired level of protein expression. The program implements the mathematical model for translation initiation described in Na et al. (Mathematical modeling of translation initiation for the estimation of its efficiency to computationally design mRNA sequences with a desired expression level in prokaryotes. BMC Syst. Biol., 4, 71). The program additionally incorporates the effect on translation efficiency of the spacer length between a Shine-Dalgarno (SD) sequence and an AUG codon, which is crucial for the incorporation of fMet-tRNA into the ribosome. RBSDesigner provides a graphical user interface (GUI) for the convenient design of synthetic RBSs. RBSDesigner is written in Python and Microsoft Visual Basic 6.0 and is publicly available as precompiled stand-alone software on the web (http://rbs.kaist.ac.kr). dhlee@kaist.ac.kr

Whole Exome Analysis of Early Onset Alzheimer’s Disease

DTIC Science & Technology

2016-04-01

Early Onset Alzheimer’s Disease 5a. CONTRACT NUMBER W81XWH-12-1-0013 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) Margaret A. Pericak...relationship between SORL1, AD, and Parkinsonism . 16 Appendix V: ABCA7 Frameshift Deletion Associated with Alzheimer’s Disease in African Americans...onset Alzheimer disease identified using whole-exome sequencing G. W. Beecham1, B. W. Kunkle1, B. Vardarajan2, P. L. Whitehead1, S . Rolati1, E. R

Synthesis, base pairing and structure studies of geranylated RNA

PubMed Central

Wang, Rui; Vangaveti, Sweta; Ranganathan, Srivathsan V.; Basanta-Sanchez, Maria; Haruehanroengra, Phensinee; Chen, Alan; Sheng, Jia

2016-01-01

Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium. The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNAGluUUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon–anticodon interaction during ribosome binding. PMID:27307604

Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs.

PubMed

Xu, Guanlong; Zhang, Xuxiao; Sun, Yipeng; Liu, Qinfang; Sun, Honglei; Xiong, Xin; Jiang, Ming; He, Qiming; Wang, Yu; Pu, Juan; Guo, Xin; Yang, Hanchun; Liu, Jinhua

2016-02-25

The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a "triple-reassortment" H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs.

Dual effect of chloramphenicol peptides on ribosome inhibition.

PubMed

Bougas, Anthony; Vlachogiannis, Ioannis A; Gatos, Dimitrios; Arenz, Stefan; Dinos, George P

2017-05-01

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Ribosomes in the sea: a window on taxon-specific lysis

NASA Astrophysics Data System (ADS)

Suttle, C.; Zhong, X.; Wirth, J.

2016-02-01

Microbes are estimated to comprise more than 90% of the biomass in the world's oceans, are major drivers of biogeochemical cycles, and have turnover rates ranging from hours to days. Despite the central role that microbes play in marine ecosystems, there is no robust method to evaluate taxon-specific mortality rates. Here, we report a method that employs extracellular free-ribosomes as a proxy to evaluate taxon-specific microbial lysis. The method was validated with laboratory cultures of the marine heterotrophic bacterium Vibrio natriegens strain PWH3a and the photoautotroph Synechococcus strain DC2, with and without grazers or viruses, to identify the origin and fate of the extracellular free-ribosomes. Our results showed both viral lysis and programmed-cell-death (PCD) contribute to free-ribosome production. Ribosomes were not released when cells were grazed, but grazers could consume free-ribosomes. We show that extracellular free-ribosomes can be used to evaluate microbial mortality caused by viral lysis and PCD. This approach was applied to environmental samples by examining the taxonomic composition and relative abundance of free 16S-ribosomes in seawater samples collected from the Strait of Georgia and Saanich Inlet, British Columbia, Canada. Based on the presence of free ribosomes, lysis was detected in 2198 out of 4013 prokaryotic taxa, representing 22 bacterial and three archaeal phyla. Of these, lysis of 140 taxa could be detected in all nine samples. Based on the ratio of free ribosomes to cellular ribosomes, some taxa associated with specific ecological niches appeared to be subject to high rates of lysis, including the genera Achromobacter, Chryseobacterium, Clostridium, Delftia, Ferruginibacter, Lactobacillus, Marinomonas, Massilia, Microbacterium, Ochrobactrum, Paenibacillus, Phyllobacterium, Pseudomonas, Rhodobacter, and Stenotrophomonas. Our results showed high-lysis coupled with low-abundance, suggesting that taxa in lower abundance are subject to higher relative rates of cell lysis, consistent with previous suggestions. The ability to estimate taxon-specific mortality as the result of cell lysis adds an important tool in our quest to explain the distribution and abundance of specific microbial taxa in nature.

Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions.

PubMed

Zhang, Jingji; Pavlov, Michael Y; Ehrenberg, Måns

2018-02-16

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

DTIC Science & Technology

2016-02-11

the White- head Genome Technology Core for sequencing . This work was supported by the UCSF Program for Breakthrough Biomedical Research (funded in...landscape of the yeast genome defined by RNA sequencing . Science 320, 1344–1349. Nedialkova, D.D., and Leidel, S.A. (2015). Optimization of Codon Translation... the CC BY license (http://creativecommons.org/licenses/by/4.0/). SUMMARY Ribosome-footprint profiling provides genome -wide snapshots of translation

Genetic progression in microsatellite instability high (MSI-H) colon cancers correlates with clinico-pathological parameters: A study of the TGRbetaRII, BAX, hMSH3, hMSH6, IGFIIR and BLM genes.

PubMed

Calin, G A; Gafà, R; Tibiletti, M G; Herlea, V; Becheanu, G; Cavazzini, L; Barbanti-Brodano, G; Nenci, I; Negrini, M; Lanza, G

2000-05-20

Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico-pathological features. This report describes the analysis of 63 "microsatellite instability-high" (MSI-H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFbetaRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFbetaRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico-pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI-H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico-pathological traits. Copyright 2000 Wiley-Liss, Inc.

Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions

PubMed Central

Zhang, Jingji

2018-01-01

Abstract We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of ‘monitoring bases’ A1492, A1493 and G530 in 16S rRNA in favor of their ‘activated’ state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the ‘intrinsic selectivity’ of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome. PMID:29267976

Four distinct immune microenvironment subtypes in gastric adenocarcinoma with special reference to microsatellite instability

PubMed Central

Heo, You Jeong; Kim, Seungtae; Kim, Nayoung KD; Park, Joon Oh; Kang, Won Ki; Lee, Jeeyun; Kim, Kyoung-Mee

2018-01-01

Introduction Programmed death-ligand 1 (PD-L1) can be overexpressed in tumours other than Epstein-Barr virus (EBV)-positive (EBV+) or microsatellite instability-high (MSI-H) gastric cancer (GC) subtypes. We aimed to determine the tumour immune microenvironment (TME) classification of GC to better understand tumour–immune interactions and help patient selection for future immunotherapy with special reference to MSI-H. Methods Immunohistochemistry (IHC) for PD-L1 and CD8+ T cells in three distinct subtypes of GC (43 EBV+, 79 MSI-H and 125 EBV−/MSS) were performed and analysed. In 66 MSI-H GC, mutation counts were compared with PD-L1 expression and survival of the patients. Results GC TME divided by PD-L1 IHC and tumour-infiltrating lymphocytes (TIL) measured by intratumoural CD8 density showed: (1) about 40% of GC are type I (PD-L1+/TIL+) consisting ~70% of MSI-H or EBV+ GC, and ~15% of EBV−/microsatellite stable (MSS) GC patients show the best survival in both disease-free (HR 2.044) and overall survival (HR 1.993); this type would respond to a checkpoint blockade therapy; (2) almost 30% of GC are type II (PD-L1−/TIL−) with the worst survival; (3) approximately 10% of GC are type III (PD-L1+/TIL−); and (4) up to 20% are type IV (PD-L1−/TIL+) and, unexpectedly, ~25% of EBV+ or MSI-H GC are within this subtype. In MSI-H GC, frequent frameshift mutations were observed in ARID1A, RNF43, NF1, MSH6, BRD3, NCOA3, BCORL1, TNKS2 and NPM1 and the numbers of frameshift mutation correlated significantly with PD-L1 expression (P<0.05). Discussion GC can be classified into four TME types based on PD-L1 and TIL, and numbers of frameshift mutation correlate well with PD-L1 expression in MSI-H GC. PMID:29636988

Four distinct immune microenvironment subtypes in gastric adenocarcinoma with special reference to microsatellite instability.

PubMed

Cho, Junhun; Chang, Young Hwan; Heo, You Jeong; Kim, Seungtae; Kim, Nayoung Kd; Park, Joon Oh; Kang, Won Ki; Lee, Jeeyun; Kim, Kyoung-Mee

2018-01-01

Programmed death-ligand 1 (PD-L1) can be overexpressed in tumours other than Epstein-Barr virus (EBV)-positive (EBV + ) or microsatellite instability-high (MSI-H) gastric cancer (GC) subtypes. We aimed to determine the tumour immune microenvironment (TME) classification of GC to better understand tumour-immune interactions and help patient selection for future immunotherapy with special reference to MSI-H. Immunohistochemistry (IHC) for PD-L1 and CD8 + T cells in three distinct subtypes of GC (43 EBV + , 79 MSI-H and 125 EBV - /MSS) were performed and analysed. In 66 MSI-H GC, mutation counts were compared with PD-L1 expression and survival of the patients. GC TME divided by PD-L1 IHC and tumour-infiltrating lymphocytes (TIL) measured by intratumoural CD8 density showed: (1) about 40% of GC are type I (PD-L1 + /TIL + ) consisting ~70% of MSI-H or EBV + GC, and ~15% of EBV - /microsatellite stable (MSS) GC patients show the best survival in both disease-free (HR 2.044) and overall survival (HR 1.993); this type would respond to a checkpoint blockade therapy; (2) almost 30% of GC are type II (PD-L1 - /TIL - ) with the worst survival; (3) approximately 10% of GC are type III (PD-L1 + /TIL - ); and (4) up to 20% are type IV (PD-L1 - /TIL + ) and, unexpectedly, ~25% of EBV + or MSI-H GC are within this subtype. In MSI-H GC, frequent frameshift mutations were observed in ARID1A , RNF43 , NF1 , MSH6 , BRD3 , NCOA3 , BCORL1 , TNKS2 and NPM1 and the numbers of frameshift mutation correlated significantly with PD-L1 expression (P<0.05). GC can be classified into four TME types based on PD-L1 and TIL, and numbers of frameshift mutation correlate well with PD-L1 expression in MSI-H GC.

A Novel Frameshift Mutation at Codons 138/139 (HBB: c.417_418insT) on the β-Globin Gene Leads to β-Thalassemia.

PubMed

Jiang, Fan; Huang, Lv-Yin; Chen, Gui-Lan; Zhou, Jian-Ying; Xie, Xing-Mei; Li, Dong-Zhi

2017-01-01

We describe a new β-thalassemic mutation in a Chinese subject. This allele develops by insertion of one nucleotide (+T) between codons 138 and 139 in the third exon of the β-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. In the heterozygote, this allele has the phenotype of classical β-thalassemia (β-thal) minor.

Premature chain termination is a unifying mechanism for COL1A1 null alleles in osteogenesis imperfecta type I cell strains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Willing, M.C.; Deschenes, S.P.; Roberts, E.J.

Nonsense and frameshift mutations, which predict premature termination of translation, often cause a dramatic reduction in the amount of transcript from the mutant allele (nonsense-mediated mRNA decay). In some genes, these mutations also influence RNA splicing and induce skipping of the exon that contains the nonsense codon. To begin to dissect how premature termination alters the metabolism of RNA from the COL1A1 gene, we studied nonsense and frameshift mutations distributed over exons 11-49 of the gene. These mutations were originally identified in 10 unrelated families with osteogenesis imperfecta (OI) type I. We observed marked reduction in steady-state amounts of mRNAmore » from the mutant allele in both total cellular and nuclear RNA extracts of cells from affected individuals, suggesting that nonsense-mediated decay of COL1A1 RNA is a nuclear phenomenon. Position of the mutation within the gene did not influence this observation. None of the mutations induced skipping of either the exon containing the mutation or, for the frameshifts, the downstream exons with the new termination sites. Our data suggest that nonsense and frameshift mutations throughout most of the COL1A1 gene result in a null allele, which is associated with the predictable mild clinical phenotype, OI type I. 42 refs., 6 figs., 1 tab.« less

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer.

PubMed

Koi, Minoru; Tseng-Rogenski, Stephanie S; Carethers, John M

2018-01-15

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1 , resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation's effect upon the DNA MMR system.

Inflammation-associated microsatellite alterations: Mechanisms and significance in the prognosis of patients with colorectal cancer

PubMed Central

Koi, Minoru; Tseng-Rogenski, Stephanie S; Carethers, John M

2018-01-01

Microsatellite alterations within genomic DNA frameshift as a result of defective DNA mismatch repair (MMR). About 15% of sporadic colorectal cancers (CRCs) manifest hypermethylation of the DNA MMR gene MLH1, resulting in mono- and di-nucleotide frameshifts to classify it as microsatellite instability-high (MSI-H) and hypermutated, and due to frameshifts at coding microsatellites generating neo-antigens, produce a robust protective immune response that can be enhanced with immune checkpoint blockade. More commonly, approximately 50% of sporadic non-MSI-H CRCs demonstrate frameshifts at di- and tetra-nucleotide microsatellites to classify it as MSI-low/elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) as a result of functional somatic inactivation of the DNA MMR protein MSH3 via a nuclear-to-cytosolic displacement. The trigger for MSH3 displacement appears to be inflammation and/or oxidative stress, and unlike MSI-H CRC patients, patients with MSI-L/EMAST CRCs show poor prognosis. These inflammatory-associated microsatellite alterations are a consequence of the local tumor microenvironment, and in theory, if the microenvironment is manipulated to lower inflammation, the microsatellite alterations and MSH3 dysfunction should be corrected. Here we describe the mechanisms and significance of inflammatory-associated microsatellite alterations, and propose three areas to deeply explore the consequences and prevention of inflammation’s effect upon the DNA MMR system. PMID:29375743

De novo nonsense and frameshift variants of TCF20 in individuals with intellectual disability and postnatal overgrowth.

PubMed

Schäfgen, Johanna; Cremer, Kirsten; Becker, Jessica; Wieland, Thomas; Zink, Alexander M; Kim, Sarah; Windheuser, Isabelle C; Kreiß, Martina; Aretz, Stefan; Strom, Tim M; Wieczorek, Dagmar; Engels, Hartmut

2016-12-01

Recently, germline variants of the transcriptional co-regulator gene TCF20 have been implicated in the aetiology of autism spectrum disorders (ASD). However, the knowledge about the associated clinical picture remains fragmentary. In this study, two individuals with de novo TCF20 sequence variants were identified in a cohort of 313 individuals with intellectual disability of unknown aetiology, which was analysed by whole exome sequencing using a child-parent trio design. Both detected variants - one nonsense and one frameshift variant - were truncating. A comprehensive clinical characterisation of the patients yielded mild intellectual disability, postnatal tall stature and macrocephaly, obesity and muscular hypotonia as common clinical signs while ASD was only present in one proband. The present report begins to establish the clinical picture of individuals with de novo nonsense and frameshift variants of TCF20 which includes features such as proportionate overgrowth and muscular hypotonia. Furthermore, intellectual disability/developmental delay seems to be fully penetrant amongst known individuals with de novo nonsense and frameshift variants of TCF20, whereas ASD is shown to be incompletely penetrant. The transcriptional co-regulator gene TCF20 is hereby added to the growing number of genes implicated in the aetiology of both ASD and intellectual disability. Furthermore, such de novo variants of TCF20 may represent a novel differential diagnosis in the overgrowth syndrome spectrum.

Klebsazolicin inhibits 70S ribosome by obstructing the peptide exit tunnel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Metelev, Mikhail; Osterman, Ilya A.; Ghilarov, Dmitry

Whereas screening of the small-molecule metabolites produced by most cultivatable microorganisms often results in the rediscovery of known compounds, genome-mining programs allow researchers to harness much greater chemical diversity, and result in the discovery of new molecular scaffolds. Here we report the genome-guided identification of a new antibiotic, klebsazolicin (KLB), from Klebsiella pneumoniae that inhibits the growth of sensitive cells by targeting ribosomes. A ribosomally synthesized post-translationally modified peptide (RiPP), KLB is characterized by the presence of a unique N-terminal amidine ring that is essential for its activity. Biochemical in vitro studies indicate that KLB inhibits ribosomes by interfering withmore » translation elongation. Structural analysis of the ribosome–KLB complex showed that the compound binds in the peptide exit tunnel overlapping with the binding sites of macrolides or streptogramin-B. KLB adopts a compact conformation and largely obstructs the tunnel. Engineered KLB fragments were observed to retain in vitro activity, and thus have the potential to serve as a starting point for the development of new bioactive compounds.« less

Synthesis, base pairing and structure studies of geranylated RNA.

PubMed

Wang, Rui; Vangaveti, Sweta; Ranganathan, Srivathsan V; Basanta-Sanchez, Maria; Haruehanroengra, Phensinee; Chen, Alan; Sheng, Jia

2016-07-27

Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNA(Glu) UUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon-anticodon interaction during ribosome binding. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Truncation of C-terminal 20 amino acids in PA-X contributes to adaptation of swine influenza virus in pigs

PubMed Central

Xu, Guanlong; Zhang, Xuxiao; Sun, Yipeng; Liu, Qinfang; Sun, Honglei; Xiong, Xin; Jiang, Ming; He, Qiming; Wang, Yu; Pu, Juan; Guo, Xin; Yang, Hanchun; Liu, Jinhua

2016-01-01

The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting, and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a “triple-reassortment” H1N2 SIV with truncated PA-X, and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs. PMID:26912401

New insights into the transposition mechanisms of IS6110 and its dynamic distribution between Mycobacterium tuberculosis Complex lineages

PubMed Central

Uranga, Santiago; Picó, Ana; Lampreave, Carlos; Cebollada, Alberto; Otal, Isabel

2018-01-01

The insertion Sequence IS6110, only present in the pathogens of the Mycobacterium tuberculosis Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS6110 transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS6110-RFLP and covering the MTBC, we remarked a lineage-specific content of IS6110 being higher in modern globally distributed strains. Once observed the IS6110 distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS6110 and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS6110 transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in M. bovis and M. tuberculosis during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in M. tuberculosis, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS6110 transposition is dynamically adapted to the host and to adverse growth conditions. PMID:29649213

Mathematical fundamentals for the noise immunity of the genetic code.

PubMed

Fimmel, Elena; Strüngmann, Lutz

2018-02-01

Symmetry is one of the essential and most visible patterns that can be seen in nature. Starting from the left-right symmetry of the human body, all types of symmetry can be found in crystals, plants, animals and nature as a whole. Similarly, principals of symmetry are also some of the fundamental and most useful tools in modern mathematical natural science that play a major role in theory and applications. As a consequence, it is not surprising that the desire to understand the origin of life, based on the genetic code, forces us to involve symmetry as a mathematical concept. The genetic code can be seen as a key to biological self-organisation. All living organisms have the same molecular bases - an alphabet consisting of four letters (nitrogenous bases): adenine, cytosine, guanine, and thymine. Linearly ordered sequences of these bases contain the genetic information for synthesis of proteins in all forms of life. Thus, one of the most fascinating riddles of nature is to explain why the genetic code is as it is. Genetic coding possesses noise immunity which is the fundamental feature that allows to pass on the genetic information from parents to their descendants. Hence, since the time of the discovery of the genetic code, scientists have tried to explain the noise immunity of the genetic information. In this chapter we will discuss recent results in mathematical modelling of the genetic code with respect to noise immunity, in particular error-detection and error-correction. We will focus on two central properties: Degeneracy and frameshift correction. Different amino acids are encoded by different quantities of codons and a connection between this degeneracy and the noise immunity of genetic information is a long standing hypothesis. Biological implications of the degeneracy have been intensively studied and whether the natural code is a frozen accident or a highly optimised product of evolution is still controversially discussed. Symmetries in the structure of degeneracy of the genetic code are essential and give evidence of substantial advantages of the natural code over other possible ones. In the present chapter we will present a recent approach to explain the degeneracy of the genetic code by algorithmic methods from bioinformatics, and discuss its biological consequences. The biologists recognised this problem immediately after the detection of the non-overlapping structure of the genetic code, i.e., coding sequences are to be read in a unique way determined by their reading frame. But how does the reading head of the ribosome recognises an error in the grouping of codons, caused by e.g. insertion or deletion of a base, that can be fatal during the translation process and may result in nonfunctional proteins? In this chapter we will discuss possible solutions to the frameshift problem with a focus on the theory of so-called circular codes that were discovered in large gene populations of prokaryotes and eukaryotes in the early 90s. Circular codes allow to detect a frameshift of one or two positions and recently a beautiful theory of such codes has been developed using statistics, group theory and graph theory. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel frameshift mutation of CHD7 in a Japanese patient with CHARGE syndrome

PubMed Central

Kohmoto, Tomohiro; Shono, Miki; Naruto, Takuya; Watanabe, Miki; Suga, Ken-ichi; Nakagawa, Ryuji; Kagami, Shoji; Masuda, Kiyoshi; Imoto, Issei

2016-01-01

CHARGE syndrome is a rare autosomal dominant developmental disorder involving multiple organs. CHD7 is a major causative gene of CHARGE syndrome. We performed targeted-exome sequencing using a next-generation sequencer for molecular diagnosis of a 4-month-old male patient who was clinically suspected to have CHARGE syndrome, and report a novel monoallelic mutation in CHD7, NM_017780.3(CHD7_v001):c.2966del causing a reading frameshift [p.(Cys989Serfs*3)]. PMID:27081570

A novel frameshift mutation of CHD7 in a Japanese patient with CHARGE syndrome.

PubMed

Kohmoto, Tomohiro; Shono, Miki; Naruto, Takuya; Watanabe, Miki; Suga, Ken-Ichi; Nakagawa, Ryuji; Kagami, Shoji; Masuda, Kiyoshi; Imoto, Issei

2016-01-01

CHARGE syndrome is a rare autosomal dominant developmental disorder involving multiple organs. CHD7 is a major causative gene of CHARGE syndrome. We performed targeted-exome sequencing using a next-generation sequencer for molecular diagnosis of a 4-month-old male patient who was clinically suspected to have CHARGE syndrome, and report a novel monoallelic mutation in CHD7, NM_017780.3(CHD7_v001):c.2966del causing a reading frameshift [p.(Cys989Serfs*3)].

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

Nucleolus-derived mediators in oncogenic stress response and activation of p53-dependent pathways.

PubMed

Stępiński, Dariusz

2016-08-01

Rapid growth and division of cells, including tumor ones, is correlated with intensive protein biosynthesis. The output of nucleoli, organelles where translational machineries are formed, depends on a rate of particular stages of ribosome production and on accessibility of elements crucial for their effective functioning, including substrates, enzymes as well as energy resources. Different factors that induce cellular stress also often lead to nucleolar dysfunction which results in ribosome biogenesis impairment. Such nucleolar disorders, called nucleolar or ribosomal stress, usually affect cellular functioning which in fact is a result of p53-dependent pathway activation, elicited as a response to stress. These pathways direct cells to new destinations such as cell cycle arrest, damage repair, differentiation, autophagy, programmed cell death or aging. In the case of impaired nucleolar functioning, nucleolar and ribosomal proteins mediate activation of the p53 pathways. They are also triggered as a response to oncogenic factor overexpression to protect tissues and organs against extensive proliferation of abnormal cells. Intentional impairment of any step of ribosome biosynthesis which would direct the cells to these destinations could be a strategy used in anticancer therapy. This review presents current knowledge on a nucleolus, mainly in relation to cancer biology, which is an important and extremely sensitive element of the mechanism participating in cellular stress reaction mediating activation of the p53 pathways in order to counteract stress effects, especially cancer development.

UV-induced reversion of his4 frameshift mutations in rad6, rev1, and rev3 mutants of yeast.

PubMed

Lawrence, C W; O'Brien, T; Bond, J

1984-01-01

The UV-induced reversion of two his4 frameshift alleles was much reduced in rad6 mutants of Saccharomyces cerevisiae, an observation that is consistent with the hypothesis that RAD6 function is required for the induction of all types of genetic alteration in misrepair mutagenesis. The reversion of these his4 alleles, together with two others of the same type, was also reduced in rev1 and rev3 mutant strains; in these, however, the extent of the reduction varied considerably with test allele used, in a manner analogous to the results in these strains for base repair substitution test alleles. The general features of UV-induced frameshift and substitution mutagenesis therefore appear quite similar, indicating that they may depend on related processes. If this conclusion is correct, greater attention must be given to integrating models which account for the production of nucleotide additions and deletions into those concerning misrepair mutagenesis.

The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome.

PubMed

Hountondji, Codjo; Bulygin, Konstantin; Créchet, Jean-Bernard; Woisard, Anne; Tuffery, Pierre; Nakayama, Jun-Ichi; Frolova, Ludmila; Nierhaus, Knud H; Karpova, Galina; Baouz, Soria

2014-01-01

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2',3'-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2'- and 3'-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process.

Structural determinants of an internal ribosome entry site that direct translational reading frame selection

PubMed Central

Ren, Qian; Au, Hilda H.T.; Wang, Qing S.; Lee, Seonghoon; Jan, Eric

2014-01-01

The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A subset of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polyprotein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demonstrates that 0 and +1 frame translation can be uncoupled. Ribonucleic acid (RNA) structural probing analyses reveal that the mutant IRESs adopt distinct conformations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ribosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate translation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome. PMID:25038250

New developmental evidence supports a homeotic frameshift of digit identity in the evolution of the bird wing

PubMed Central

2014-01-01

Background The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. Results We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Conclusion Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear. PMID:24725625

New developmental evidence supports a homeotic frameshift of digit identity in the evolution of the bird wing.

PubMed

Salinas-Saavedra, Miguel; Gonzalez-Cabrera, Cristian; Ossa-Fuentes, Luis; Botelho, Joao F; Ruiz-Flores, Macarena; Vargas, Alexander O

2014-04-12

The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.

A Frameshift Mutation in the Cubilin Gene (CUBN) in Border Collies with Imerslund-Gräsbeck Syndrome (Selective Cobalamin Malabsorption)

PubMed Central

Owczarek-Lipska, Marta; Jagannathan, Vidhya; Drögemüller, Cord; Lutz, Sabina; Glanemann, Barbara

2013-01-01

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies. PMID:23613799

An MSI tumor specific frameshift mutation in a coding microsatellite of MSH3 encodes for HLA-A0201-restricted CD8+ cytotoxic T cell epitopes.

PubMed

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI(+) colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. FSP-specific CD8(+) T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI(+) colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were (386)-FLLALWECSL (FSP18) and (387)-LLALWECSL (FSP19) as well as (403)-IVSRTLLLV (FSP23) and (402)-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI(+)-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or--even more important--preventive purposes.

A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma

PubMed Central

Liu, Chunqiao; Widen, Sonya A.; Williamson, Kathleen A.; Ratnapriya, Rinki; Gerth-Kahlert, Christina; Rainger, Joe; Alur, Ramakrishna P.; Strachan, Erin; Manjunath, Souparnika H.; Balakrishnan, Archana; Floyd, James A.; Li, Tiansen; Waskiewicz, Andrew; Brooks, Brian P.; Lehmann, Ordan J.; FitzPatrick, David R.; Swaroop, Anand

2016-01-01

Ocular coloboma is a common eye malformation resulting from incomplete fusion of the optic fissure during development. Coloboma is often associated with microphthalmia and/or contralateral anophthalmia. Coloboma shows extensive locus heterogeneity associated with causative mutations identified in genes encoding developmental transcription factors or components of signaling pathways. We report an ultra-rare, heterozygous frameshift mutation in FZD5 (p.Ala219Glufs*49) that was identified independently in two branches of a large family with autosomal dominant non-syndromic coloboma. FZD5 has a single-coding exon and consequently a transcript with this frameshift variant is not a canonical substrate for nonsense-mediated decay. FZD5 encodes a transmembrane receptor with a conserved extracellular cysteine rich domain for ligand binding. The frameshift mutation results in the production of a truncated protein, which retains the Wingless-type MMTV integration site family member-ligand-binding domain, but lacks the transmembrane domain. The truncated protein was secreted from cells, and behaved as a dominant-negative FZD5 receptor, antagonizing both canonical and non-canonical WNT signaling. Expression of the resultant mutant protein caused coloboma and microphthalmia in zebrafish, and disruption of the apical junction of the retinal neural epithelium in mouse, mimicking the phenotype of Fz5/Fz8 compound conditional knockout mutants. Our studies have revealed a conserved role of Wnt–Frizzled (FZD) signaling in ocular development and directly implicate WNT–FZD signaling both in normal closure of the human optic fissure and pathogenesis of coloboma. PMID:26908622

Somatic frameshift mutations in the Bloom syndrome BLM gene are frequent in sporadic gastric carcinomas with microsatellite mutator phenotype

PubMed Central

Calin, George; Ranzani, Guglielmina N; Amadori, Dino; Herlea, Vlad; Matei, Irina; Barbanti-Brodano, Giuseppe; Negrini, Massimo

2001-01-01

Background Genomic instability has been reported at microsatellite tracts in few coding sequences. We have shown that the Bloom syndrome BLM gene may be a target of microsatelliteinstability (MSI) in a short poly-adenine repeat located in its coding region. To further characterize the involvement of BLM in tumorigenesis, we have investigated mutations in nine genes containing coding microsatellites in microsatellite mutator phenotype (MMP) positive and negative gastric carcinomas (GCs). Methods We analyzed 50 gastric carcinomas (GCs) for mutations in the BLM poly(A) tract aswell as in the coding microsatellites of the TGFβ1-RII, IGFIIR, hMSH3, hMSH6, BAX, WRN, RECQL and CBL genes. Results BLM mutations were found in 27% of MMP+ GCs (4/15 cases) but not in any of the MMP negative GCs (0/35 cases). The frequency of mutations in the other eight coding regions microsatellite was the following: TGFβ1-RII (60 %), BAX (27%), hMSH6 (20%),hMSH3 (13%), CBL (13%), IGFIIR (7%), RECQL (0%) and WRN (0%). Mutations in BLM appear to be more frequently associated with frameshifts in BAX and in hMSH6and/or hMSH3. Tumors with BLM alterations present a higher frequency of unstable mono- and trinucleotide repeats located in coding regions as compared with mutator phenotype tumors without BLM frameshifts. Conclusions BLM frameshifts are frequent alterations in GCs specifically associated with MMP+tumors. We suggest that BLM loss of function by MSI may increase the genetic instability of a pre-existent unstable genotype in gastric tumors. PMID:11532193

Herpes Simplex Virus Type 2 Glycoprotein G-Negative Clinical Isolates Are Generated by Single Frameshift Mutations

PubMed Central

Liljeqvist, Jan-Åke; Svennerholm, Bo; Bergström, Tomas

1999-01-01

Herpes simplex virus (HSV) codes for several envelope glycoproteins, including glycoprotein G-2 (gG-2) of HSV type 2 (HSV-2), which are dispensable for replication in cell culture. However, clinical isolates which are deficient in such proteins occur rarely. We describe here five clinical HSV-2 isolates which were found to be unreactive to a panel of anti-gG-2 monoclonal antibodies and therefore considered phenotypically gG-2 negative. These isolates were further examined for expression of the secreted amino-terminal and cell-associated carboxy-terminal portions of gG-2 by immunoblotting and radioimmunoprecipitation. The gG-2 gene was completely inactivated in four isolates, with no expression of the two protein products. For one isolate a normally produced secreted portion and a truncated carboxy-terminal portion of gG-2 were detected in virus-infected cell medium. Sequencing of the complete gG-2 gene identified a single insertion or deletion of guanine or cytosine nucleotides in all five strains, resulting in a premature termination codon. The frameshift mutations were localized within runs of five or more guanine or cytosine nucleotides and were dispersed throughout the gene. For the isolate for which a partially inactivated gG-2 gene was detected, the frameshift mutation was localized upstream of but adjacent to the nucleotides coding for the transmembranous region. Thus, this study demonstrates the existence of clinical HSV-2 isolates which do not express an envelope glycoprotein and identifies the underlying molecular mechanism to be a single frameshift mutation. PMID:10559290

Ribosomal proteins: functions beyond the ribosome.

PubMed

Zhou, Xiang; Liao, Wen-Juan; Liao, Jun-Ming; Liao, Peng; Lu, Hua

2015-04-01

Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation, their ribosome-independent functions have also been greatly appreciated. Over the past decade, more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress. In addition, these ribosomal proteins are involved in various physiological and pathological processes. This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins, as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis, immune signaling, and development. We also propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

De novo frameshift mutation in fibroblast growth factor 8 in a male patient with gonadotropin deficiency.

PubMed

Suzuki, Erina; Yatsuga, Shuichi; Igarashi, Maki; Miyado, Mami; Nakabayashi, Kazuhiko; Hayashi, Keiko; Hata, Kenichirou; Umezawa, Akihiro; Yamada, Gen; Ogata, Tsutomu; Fukami, Maki

2014-01-01

Missense, nonsense, and splice mutations in the Fibroblast Growth Factor 8(FGF8) have recently been identified in patients with hypothalamo-pituitary dysfunction and craniofacial anomalies. Here, we report a male patient with a frameshift mutation in FGF8. The patient exhibited micropenis, craniofacial anomalies, and ventricular septal defect at birth. Clinical evaluation at 16 years and 8 months of age revealed delayed puberty, hyposmia, borderline mental retardation, and mild hearing difficulty. Endocrine findings included gonadotropin deficiency and primary hypothyroidism. Molecular analysis identified a de novo heterozygous p.S192fsX204 mutation in the last exon of FGF8. RT-PCR analysis of normal human tissues detected FGF8 expression in the genital skin, and whole-mount in situ hybridization analysis of mouse embryos revealed Fgf8 expression in the anlage of the penis. The results indicate that frameshift mutations in FGF8 account for a part of the etiology of hypothalamo-pituitary dysfunction. Micropenis in patients with FGF8 abnormalities appears to be caused by gonadotropin deficiency and defective outgrowth of the anlage of the penis.

Two new mutations in the 3' coding region of the glycogen debranching enzyme in a glycogen storage disease type IIIa Ashkenazi Jewish patient.

PubMed

Parvari, R; Shen, J; Hershkovitz, E; Chen, Y T; Moses, S W

1998-04-01

Glycogen storage disease type III (GSD III) is an autosomal recessive disease caused by the deficiency of glycogen debranching enzyme (AGL). We report the finding of two new mutations in a GSD IIIa Ashkenazi Jewish patient. Both mutations are insertion of an adenine into a stretch of 8 adenines towards the 3' end of the coding region, one at position 3904 (3904insA) in exon 30, the second at position 4214 (4214insA) in exon 32. The mutations cause frameshifts and premature terminations of the glycogen debranching enzyme, the first causing a frameshift at amino acid 1304, the second causing a frameshift at amino acid 1408 of the total of 1532. These mutations demonstrate the importance of the 125 amino acids at the carboxy-terminus of the debrancher enzyme for its activity and support the suggestion that the putative glycogen binding domain is located in the carboxy-terminus of the AGL. The mutations cause distinctive single-strand conformation polymorphism (SSCP) patterns enabling easy detection.

Ribosomal vaccines. I. Immunogenicity of ribosomal fractions isolated from Salmonella typhimurium and Yersinia pestis.

PubMed

Johnson, W

1972-06-01

The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein.

Ribosomal Vaccines I. Immunogenicity of Ribosomal Fractions Isolated from Salmonella typhimurium and Yersinia pestis

PubMed Central

Johnson, William

1972-01-01

The immunogenicity of ribosomes and ribosomal subfractions isolated from Yersina pestis and Salmonella typhimurium has been studied. Ribosomes and ribosomal protein isolated from S. typhimurium protected mice against lethal challenge. Ribosomal ribonucleic acid isolated by phenol extraction failed to induce any significant level of protection in mice. None of the ribosomes or ribosomal subfractions isolated from Y. pestis were effective in inducing immunity to lethal challenge. These results suggest that the immunogen of the ribosomal vaccine is protein. Images PMID:4564407

Cryptic tRNAs in chaetognath mitochondrial genomes.

PubMed

Barthélémy, Roxane-Marie; Seligmann, Hervé

2016-06-01

The chaetognaths constitute a small and enigmatic phylum of little marine invertebrates. Both nuclear and mitochondrial genomes have numerous originalities, some phylum-specific. Until recently, their mitogenomes seemed containing only one tRNA gene (trnMet), but a recent study found in two chaetognath mitogenomes two and four tRNA genes. Moreover, apparently two conspecific mitogenomes have different tRNA gene numbers (one and two). Reanalyses by tRNAscan-SE and ARWEN softwares of the five available complete chaetognath mitogenomes suggest numerous additional tRNA genes from different types. Their total number never reaches the 22 found in most other invertebrates using that genetic code. Predicted error compensation between codon-anticodon mismatch and tRNA misacylation suggests translational activity by tRNAs predicted solely according to secondary structure for tRNAs predicted by tRNAscan-SE, not ARWEN. Numbers of predicted stop-suppressor (antitermination) tRNAs coevolve with predicted overlapping, frameshifted protein coding genes including stop codons. Sequence alignments in secondary structure prediction with non-chaetognath tRNAs suggest that the most likely functional tRNAs are in intergenic regions, as regular mt-tRNAs. Due to usually short intergenic regions, generally tRNA sequences partially overlap with flanking genes. Some tRNA pairs seem templated by sense-antisense strands. Moreover, 16S rRNA genes, but not 12S rRNAs, appear as tRNA nurseries, as previously suggested for multifunctional ribosomal-like protogenomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus

PubMed Central

Salem, Nida’ M.; Miller, W. Allen; Rowhani, Adib; Golino, Deborah A.; Moyne, Anne-Laure; Falk, Bryce W.

2015-01-01

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5′- and 3′-RACE showed the RSDaV genomic RNA to be 5,808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3′-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5′ ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5′ end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3′ cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae. PMID:18329064

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

PubMed

Salem, Nida' M; Miller, W Allen; Rowhani, Adib; Golino, Deborah A; Moyne, Anne-Laure; Falk, Bryce W

2008-06-05

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

The CCA-end of P-tRNA Contacts Both the Human RPL36AL and the A-site Bound Translation Termination Factor eRF1 at the Peptidyl Transferase Center of the Human 80S Ribosome

PubMed Central

Hountondji, Codjo; Bulygin, Konstantin; Créchet, Jean-Bernard; Woisard, Anne; Tuffery, Pierre; Nakayama, Jun-ichi; Frolova, Ludmila; Nierhaus, Knud H; Karpova, Galina; Baouz, Soria

2014-01-01

We have demonstrated previously that the E-site specific protein RPL36AL present in human ribosomes can be crosslinked with the CCA-end of a P-tRNA in situ. Here we report the following: (i) We modeled RPL36AL into the structure of the archaeal ortholog RPL44E extracted from the known X-ray structure of the 50S subunit of Haloarcula marismortui. Superimposing the obtained RPL36AL structure with that of P/E tRNA observed in eukaryotic 80S ribosomes suggested that RPL36AL might in addition to its CCA neighbourhood interact with the inner site of the tRNA elbow similar to an interaction pattern known from tRNA•synthetase pairs. (ii) Accordingly, we detected that the isolated recombinant protein RPL36AL can form a tight binary complex with deacylated tRNA, and even tRNA fragments truncated at their CCA end showed a high affinity in the nanomolar range supporting a strong interaction outside the CCA end. (iii) We constructed programmed 80S complexes containing the termination factor eRF1 (stop codon UAA at the A-site) and a 2’,3’-dialdehyde tRNA (tRNAox) analog at the P-site. Surprisingly, we observed a crosslinked ternary complex containing the tRNA, eRF1 and RPL36AL crosslinked both to the aldehyde groups of tRNAox at the 2’- and 3’-positions of the ultimate A. We also demonstrated that, upon binding to the ribosomal A-site, eRF1 induces an alternative conformation of the ribosome and/or the tRNA, leading to a novel crosslink of tRNAox to another large-subunit ribosomal protein (namely L37) rather than to RPL36AL, both ribosomal proteins being labeled in a mutually exclusive fashion. Since the human 80S ribosome in complex with P-site bound tRNAox and A-site bound eRF1 corresponds to the post-termination state of the ribosome, the results represent the first biochemical evidence for the positioning of the CCA-arm of the P-tRNA in close proximity to both RPL36AL and eRF1 at the end of the translation process. PMID:25191528

Creation of a data base for sequences of ribosomal nucleic acids and detection of conserved restriction endonucleases sites through computerized processing.

PubMed Central

Patarca, R; Dorta, B; Ramirez, J L

1982-01-01

As part of a project pertaining the organization of ribosomal genes in Kinetoplastidae, we have created a data base for published sequences of ribosomal nucleic acids, with information in Spanish. As a first step in their processing, we have written a computer program which introduces the new feature of determining the length of the fragments produced after single or multiple digestion with any of the known restriction enzymes. With this information we have detected conserved SAU 3A sites: (i) at the 5' end of the 5.8S rRNA and at the 3' end of the small subunit rRNA, both included in similar larger sequences; (ii) in the 5.8S rRNA of vertebrates (a second one), which is not present in lower eukaryotes, showing a clear evolutive divergence; and, (iii) at the 5' terminal of the small subunit rRNA, included in a larger conserved sequence. The possible biological importance of these sequences is discussed. PMID:6278402

Proteomic analysis of rodent ribosomes revealed heterogeneity including ribosomal proteins L10-like, L22-like 1, and L39-like.

PubMed

Sugihara, Yoshihiko; Honda, Hiroki; Iida, Tomoharu; Morinaga, Takuma; Hino, Shingo; Okajima, Tetsuya; Matsuda, Tsukasa; Nadano, Daita

2010-03-05

Heterogeneity of ribosome structure, due to variations in ribosomal protein composition, has been shown to be of physiological significance in plants and yeast. Mammalian genomics have demonstrated numerous genes that are paralogous to genes encoding ribosomal proteins. Although the vast majority are considered to be pseudogenes, mRNA expression of a few paralogues, such as human ribosomal protein L39-like/L39-2, has been reported. In the present study, ribosomes from the liver, mammary gland, and testis of rodents were analyzed using a combination of two-dimensional gel electrophoresis under radical-free and highly reducing conditions, and mass spectrometry. This system allowed identification of 78 ribosomal proteins and Rack1 from a single gel. The degree of heterogeneity was far less than that reported for plant and yeast ribosomes, and was in accord with published biochemical and genetic data for mammalian ribosomes. Nevertheless, an uncharacterized paralogue of ribosomal protein L22, ribosomal protein L22-like 1, was identified as a minor ribosomal component. Ribosomal proteins L10-like and L39-like, paralogues of ribosomal proteins L10 and L39, respectively, were found in ribosomes only from the testis. Reverse transcription-polymerase chain reaction yielded supportive evidence for specific expression of L10-like and L39-like in the testis. Newly synthesized L39-like is likely to be transported to the nucleolus, where ribosome biosynthesis occurs, and then incorporated into translating ribosomes in the cytoplasm. Heterogeneity of mammalian testicular ribosomes is structurally non-negligible, and may offer valuable insights into the function of the customized ribosome.

An MSI Tumor Specific Frameshift Mutation in a Coding Microsatellite of MSH3 Encodes for HLA-A0201-Restricted CD8+ Cytotoxic T Cell Epitopes

PubMed Central

Garbe, Yvette; Maletzki, Claudia; Linnebacher, Michael

2011-01-01

Background Microsatellite instability (MSI) resulting from inactivation of the DNA mismatch repair system (MMR) characterizes a highly immunological subtype of colorectal carcinomas. Those tumors express multiple frameshift-mutated proteins which present a unique pool of tumor-specific antigens. The DNA MMR protein MSH3 is frequently mutated in MSI+ colorectal tumors, thus making it an attractive candidate for T cell-based immunotherapies. Methodology/Principal Findings FSP-specific CD8+ T cells were generated from a healthy donor using reverse immunology. Those T cells specifically recognized T2 cells sensitized with the respective peptides. Specific recognition and killing of MSI+ colorectal carcinoma cells harbouring the mutated reading frame was observed. The results obtained with T cell bulk cultures could be reproduced with T cell clones obtained from the same cultures. Blocking experiments (using antibodies and cold target inhibition) confirmed peptide as well as HLA-A0201-specificity. Conclusions We identified two novel HLA-A0201-restricted cytotoxic T cell epitopes derived from a (-1) frameshift mutation of a coding A(8) tract within the MSH3 gene. These were 386-FLLALWECSL (FSP18) and 387-LLALWECSL (FSP19) as well as 403-IVSRTLLLV (FSP23) and 402-LIVSRTLLLV (FSP31), respectively. These results suggest that MSH3(-1) represents another promising MSI+-induced target antigen. By identifying two distinct epitopes within MSH3(-1), the sustained immunogenicity of the frameshift mutated sequence was confirmed. Our data therefore encourage further exploitation of MSH3 as a piece for peptide-based vaccines either for therapeutic or –even more important– preventive purposes. PMID:22110587

Kinetic modeling predicts a stimulatory role for ribosome collisions at elongation stall sites in bacteria

PubMed Central

Ferrin, Michael A; Subramaniam, Arvind R

2017-01-01

Ribosome stalling on mRNAs can decrease protein expression. To decipher ribosome kinetics at stall sites, we induced ribosome stalling at specific codons by starving the bacterium Escherichia coli for the cognate amino acid. We measured protein synthesis rates from a reporter library of over 100 variants that encoded systematic perturbations of translation initiation rate, the number of stall sites, and the distance between stall sites. Our measurements are quantitatively inconsistent with two widely-used kinetic models for stalled ribosomes: ribosome traffic jams that block initiation, and abortive (premature) termination of stalled ribosomes. Rather, our measurements support a model in which collision with a trailing ribosome causes abortive termination of the stalled ribosome. In our computational analysis, ribosome collisions selectively stimulate abortive termination without fine-tuning of kinetic rate parameters at ribosome stall sites. We propose that ribosome collisions serve as a robust timer for translational quality control pathways to recognize stalled ribosomes. DOI: http://dx.doi.org/10.7554/eLife.23629.001 PMID:28498106

Novel bandlike signal abnormality suggestive of heterotopia in patient with a KCNQ1 frameshift mutation.

PubMed

Sabharwal, Priyanka; Devinsky, Orrin; M Shepherd, Timothy

2017-12-01

Malformations of cortical development are associated with epilepsy and cognitive dysfunction, and can occur in patients with SCN1A ion channel mutations. We report a novel and subtle bandlike subcortical heterotopia on integrated positron emission tomography-magnetic resonance imaging ( PET-MRI) in a patient with treatment-resistant epilepsy due to a de novo KCNQ1 frameshift mutation. Our case highlights the potential for other channel mutations to cause both epilepsy and cortical malformations. Further scrutiny of high contrast resolution MRI studies is warranted for patients with KCNQ1 and other epilepsy genes to further define their extended phenotype.

Atrial Natriuretic Peptide Frameshift Mutation in Familial Atrial Fibrillation

PubMed Central

Hodgson-Zingman, Denice M.; Karst, Margaret L.; Zingman, Leonid V.; Heublein, Denise M.; Darbar, Dawood; Herron, Kathleen J.; Ballew, Jeffrey D.; de Andrade, Mariza; Burnett, John C.; Olson, Timothy M.

2008-01-01

Summary Atrial fibrillation is a common arrhythmia that is hereditary in a small subgroup of patients. In a family with 11 clinically affected members, we mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide–cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability. PMID:18614783

Embryonal rhabdomyosarcoma in a patient with a heterozygous frameshift variant in the DICER1 gene and additional manifestations of the DICER1 syndrome.

PubMed

Fremerey, Julia; Balzer, Stefan; Brozou, Triantafyllia; Schaper, Joerg; Borkhardt, Arndt; Kuhlen, Michaela

2017-07-01

Germline mutations in the DICER1 gene are associated with an inherited cancer predisposition syndrome also known as the DICER1-syndrome, which is implicated in a broad range of tumors including pleuropulmonary blastoma, ovarian Sertoli-Leydig cell tumors, ciliary body medulloepithelioma (CBME), pituitary blastoma, embryonal rhabdomyosarcoma (eRMS), anaplastic renal sarcoma as well as ocular, sinonasal tumors ovarian sex-cord tumors, thyroid neoplasia and cystic nephroma. This study describes a novel, heterozygous frameshift DICER1 mutation in a patient, who is affected by different tumors of the DICER1-syndrome, including eRMS, CBME and suspected pleuropulmonary blastoma type I. By whole-exome sequencing of germline material using peripheral blood-derived DNA, we identified a single base pair duplication within the DICER1 gene (c.3405 dupA) that leads to a frameshift and results in a premature stop in exon 21 (p.Gly1136Arg). The metachronous occurrence of two unrelated tumor entities (eRMS and CBME) in a very young child within a short timeframe should have raised the suspicion of an underlying cancer susceptibility syndrome and should be prompt tested for DICER1.

Novel XLRS1 gene mutations cause X-linked juvenile retinoschisis in Chinese families.

PubMed

Ma, Xiang; Li, Xiaoxin; Wang, Lihua

2008-01-01

To investigate various XLRS1 (RS1) gene mutations in Chinese families with X-linked juvenile retinoschisis (XLRS or RS). Genomic DNA was isolated from leukocytes of 29 male patients with X-linked juvenile retinoschisis, 38 female carriers, and 100 normal controls. All 6 exons of the RS1 gene were amplified by polymerase chain reaction, and the RS1 gene mutations were determined by direct sequencing. Eleven different RS1 mutations in 12 families were identified in the 29 male patients. The mutations comprised eight missense, two frameshift, and one splice donor site mutation. Four of these mutations, one frameshift mutation (26 del T) in exon 1, one frameshift mutation (488 del G) in exon 5, Asp145His and Arg156Gly in exon 5, have not been previously described. One novel non-disease-related polymorphism, 576C to T (Pro192Pro) in exon 6, was also found. Six recurrent mutations, Ser73Pro and Arg102Gln mutations in exon 4 and Arg200Cys, Arg209His, Arg213Gln, and Cys223Arg mutations in exon 6, were also identified in this study. RS1 gene mutations caused X-linked juvenile retinoschisis in these Chinese families.

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

Distribution and diversity of ribosome binding sites in prokaryotic genomes.

PubMed

Omotajo, Damilola; Tate, Travis; Cho, Hyuk; Choudhary, Madhusudan

2015-08-14

Prokaryotic translation initiation involves the proper docking, anchoring, and accommodation of mRNA to the 30S ribosomal subunit. Three initiation factors (IF1, IF2, and IF3) and some ribosomal proteins mediate the assembly and activation of the translation initiation complex. Although the interaction between Shine-Dalgarno (SD) sequence and its complementary sequence in the 16S rRNA is important in initiation, some genes lacking an SD ribosome binding site (RBS) are still well expressed. The objective of this study is to examine the pattern of distribution and diversity of RBS in fully sequenced bacterial genomes. The following three hypotheses were tested: SD motifs are prevalent in bacterial genomes; all previously identified SD motifs are uniformly distributed across prokaryotes; and genes with specific cluster of orthologous gene (COG) functions differ in their use of SD motifs. Data for 2,458 bacterial genomes, previously generated by Prodigal (PROkaryotic DYnamic programming Gene-finding ALgorithm) and currently available at the National Center for Biotechnology Information (NCBI), were analyzed. Of the total genes examined, ~77.0% use an SD RBS, while ~23.0% have no RBS. Majority of the genes with the most common SD motifs are distributed in a manner that is representative of their abundance for each COG functional category, while motifs 13 (5'-GGA-3'/5'-GAG-3'/5'-AGG-3') and 27 (5'-AGGAGG-3') appear to be predominantly used by genes for information storage and processing, and translation and ribosome biogenesis, respectively. These findings suggest that an SD sequence is not obligatory for translation initiation; instead, other signals, such as the RBS spacer, may have an overarching influence on translation of mRNAs. Subsequent analyses of the 5' secondary structure of these mRNAs may provide further insight into the translation initiation mechanism.

High-resolution structure of the Escherichia coli ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

High-resolution structure of the Escherichia coli ribosome

DOE PAGES

Noeske, Jonas; Wasserman, Michael R.; Terry, Daniel S.; ...

2015-03-16

Protein synthesis by the ribosome is highly dependent on the ionic conditions in the cellular environment, but the roles of ribosome solvation remain poorly understood. Moreover, the function of modifications to ribosomal RNA and ribosomal proteins are unclear. Here we present the structure of the Escherichia coli 70S ribosome to 2.4 Å resolution. The structure reveals details of the ribosomal subunit interface that are conserved in all domains of life, and suggest how solvation contributes to ribosome integrity and function. The structure also suggests how the conformation of ribosomal protein uS12 likely impacts its contribution to messenger RNA decoding. Inmore » conclusion, this structure helps to explain the phylogenetic conservation of key elements of the ribosome, including posttranscriptional and posttranslational modifications and should serve as a basis for future antibiotic development.« less

Getting ready to translate: cytoplasmic maturation of eukaryotic ribosomes.

PubMed

Panse, Vikram Govind

2011-01-01

The ribosome is the 'universal ribozyme' that is responsible for the final step of decoding genetic information into proteins. While the function of the ribosome is being elucidated at the atomic level, in comparison, little is known regarding its assembly in vivo and intracellular transport. In contrast to prokaryotic ribosomes, the construction of eukaryotic ribosomes, which begins in the nucleolus, requires >200 evolutionary conserved non-ribosomal trans-acting factors, which transiently associate with pre-ribosomal subunits at distinct assembly stages and perform specific maturation steps. Notably, pre-ribosomal subunits are transported to the cytoplasm in a functionally inactive state where they undergo maturation prior to entering translation. In this review, I will summarize our current knowledge of the eukaryotic ribosome assembly pathway with emphasis on cytoplasmic maturation events that render pre-ribosomal subunits translation competent.

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

PubMed Central

Konikkat, Salini; Woolford, John L.

2017-01-01

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ~76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly. PMID:28062837

Cucumber as a Model for Organellar Genetics

USDA-ARS?s Scientific Manuscript database

Mitochondria are found in the cells of all eukaryotes, are imperative for energy production, and play important roles in programmed cell death, ageing, and disease development. Mitochondria possess their own DNA and encode for approximately 20 proteins, as well as their own ribosomal and transfer R...

Detection and Quantification of Ribosome Inhibition by Aminoglycoside Antibiotics in Living Bacteria Using an Orthogonal Ribosome-Controlled Fluorescent Reporter.

PubMed

Huang, Shijie; Zhu, Xuechen; Melançon, Charles E

2016-01-15

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome-controlled green fluorescent protein (GFP) reporter that produce fluorescent signal when the orthogonal ribosome is inhibited. As a proof of concept, we demonstrate that these strains, when coexpressing homogeneous populations of aminoglycoside resistant ribosomes, act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse aminoglycoside antibiotics. We suggest that this strategy can be extended to quantifying ribosome inhibition by other drug classes.

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

PubMed Central

Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

2016-01-01

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

Entrainment to periodic initiation and transition rates in a computational model for gene translation.

PubMed

Margaliot, Michael; Sontag, Eduardo D; Tuller, Tamir

2014-01-01

Periodic oscillations play an important role in many biomedical systems. Proper functioning of biological systems that respond to periodic signals requires the ability to synchronize with the periodic excitation. For example, the sleep/wake cycle is a manifestation of an internal timing system that synchronizes to the solar day. In the terminology of systems theory, the biological system must entrain or phase-lock to the periodic excitation. Entrainment is also important in synthetic biology. For example, connecting several artificial biological systems that entrain to a common clock may lead to a well-functioning modular system. The cell-cycle is a periodic program that regulates DNA synthesis and cell division. Recent biological studies suggest that cell-cycle related genes entrain to this periodic program at the gene translation level, leading to periodically-varying protein levels of these genes. The ribosome flow model (RFM) is a deterministic model obtained via a mean-field approximation of a stochastic model from statistical physics that has been used to model numerous processes including ribosome flow along the mRNA. Here we analyze the RFM under the assumption that the initiation and/or transition rates vary periodically with a common period T. We show that the ribosome distribution profile in the RFM entrains to this periodic excitation. In particular, the protein synthesis pattern converges to a unique periodic solution with period T. To the best of our knowledge, this is the first proof of entrainment in a mathematical model for translation that encapsulates aspects such as initiation and termination rates, ribosomal movement and interactions, and non-homogeneous elongation speeds along the mRNA. Our results support the conjecture that periodic oscillations in tRNA levels and other factors related to the translation process can induce periodic oscillations in protein levels, and may suggest a new approach for re-engineering genetic systems to obtain a desired, periodic, protein synthesis rate.

High performance computing in biology: multimillion atom simulations of nanoscale systems

PubMed Central

Sanbonmatsu, K. Y.; Tung, C.-S.

2007-01-01

Computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). Of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. Breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulations of large biomolecular complexes. Here, we report simulation results for the ribosome, using approximately 2.64 million atoms, the largest all-atom biomolecular simulation published to date. Several other nanoscale systems with different numbers of atoms were studied to measure the performance of the NAMD molecular dynamics simulation program on the Los Alamos National Laboratory Q Machine. We demonstrate that multimillion atom systems represent a 'sweet spot' for the NAMD code on large supercomputers. NAMD displays an unprecedented 85% parallel scaling efficiency for the ribosome system on 1024 CPUs. We also review recent targeted molecular dynamics simulations of the ribosome that prove useful for studying conformational changes of this large biomolecular complex in atomic detail. PMID:17187988

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

PubMed Central

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

2017-01-01

Abstract 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. PMID:27986857

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

PubMed

Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Mechanisms of In Vivo Ribosome Maintenance Change in Response to Nutrient Signals*

PubMed Central

Mathis, Andrew D.; Naylor, Bradley C.; Carson, Richard H.; Evans, Eric; Harwell, Justin; Knecht, Jared; Hexem, Eric; Peelor, Fredrick F.; Miller, Benjamin F.; Hamilton, Karyn L.; Transtrum, Mark K.; Bikman, Benjamin T.; Price, John C.

2017-01-01

Control of protein homeostasis is fundamental to the health and longevity of all organisms. Because the rate of protein synthesis by ribosomes is a central control point in this process, regulation, and maintenance of ribosome function could have amplified importance in the overall regulatory circuit. Indeed, ribosomal defects are commonly associated with loss of protein homeostasis, aging, and disease (1–4), whereas improved protein homeostasis, implying optimal ribosomal function, is associated with disease resistance and increased lifespan (5–7). To maintain a high-quality ribosome population within the cell, dysfunctional ribosomes are targeted for autophagic degradation. It is not known if complete degradation is the only mechanism for eukaryotic ribosome maintenance or if they might also be repaired by replacement of defective components. We used stable-isotope feeding and protein mass spectrometry to measure the kinetics of turnover of ribosomal RNA (rRNA) and 71 ribosomal proteins (r-proteins) in mice. The results indicate that exchange of individual proteins and whole ribosome degradation both contribute to ribosome maintenance in vivo. In general, peripheral r-proteins and those with more direct roles in peptide-bond formation are replaced multiple times during the lifespan of the assembled structure, presumably by exchange with a free cytoplasmic pool, whereas the majority of r-proteins are stably incorporated for the lifetime of the ribosome. Dietary signals impact the rates of both new ribosome assembly and component exchange. Signal-specific modulation of ribosomal repair and degradation could provide a mechanistic link in the frequently observed associations among diminished rates of protein synthesis, increased autophagy, and greater longevity (5, 6, 8, 9). PMID:27932527

A novel two-nucleotide deletion in the ATP7A gene associated with delayed infantile onset of Menkes disease.

PubMed

Wada, Takahito; Haddad, Marie Reine; Yi, Ling; Murakami, Tomomi; Sasaki, Akiko; Shimbo, Hiroko; Kodama, Hiroko; Osaka, Hitoshi; Kaler, Stephen G

2014-04-01

Determining the relationship between clinical phenotype and genotype in genetic diseases is important in clinical practice. In general, frameshift mutations are expected to produce premature termination codons, leading to production of mutant transcripts destined for degradation by nonsense-mediated decay. In X-linked recessive diseases, male patients with frameshift mutations typically have a severe or even lethal phenotype. We report a case of a 17-month-old boy with Menkes disease (NIM #309400), an X-linked recessive copper metabolism disorder caused by mutations in the ATP7A copper transporter gene. He exhibited an unexpectedly late onset and experienced milder symptoms. His genomic DNA showed a de novo two-nucleotide deletion in exon 4 of ATP7A, predicting a translational frameshift and premature stop codon, and a classic severe phenotype. Characterization of his ATP7A mRNA showed no abnormal splicing. We speculate that translation reinitiation could occur downstream to the premature termination codon and produce a partially functional ATP7A protein. Study of the child's fibroblasts found no evidence of translation reinitiation; however, the possibility remains that this phenomenon occurred in neural tissues and influenced the clinical phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

Genetic Characterization of the SufJ Frameshift Suppressor in SALMONELLA TYPHIMURIUM

PubMed Central

Bossi, Lionello; Kohno, Tadahiko; Roth, John R.

1983-01-01

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.—The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.—Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA). PMID:6188650

Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer.

PubMed

Saeterdal, I; Bjørheim, J; Lislerud, K; Gjertsen, M K; Bukholm, I K; Olsen, O C; Nesland, J M; Eriksen, J A; Møller, M; Lindblom, A; Gaudernack, G

2001-11-06

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch-repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGF beta RII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGF beta RII, in TIL and peripheral blood lymphocytes from patients with MSI(+) tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI(+) colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.

Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer

PubMed Central

Sæterdal, Ingvil; Bjørheim, Jens; Lislerud, Kari; Gjertsen, Marianne K.; Bukholm, Ida K.; Olsen, Ole Christian; Nesland, Jahn M.; Eriksen, Jon Amund; Møller, Mona; Lindblom, Annika; Gaudernack, Gustav

2001-01-01

The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch–repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGFβRII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGFβRII, in TIL and peripheral blood lymphocytes from patients with MSI+ tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI+ colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC. PMID:11687624

The Saccharomyces cerevisiae MLH3 gene functions in MSH3-dependent suppression of frameshift mutations.

PubMed

Flores-Rozas, H; Kolodner, R D

1998-10-13

The Saccharomyces cerevisiae genome encodes four MutL homologs. Of these, MLH1 and PMS1 are known to act in the MSH2-dependent pathway that repairs DNA mismatches. We have investigated the role of MLH3 in mismatch repair. Mutations in MLH3 increased the rate of reversion of the hom3-10 allele by increasing the rate of deletion of a single T in a run of 7 Ts. Combination of mutations in MLH3 and MSH6 caused a synergistic increase in the hom3-10 reversion rate, whereas the hom3-10 reversion rate in an mlh3 msh3 double mutant was the same as in the respective single mutants. Similar results were observed when the accumulation of mutations at frameshift hot spots in the LYS2 gene was analyzed, although mutation of MLH3 did not cause the same extent of affect at every LYS2 frameshift hot spot. MLH3 interacted with MLH1 in a two-hybrid system. These data are consistent with the idea that a proportion of the repair of specific insertion/deletion mispairs by the MSH3-dependent mismatch repair pathway uses a heterodimeric MLH1-MLH3 complex in place of the MLH1-PMS1 complex.

A novel frameshift variant in the CADASIL gene NOTCH3: pathogenic or not?

PubMed

Schubert, V; Bender, B; Kinzel, M; Peters, N; Freilinger, T

2018-06-01

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) represents the most common monogenic cause of adult-onset ischemic stroke and vascular dementia. It is caused by heterozygous missense mutations in the NOTCH3 gene, encoding a transmembrane receptor protein on vascular smooth muscle cells. Classical CADASIL mutations affect conserved cysteine residues of the Notch3 protein. By contrast, the role of non-canonical genetic variation in NOTCH3, in particular of variants causing a hypomorphic Notch3 protein, is subject to an ongoing scientific debate. In this context, we here report a novel NOTCH3 frameshift variant in exon 18 (NM_000435.2: c.2853_2857delTCCCG), causing a frameshift and introducing a premature stop codon, which was detected in a 43-year-old woman and her father. Both carriers of the variant were carefully evaluated, including serial follow-up in the index. Neither clinical nor imaging features provided convincing evidence for a classical CADASIL phenotype, thus reinforcing the concept of hypomorphic NOTCH3 variants most likely not being causative for CADASIL. Our finding, which is discussed in the light of the published literature, has practical implications for interpreting results of NOTCH3 molecular genetic testing as well as patient counseling.

Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy.

PubMed

Fischer, Niels; Konevega, Andrey L; Wintermeyer, Wolfgang; Rodnina, Marina V; Stark, Holger

2010-07-15

The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.

Superresolution Imaging of Ribosomes and RNA Polymerase in Live Escherichia coli Cells

PubMed Central

Bakshi, Somenath; Siryaporn, Albert; Goulian, Mark; Weisshaar, James C.

2012-01-01

Summary Quantitative spatial distributions of ribosomes (S2-YFP) and RNA polymerase (β′-yGFP) in live E. coli are measured by superresolution fluorescence microscopy. In moderate growth conditions, Nucleoid-ribosome segregation is strong, and RNAP localizes to the nucleoid lobes. The mean copy numbers per cell are 4600 RNAPs and 55,000 ribosomes. Only 10–15% of the ribosomes lie within the densest part of the nucleoid lobes, and at most 4% of the RNAPs lie in the two ribosome-rich endcaps. The predominant observed diffusion coefficient of ribosomes is Dribo = 0.04 μm2/s, attributed to free mRNA being translated by one or more 70S ribosomes. We find no clear evidence of sub-diffusion, as would arise from tethering of ribosomes. The degree of DNA-ribosome segregation strongly suggests that in E. coli most translation occurs on free mRNA transcripts that have diffused into the ribosome-rich regions. Both RNAP and ribosome radial distributions extend to the cytoplasmic membrane, consistent with the transertion hypothesis. However, few if any RNAP copies lie near the membrane of the endcaps. This suggests that if transertion occurs, it exerts a direct radially expanding force on the nucleoid, but not a direct axially expanding force. PMID:22624875

Fitness advantages conferred by the L20-interacting RNA cis-regulator of ribosomal protein synthesis in Bacillus subtilis.

PubMed

Babina, Arianne M; Parker, Darren J; Li, Gene-Wei; Meyer, Michelle M

2018-06-20

In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5'-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in non-coding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or mis-assembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Germ-line mutations in the neurofibromatosis 2 gene: Correlations with disease severity and retinal abnormalities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parry, D.M.; Kaiser-Kupfer, M.; Eldridge, R.

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsensemore » mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P {le} .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study. 58 refs., 2 tabs.« less

Ghrelin gene: identification of missense variants and a frameshift mutation in extremely obese children and adolescents and healthy normal weight students.

PubMed

Hinney, Anke; Hoch, Anne; Geller, Frank; Schäfer, Helmut; Siegfried, Wolfgang; Goldschmidt, Hanspeter; Remschmidt, Helmut; Hebebrand, Johannes

2002-06-01

Ghrelin induces obesity via central and peripheral mechanisms. Administration of ghrelin leads to increased food intake and decreased fat utilisation in rodents. Ghrelin levels are decreased in obese individuals. Recently, a polymorphism (Arg-51-Gln) within the ghrelin gene (GHRL) was described to be associated with obesity. We screened the GHRL coding region in 215 extremely obese German Children and adolescents (study group 1) and 93 normal weight students (study group 2) by single strand conformation polymorphism analysis (SSCP). We found the two previously described single nucleotide polymorphisms (SNP: Arg-51-Gln and Leu-72-Met) in similar frequencies in study groups 1 and 2 (allele frequencies were: 0.019 and 0.016 for the 51-Gln allele and 0.091 and 0.086 for the 72-Met allele, respectively). Hence, we could not confirm the previous finding. Additionally, two novel variants were identified within the coding region: (1) We detected one healthy normal weight individual with a frameshift mutation (2bp deletion at codon 34). This frameshift mutation affects the coding region of the mature ghrelin. Hence, it is highly likely that the normal weight student is haplo-insufficient for ghrelin. (2) An A to T transversion leads to an amino acid exchange from Gln to Leu at amino acid position 90. The frequency of the 90-Leu allele was significantly higher in the extremely obese children and adolescents (0.063) than in the normal weight students (0.016; nominal p = 0.011). Additionally, we genotyped 134 underweight students and 44 normal weight adults for this SNP. Genotype frequencies were similar in extremely obese children and adolescents, underweight students and normal weight adults (p > 0.8). In conclusion, we identified four sequence variants in the coding region of the ghrelin gene in individuals belonging to different weight extremes. A frameshift mutation was detected in a normal weight individual. None of the variants seem to influence weight regulation.

Loss of MSH3 protein expression is frequent in MLH1-deficient colorectal cancer and is associated with disease progression.

PubMed

Plaschke, Jens; Krüger, Stefan; Jeske, Birgit; Theissig, Franz; Kreuz, Friedmar R; Pistorius, Steffen; Saeger, Hans D; Iaccarino, Ingram; Marra, Giancarlo; Schackert, Hans K

2004-02-01

Mononucleotide repeat sequences are particularly prone to frameshift mutations in tumors with biallelic inactivation of the mismatch repair (MMR) genes MLH1 or MSH2. In these tumors, several genes harboring mononucleotide repeats in their coding region have been proposed as targets involved in tumor progression, among which are also the MMR genes MSH3 and MSH6. We have analyzed the expression of the MSH3 and MSH6 proteins by immunohistochemistry in 31 colorectal carcinomas in which MLH1 was inactivated. Loss of MSH3 expression was identified in 15 tumors (48.5%), whereas all tumors expressed MSH6. Frameshift mutations at coding microsatellites were more frequent in MSH3 (16 of 31) than in MSH6 (3 of 31; Fisher's exact test, P < 0.001). Frameshift mutations and allelic losses of MSH3 were more frequent in MSH3-negative tumors compared with those with normal expression (22 mutations in 30 alleles versus 8 mutations in 28 alleles; chi(2), P = 0.001). Biallelic inactivation was evident or inferred for 60% of MSH3-negative tumors but none of the tumors with normal MSH3 expression. In contrast, we did not identify frameshift mutations in the (A)8 tract of MSH3 in a control group of 18 colorectal carcinomas in which the MMR deficiency was based on the inactivation of MSH2. As it has been suggested that mutations of MSH3 might play a role in tumor progression, we studied the association between MSH3 expression and disease stage assessed by lymph node and distant metastases status. Dukes stages C and D were more frequent in primary tumors with loss of MSH3 expression (9 of 13), compared with tumors with retained expression (1 of 14; Fisher's exact test, P = 0.001), suggesting that MSH3 abrogation may be a predictor of metastatic disease or even favor tumor cell spread in MLH1-deficient colorectal cancers.

Mutation analysis of BRCA1/2 mutations with special reference to polymorphic SNPs in Indian breast cancer patients.

PubMed

Shah, Nidhi D; Shah, Parth S; Panchal, Yash Y; Katudia, Kalpesh H; Khatri, Nikunj B; Ray, Hari Shankar P; Bhatiya, Upti R; Shah, Sandip C; Shah, Bhavini S; Rao, Mandava V

2018-01-01

Germline mutations BRCA1 and BRCA2 contribute almost equally in the causation of breast cancer (BC). The type of mutations in the Indian population that cause this condition is largely unknown. In this cohort, 79 randomized BC patients were screened for various types of BRCA1 and BRCA2 mutations including frameshift, nonsense, missense, in-frame and splice site types. The purified extracted DNA of each referral patient was subjected to Sanger gene sequencing using Codon Code Analyzer and Mutation Surveyor and next-generation sequencing (NGS) methods with Ion torrent software, after appropriate care. The data revealed that 35 cases were positive for BRCA1 or BRCA2 (35/79: 44.3%). BRCA2 mutations were higher (52.4%) than BRCA1 mutations (47.6%). Five novel mutations detected in this study were p.pro163 frameshift, p.asn997 frameshift, p.ser148 frameshift and two splice site single-nucleotide polymorphisms (SNPs). Additionally, four nonsense and one in-frame deletion were identified, which all seemed to be pathogenic. Polymorphic SNPs contributed the highest percentage of mutations (72/82: 87.8%) and contributed to pathogenic, likely pathogenic, likely benign, benign and variant of unknown significance (VUS). Young age groups (20-60 years) had a high frequency of germline mutations (62/82;75.6%) in the Indian population. This study suggested that polymorphic SNPs contributed a high percentage of mutations along with five novel types. Younger age groups are prone to having BC with a higher mutational rate. Furthermore, the SNPs detected in exons 10, 11 and 16 of BRCA1 and BRCA2 were higher than those in other exons 2, 3 and 9 polymorphic sites in two germline genes. These may be contributory for BC although missense types are known to be susceptible for cancer depending on the type of amino acid replaced in the protein and associated with pathologic events. Accordingly, appropriate counseling and treatment may be suggested.

Naturally Occurring Frameshift Mutations in the tvb Receptor Gene Are Responsible for Decreased Susceptibility of Chicken to Infection with Avian Leukosis Virus Subgroups B, D, and E.

PubMed

Li, Xinjian; Chen, Weiguo; Zhang, Huanmin; Li, Aijun; Shu, Dingming; Li, Hongxing; Dai, Zhenkai; Yan, Yiming; Zhang, Xinheng; Lin, Wencheng; Ma, Jingyun; Xie, Qingmei

2018-04-15

The group of highly related avian leukosis viruses (ALVs) in chickens are thought to have evolved from a common retroviral ancestor into six subgroups, A to E and J. These ALV subgroups use diverse cellular proteins encoded by four genetic loci in chickens as receptors to gain entry into host cells. Hosts exposed to ALVs might be under selective pressure to develop resistance to ALV infection. Indeed, resistance alleles have previously been identified in all four receptor loci in chickens. The tvb gene encodes a receptor, which determines the susceptibility of host cells to ALV subgroup B (ALV-B), ALV-D, and ALV-E. Here we describe the identification of two novel alleles of the tvb receptor gene, which possess independent insertions each within exon 4. The insertions resulted in frameshift mutations that reveal a premature stop codon that causes nonsense-mediated decay of the mutant mRNA and the production of truncated Tvb protein. As a result, we observed that the frameshift mutations in the tvb gene significantly lower the binding affinity of the truncated Tvb receptors for the ALV-B, ALV-D, and ALV-E envelope glycoproteins and significantly reduce susceptibility to infection by ALV-B, ALV-D and ALV-E in vitro and in vivo Taken together, these findings suggest that frameshift mutation can be a molecular mechanism of reducing susceptibility to ALV and enhance our understanding of virus-host coevolution. IMPORTANCE Avian leukosis virus (ALV) once caused devastating economic loss to the U.S. poultry industry prior the current eradication schemes in place, and it continues to cause severe calamity to the poultry industry in China and Southeast Asia, where deployment of a complete eradication scheme remains a challenge. The tvb gene encodes the cellular receptor necessary for subgroup B, D, and E ALV infection. Two tvb allelic variants that resulted from frameshift mutations have been identified in this study, which have been shown to have significantly reduced functionality in mediating subgroup B, D, and E ALV infection. Unlike the control of herpesvirus-induced diseases by vaccination, the control of avian leukosis in chickens has relied totally on virus eradication measures and host genetic resistance. This finding enriches the allelic pool of the tvb gene and expands the potential for genetic improvement of ALV resistance in varied chicken populations by selection. Copyright © 2018 American Society for Microbiology.

The Influence of Local DNA Sequence and DNA Repair Background on the Mutational Specificity of 1-Nitroso-8-Nitropyrene in Escherichia Coli: Inferences for Mutagenic Mechanisms

PubMed Central

Lambert, I. B.; Gordon, AJE.; Glickman, B. W.; McCalla, D. R.

1992-01-01

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/-ΔuvrB) and MucA/B-mediated error-prone translesion synthesis (+/-pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (>92%) which occurred at G·C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G·C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G·C -> T·A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G·C -> T·A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G·C -> T·A transversions by a factor of 30-60. In contrast, the -(G·C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G·C -> T·A transversions and -(G·C) frameshifts. Deletion mutations were induced in the Δuvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G·C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell. PMID:1459443

The ribosomal subunit assembly line

PubMed Central

Dlakić, Mensur

2005-01-01

Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

Ribosome Biogenesis in the Yeast Saccharomyces cerevisiae

PubMed Central

Woolford, John L.; Baserga, Susan J.

2013-01-01

Ribosomes are highly conserved ribonucleoprotein nanomachines that translate information in the genome to create the proteome in all cells. In yeast these complex particles contain four RNAs (>5400 nucleotides) and 79 different proteins. During the past 25 years, studies in yeast have led the way to understanding how these molecules are assembled into ribosomes in vivo. Assembly begins with transcription of ribosomal RNA in the nucleolus, where the RNA then undergoes complex pathways of folding, coupled with nucleotide modification, removal of spacer sequences, and binding to ribosomal proteins. More than 200 assembly factors and 76 small nucleolar RNAs transiently associate with assembling ribosomes, to enable their accurate and efficient construction. Following export of preribosomes from the nucleus to the cytoplasm, they undergo final stages of maturation before entering the pool of functioning ribosomes. Elaborate mechanisms exist to monitor the formation of correct structural and functional neighborhoods within ribosomes and to destroy preribosomes that fail to assemble properly. Studies of yeast ribosome biogenesis provide useful models for ribosomopathies, diseases in humans that result from failure to properly assemble ribosomes. PMID:24190922

5S rRNA and ribosome.

PubMed

Gongadze, G M

2011-12-01

5S rRNA is an integral component of the ribosome of all living organisms. It is known that the ribosome without 5S rRNA is functionally inactive. However, the question about the specific role of this RNA in functioning of the translation apparatus is still open. This review presents a brief history of the discovery of 5S rRNA and studies of its origin and localization in the ribosome. The previously expressed hypotheses about the role of this RNA in the functioning of the ribosome are discussed considering the unique location of 5S rRNA in the ribosome and its intermolecular contacts. Based on analysis of the current data on ribosome structure and its functional complexes, the role of 5S rRNA as an intermediary between ribosome functional domains is discussed.

Mechanistic Insight into the Reactivation of BCAII Enzyme from Denatured and Molten Globule States by Eukaryotic Ribosomes and Domain V rRNAs

PubMed Central

Chakraborty, Biprashekhar; Bhakta, Sayan; Sengupta, Jayati

2016-01-01

In all life forms, decoding of messenger-RNA into polypeptide chain is accomplished by the ribosome. Several protein chaperones are known to bind at the exit of ribosomal tunnel to ensure proper folding of the nascent chain by inhibiting their premature folding in the densely crowded environment of the cell. However, accumulating evidence suggests that ribosome may play a chaperone role in protein folding events in vitro. Ribosome-mediated folding of denatured proteins by prokaryotic ribosomes has been studied extensively. The RNA-assisted chaperone activity of the prokaryotic ribosome has been attributed to the domain V, a span of 23S rRNA at the intersubunit side of the large subunit encompassing the Peptidyl Transferase Centre. Evidently, this functional property of ribosome is unrelated to the nascent chain protein folding at the exit of the ribosomal tunnel. Here, we seek to scrutinize whether this unique function is conserved in a primitive kinetoplastid group of eukaryotic species Leishmania donovani where the ribosome structure possesses distinct additional features and appears markedly different compared to other higher eukaryotic ribosomes. Bovine Carbonic Anhydrase II (BCAII) enzyme was considered as the model protein. Our results manifest that domain V of the large subunit rRNA of Leishmania ribosomes preserves chaperone activity suggesting that ribosome-mediated protein folding is, indeed, a conserved phenomenon. Further, we aimed to investigate the mechanism underpinning the ribosome-assisted protein reactivation process. Interestingly, the surface plasmon resonance binding analyses exhibit that rRNA guides productive folding by directly interacting with molten globule-like states of the protein. In contrast, native protein shows no notable affinity to the rRNA. Thus, our study not only confirms conserved, RNA-mediated chaperoning role of ribosome but also provides crucial insight into the mechanism of the process. PMID:27099964

Purification and crystallization of components of the protein-synthesizing system from Thermus thermophilus

NASA Astrophysics Data System (ADS)

Garber, M. B.; Agalarov, S. Ch.; Eliseikina, I. A.; Sedelnikova, S. E.; Tishchenko, S. V.; Shirokov, V. A.; Yusupov, M. M.; Reshetnikova, L. S.; Trakhanov, S. D.; Tukalo, M. A.; Yaremchuk, A. D.

1991-03-01

An extreme thermophilic bacterium Thermus thermophilus has been chosen as a source for the isolation of components of the protein-synthesizing system to investigate their structures by X-ray crystallographic methods. The scheme of simultaneous isolation of ribosomes, tRNA, three elongation factors, several aminoacyl-tRNA synthetases and several enzymes has been developed. Methods of purification of ribosomes and individual ribosomal proteins without denaturation were elaborated. Crystals of the elongation factor G, the 70S ribosome, the 30S ribosomal subunit, six ribosomal proteins and three aminoacyl-tRNA synthetases have been obtained. Structural investigations of EF-G and the 70S ribosome are underway.

Paternal Somatic Mosaicism of a Novel Frameshift Mutation in ELANE Causing Severe Congenital Neutropenia.

PubMed

Kim, Hee-Jung; Song, Min-Jung; Lee, Ki-O; Kim, Sun-Hee; Kim, Hee-Jin

2015-12-01

Severe congenital neutropenia (SCN) is a bone marrow failure disease with an autosomal dominant inheritance from mutations in ELANE. Here, we report a 7-week-old Korean male with SCN. His elder sister died from pneumonia at 2 years. Direct sequencing of ELANE in the proband identified a heterozygous novel frameshift mutation: c.658delC (p.Arg220Glyfs20*). Family study involving his asymptomatic parents with normal cell counts revealed that his father had the same mutation, but at a lower burden than expected in a typical heterozygous state. Further molecular investigation demonstrated somatic mosaicism with ~18% mutant alleles. We concluded the proband inherited the mutation from his somatic mosaic father. © 2015 Wiley Periodicals, Inc.

High MRPS23 expression contributes to hepatocellular carcinoma proliferation and indicates poor survival outcomes.

PubMed

Pu, Meng; Wang, Jianlin; Huang, Qike; Zhao, Ge; Xia, Congcong; Shang, Runze; Zhang, Zhuochao; Bian, Zhenyuan; Yang, Xishegn; Tao, Kaishan

2017-07-01

Hepatocellular carcinoma is one of the most prevalent neoplasms and the leading cause of cancer-related mortality worldwide. Mitochondrial ribosomal protein S23 is encoded by a nuclear gene and participates in mitochondrial protein translation. Mitochondrial ribosomal protein S23 overexpression has been found in many types of cancer. In this study, we explored mitochondrial ribosomal protein S23 expression in primary hepatocellular carcinoma tissues compared with matched adjacent non-tumoral liver tissues using mitochondrial ribosomal protein S23 messenger RNA and protein levels collected from public databases and clinical samples. Immunohistochemistry was performed to analyze the relationship between mitochondrial ribosomal protein S23 and various clinicopathological features. The results indicated that mitochondrial ribosomal protein S23 was significantly overexpressed in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 expression was correlated with the tumor size and tumor-metastasis-node stage. Moreover, patients with high mitochondrial ribosomal protein S23 expression levels presented poorer survival rates. Mitochondrial ribosomal protein S23 was an independent prognostic factor for survival, especially at the early stage of hepatocellular carcinoma. In addition, the downregulation of mitochondrial ribosomal protein S23 decreased the proliferation of hepatocellular carcinoma in vitro and in vivo. In conclusion, we verified for the first time that mitochondrial ribosomal protein S23 expression was upregulated in hepatocellular carcinoma. High mitochondrial ribosomal protein S23 levels can predict poor clinical outcomes in hepatocellular carcinoma, and this protein plays a key role in tumor proliferation. Therefore, mitochondrial ribosomal protein S23 may be a potential therapeutic target for hepatocellular carcinoma.

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

Polar bears, antibiotics, and the evolving ribosome (Nobel Lecture).

PubMed

Yonath, Ada

2010-06-14

High-resolution structures of ribosomes, the cellular machines that translate the genetic code into proteins, revealed the decoding mechanism, detected the mRNA path, identified the sites of the tRNA molecules in the ribosome, elucidated the position and the nature of the nascent proteins exit tunnel, illuminated the interactions of the ribosome with non-ribosomal factors, such as the initiation, release and recycling factors, and provided valuable information on ribosomal antibiotics, their binding sites, modes of action, principles of selectivity and the mechanisms leading to their resistance. Notably, these structures proved that the ribosome is a ribozyme whose active site, namely where the peptide bonds are being formed, is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this symmetrical region is highly conserved and provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric prebiotic machine that formed peptide bonds and non-coded polypeptide chains. Structures of complexes of ribosomes with antibiotics targeting them revealed the principles allowing for their clinical use, identified resistance mechanisms and showed the structural bases for discriminating pathogenic bacteria from hosts, hence providing valuable structural information for antibiotics improvement and for the design of novel compounds that can serve as antibiotics.

Ribosome A and P sites revealed by length analysis of ribosome profiling data

PubMed Central

Martens, Andrew T.; Taylor, James; Hilser, Vincent J.

2015-01-01

The high-throughput sequencing of nuclease-protected mRNA fragments bound to ribosomes, a technique known as ribosome profiling, quantifies the relative frequencies with which different regions of transcripts are translated. This technique has revealed novel translation initiation sites with unprecedented scope and has furthered investigations into the connections between codon biases and translation rates. Yet the location of the codon being decoded in ribosome footprints is still unknown, and has been complicated by the recent observation of footprints with non-canonical lengths. Here we show how taking into account the variations in ribosome footprint lengths can reveal the ribosome aminoacyl (A) and peptidyl (P) site locations. These location assignments are in agreement with the proposed mechanisms for various ribosome pauses and further enhance the resolution of the profiling data. We also show that GC-rich motifs at the 5′ ends of footprints are found in yeast, calling into question the anti-Shine-Dalgarno effect's role in ribosome pausing. PMID:25805170

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

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation.

PubMed

Rodnina, Marina V; Wintermeyer, Wolfgang

2011-04-01

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

Molecular cloning of low-temperature-inducible ribosomal proteins from soybean.

PubMed

Kim, Kee-Young; Park, Seong-Whan; Chung, Young-Soo; Chung, Chung-Han; Kim, Jung-In; Lee, Jai-Heon

2004-05-01

Three ribosomal protein genes induced by low-temperature treatment were isolated from soybean. GmRPS13 (742 bp) encodes a 17.1 kDa protein which has 95% identity with the 40S ribosomal protein S13 of Panax ginseng (AB043974). GmRPS6 (925 bp) encodes a 28.1 kDa protein which has 94% identity with the 40S ribosomal protein S6 of Asparagus officinalis (AJ277533). GmRPL37 (494 bp) encodes a 10.7 kDa protein which has 85% identity with the 60S ribosomal protein L37 of Arabidopsis thaliana (AF370216). The expression of these ribosomal protein genes started to increase 3 d after low-temperature treatment, whereas the cold-stress protein src1 was highly induced from the first day. Such late response of these ribosomal protein genes may be due to secondary signals during cold adaptation. The induction of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under low-temperature conditions.

Regulation of the protein-conducting channel by a bound ribosome

PubMed Central

Gumbart, James; Trabuco, Leonardo G.; Schreiner, Eduard; Villa, Elizabeth; Schulten, Klaus

2009-01-01

Summary During protein synthesis, it is often necessary for the ribosome to form a complex with a membrane-bound channel, the SecY/Sec61 complex, in order to translocate nascent proteins across a cellular membrane. Structural data on the ribosome-channel complex are currently limited to low-resolution cryo-electron microscopy maps, including one showing a bacterial ribosome bound to a monomeric SecY complex. Using that map along with available atomic-level models of the ribosome and SecY, we have determined, through molecular dynamics flexible fitting (MDFF), an atomic-resolution model of the ribosome-channel complex. We characterized computationally the sites of ribosome-SecY interaction within the complex and determined the effect of ribosome binding on the SecY channel. We also constructed a model of a ribosome in complex with a SecY dimer by adding a second copy of SecY to the MDFF-derived model. The study involved 2.7-million-atom simulations over altogether nearly 50 ns. PMID:19913480

In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

PubMed Central

Lalucque, Hervé; Silar, Philippe

2000-01-01

Background To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible. Results Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase. Conclusion Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division. PMID:11112985

Ribosomal targets for antibiotic drug discovery

DOEpatents

Blanchard, Scott C.; Feldman, Michael Brian; Wang, Leyi; Doudna Cate, James H.; Pulk, Arto; Altman, Roger B.; Wasserman, Michael R

2016-09-13

The present invention relates to methods to identify molecules that binds in the neomycin binding pocket of a bacterial ribosome using structures of an intact bacterial ribosome that reveal how the ribosome binds tRNA in two functionally distinct states, determined by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. Additionally, the invention relates to various assays, including single-molecule assay for ribosome recycling, and methods to identify compounds that interfere with ribosomal function by detecting newly identified intermediate FRET states using known and novel FRET pairs on the ribosome. The invention also provides vectors and compositions with an N-terminally tagged S13 protein.

Effects of S-adenosylmethionine decarboxylase, polyamines, amino acids, and weak bases (amines and ammonia) on development and ribosomal RNA synthesis in Xenopus embryos.

PubMed

Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Takai, Jun-Ichi; Yoshida, Junki; Mishina, Takamichi; Fuchimukai, Kota; Ogasawara, Tsukasa; Kariya, Taro; Tashiro, Kosuke; Igarashi, Kazuei

2010-02-01

We have been studying control mechanisms of gene expression in early embryogenesis in a South African clawed toad Xenopus laevis, especially during the period of midblastula transition (MBT), or the transition from the phase of active cell division (cleavage stage) to the phase of extensive morphogenesis (post-blastular stages). We first found that ribosomal RNA synthesis is initiated shortly after MBT in Xenopus embryos and those weak bases, such as amines and ammonium ion, selectively inhibit the initiation and subsequent activation of rRNA synthesis. We then found that rapidly labeled heterogeneous mRNA-like RNA is synthesized in embryos at pre-MBT stage. We then performed cloning and expression studies of several genes, such as those for activin receptors, follistatin and aldolases, and then reached the studies of S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine metabolism. Here, we cloned a Xenopus SAMDC cDNA and performed experiments to overexpress the in vitro-synthesized SAMDC mRNA in Xenopus early embryos, and found that the maternally preset program of apoptosis occurs in cleavage stage embryos, which is executed when embryos reach the stage of MBT. In the present article, we first summarize results on SAMDC and the maternal program of apoptosis, and then describe our studies on small-molecular-weight substances like polyamines, amino acids, and amines in Xenopus embryos. Finally, we summarize our studies on weak bases, especially on ammonium ion, as the specific inhibitor of ribosomal RNA synthesis in Xenopus embryonic cells.

The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate.

PubMed

Paszkiewicz-Gadek, A; Porowska, H; Gałasiński, W

1992-01-01

UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.

Complete kinetic mechanism for recycling of the bacterial ribosome

PubMed Central

Borg, Anneli; Pavlov, Michael

2016-01-01

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec−1, is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec−1, are discussed in the perspective of rapidly growing bacterial cells. PMID:26527791

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

PubMed

Mikulík, K; Janda, I; Weiser, J; Stastná, J; Jiránová, A

1984-12-03

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

Detection of abrin holotoxin using novel monoclonal antibodies

USDA-ARS?s Scientific Manuscript database

Abrin, a member of ribosome-inactivating protein family, is produced by the Abrus precatorius plant. It has been determined to have the potential to pose a severe threat to both human and animal health and is listed as a select agent by the U.S. Federal Select Agent Program. However, an immunoassay ...

Attachment of UDP-hexosamines to the ribosomes isolated from rat liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kopacz-Jodczyk, T.; Paszkiewicz-Gadek, A.; Galasinski, W.

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

The attachment of UDP-hexosamines to the ribosomes isolated from rat liver.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Gałasiński, W

1988-06-01

The binding of UDP-N-acetylhexosamines with purified ribosomes was studied and it was found that the radioactive nucleotides can be attached to these particles. The radioactivity of the purified ribosomal pellet depends on the amounts of ribosomes and UDP-N-acetylhexosamines. Some characteristics of the binding system indicate that the attachment of UDP-sugar to ribosome does not require the participation of glycosyltransferases. The results of the competition experiment would suggest that there are specific sites on ribosomes for the binding of UDP-N-acetylglucosamine.

A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers.

PubMed

Jensen, Line; Hegelund, Josefine Nymark; Olsen, Andreas; Lütken, Henrik; Müller, Renate

2016-05-23

The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers are sensitive to even minute concentrations of ethylene. Monitoring flower longevity in three Campanula species revealed C. portenschlagiana (Cp) as ethylene sensitive, C. formanekiana (Cf) with intermediate sensitivity and C. medium (Cm) as ethylene insensitive. We identified key elements in ethylene signal transduction, specifically in Ethylene Response Sensor 2 (ERS2), Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 3- Like 1 and 2 (EIL1 and EIL2) homologous. Transcripts of ERS2, CTR1 and EIL1 were constitutively expressed in all species both throughout flower development and in response to ethylene. In contrast, EIL2 was found only in Cf and Cm. We identified a natural mutation in Cmeil2 causing a frameshift which resulted in difference in expression levels of EIL2, with more than 100-fold change between Cf and Cm in young flowers. This study shows that the naturally occurring 7 bp frameshift discovered in Cmeil2, a key gene in the ethylene signaling pathway, correlates with ethylene insensitivity in flowers. We suggest that transfer of the eil2 mutation to other plant species will provide a novel tool to engineer ethylene insensitive flowers.

Genetic Mutations in Cancer

Cancer.gov

Many different types of genetic mutations are found in cancer cells. This infographic outlines certain types of alterations that are present in cancer, such as missense, nonsense, frameshift, and chromosome rearrangements.

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

A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

PubMed Central

Hurt, Ed; Hannus, Stefan; Schmelzl, Birgit; Lau, Denise; Tollervey, David; Simos, George

1999-01-01

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes. PMID:9971735

Analysis of Ribosome Stalling and Translation Elongation Dynamics by Deep Learning.

PubMed

Zhang, Sai; Hu, Hailin; Zhou, Jingtian; He, Xuan; Jiang, Tao; Zeng, Jianyang

2017-09-27

Ribosome stalling is manifested by the local accumulation of ribosomes at specific codon positions of mRNAs. Here, we present ROSE, a deep learning framework to analyze high-throughput ribosome profiling data and estimate the probability of a ribosome stalling event occurring at each genomic location. Extensive validation tests on independent data demonstrated that ROSE possessed higher prediction accuracy than conventional prediction models, with an increase in the area under the receiver operating characteristic curve by up to 18.4%. In addition, genome-wide statistical analyses showed that ROSE predictions can be well correlated with diverse putative regulatory factors of ribosome stalling. Moreover, the genome-wide ribosome stalling landscapes of both human and yeast computed by ROSE recovered the functional interplays between ribosome stalling and cotranslational events in protein biogenesis, including protein targeting by the signal recognition particles and protein secondary structure formation. Overall, our study provides a novel method to complement the ribosome profiling techniques and further decipher the complex regulatory mechanisms underlying translation elongation dynamics encoded in the mRNA sequence. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of ribosomopathy genes during Xenopus tropicalis embryogenesis.

PubMed

Robson, Andrew; Owens, Nick D L; Baserga, Susan J; Khokha, Mustafa K; Griffin, John N

2016-10-26

Because ribosomes are ubiquitously required for protein production, it was long assumed that any inherited defect in ribosome manufacture would be embryonically lethal. However, several human congenital diseases have been found to be associated with mutations in ribosome biogenesis factors. Surprisingly, despite the global requirement for ribosomes, these "ribosomopathies" are characterized by distinct and tissue specific phenotypes. The reasons for such tissue proclivity in ribosomopathies remain mysterious but may include differential expression of ribosome biogenesis factors in distinct tissues. Here we use in situ hybridization of labeled antisense mRNA probes and ultra high temporal resolution RNA-Seq data to examine and compare expression of 13 disease associated ribosome biogenesis factors at six key stages in Xenopus tropicalis development. Rather than being ubiquitously expressed during development, mRNAs of all examined ribosome biogenesis factors were highly enriched in specific tissues, including the cranial neural crest and ventral blood islands. Interestingly, expression of ribosome biogenesis factors demonstrates clear differences in timing, transcript number and tissue localization. Ribosome biogenesis factor expression is more spatiotemporally regulated during embryonic development than previously expected and correlates closely with many of the common ribosomopathy phenotypes. Our findings provide information on the dynamic use of ribosome production machinery components during development and advance our understanding of their roles in disease.

A novel de novo POGZ mutation in a patient with intellectual disability.

PubMed

Tan, Bo; Zou, Yongyi; Zhang, Yue; Zhang, Rui; Ou, Jianjun; Shen, Yidong; Zhao, Jingping; Luo, Xiaomei; Guo, Jing; Zeng, Lanlan; Hu, Yiqiao; Zheng, Yu; Pan, Qian; Liang, Desheng; Wu, Lingqian

2016-04-01

POGZ, the gene encoding pogo transposable element-derived protein with zinc-finger domain, has been implicated in autism spectrum disorder and it is widely expressed in the human tissues, including the brain. Intellectual disability (ID) is highly heterogeneous neurodevelopment disorder and affects ~2-3% of the general population. Here we report the identification of a novel frameshift mutation in the coding region of the POGZ gene (c.1277_1278insC), which occurred de novo in a Chinese patient with ID. In silico analysis and western blotting revealed this frameshift mutation generating truncated protein in peripheral blood lymphocytes, and this may disrupt several important domains of POGZ gene. Our finding broadens the spectrum of POGZ mutations and may help to understand the molecular basis of ID and aid genetic counseling.

Toward a Whole-Cell Model of Ribosome Biogenesis: Kinetic Modeling of SSU Assembly

PubMed Central

Earnest, Tyler M.; Lai, Jonathan; Chen, Ke; Hallock, Michael J.; Williamson, James R.; Luthey-Schulten, Zaida

2015-01-01

Central to all life is the assembly of the ribosome: a coordinated process involving the hierarchical association of ribosomal proteins to the RNAs forming the small and large ribosomal subunits. The process is further complicated by effects arising from the intracellular heterogeneous environment and the location of ribosomal operons within the cell. We provide a simplified model of ribosome biogenesis in slow-growing Escherichia coli. Kinetic models of in vitro small-subunit reconstitution at the level of individual protein/ribosomal RNA interactions are developed for two temperature regimes. The model at low temperatures predicts the existence of a novel 5′→3′→central assembly pathway, which we investigate further using molecular dynamics. The high-temperature assembly network is incorporated into a model of in vivo ribosome biogenesis in slow-growing E. coli. The model, described in terms of reaction-diffusion master equations, contains 1336 reactions and 251 species that dynamically couple transcription and translation to ribosome assembly. We use the Lattice Microbes software package to simulate the stochastic production of mRNA, proteins, and ribosome intermediates over a full cell cycle of 120 min. The whole-cell model captures the correct growth rate of ribosomes, predicts the localization of early assembly intermediates to the nucleoid region, and reproduces the known assembly timescales for the small subunit with no modifications made to the embedded in vitro assembly network. PMID:26333594

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export

PubMed Central

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms. PMID:26872259

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

PubMed

Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

PubMed Central

Jakovljevic, Jelena; Ohmayer, Uli; Gamalinda, Michael; Talkish, Jason; Alexander, Lisa; Linnemann, Jan; Milkereit, Philipp; Woolford, John L.

2012-01-01

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA3 to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA3 pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A3 assembly factors and for proper assembly of the neighborhoods containing domains I and II. PMID:22893726

Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryo-EM

PubMed Central

Chen, Bo; Kaledhonkar, Sandip; Sun, Ming; Shen, Bingxin; Lu, Zonghuan; Barnard, David; Lu, Toh-Ming; Gonzalez, Ruben L.; Frank, Joachim

2015-01-01

Ribosomal subunit association is a key checkpoint in translation initiation, but its structural dynamics are poorly understood. Here, we used a recently developed mixing-spraying, time-resolved, cryogenic electron microscopy (cryo-EM) method to study ribosomal subunit association in the sub-second time range. We have improved this method and increased the cryo-EM data yield by tenfold. Pre-equilibrium states of the association reaction were captured by reacting the mixture of ribosomal subunits for 60 ms and 140 ms. We also identified three distinct ribosome conformations in the associated ribosomes. The observed proportions of these conformations are the same in these two time points, suggesting that ribosomes equilibrate among the three conformations within less than 60 ms upon formation. Our results demonstrate that the mixing-spraying method can capture multiple states of macromolecules during a sub-second reaction. Other fast processes, such as translation initiation, decoding and ribosome recycling, are amenable to study with this method. PMID:26004440

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression

PubMed Central

Parks, Matthew M.; Kurylo, Chad M.; Dass, Randall A.; Bojmar, Linda; Lyden, David; Vincent, C. Theresa; Blanchard, Scott C.

2018-01-01

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome’s molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5S, 5.8S, 18S, and 28S ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease. PMID:29503865

Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin

PubMed Central

Montanaro, Lucio; Sperti, Simonetta; Stirpe, Fiorenzo

1973-01-01

1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH4Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin. PMID:4780693

Positively Charged Residues Are the Major Determinants of Ribosomal Velocity

PubMed Central

Charneski, Catherine A.; Hurst, Laurence D.

2013-01-01

Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data. PMID:23554576

The RNA-binding protein Hfq is important for ribosome biogenesis and affects translation fidelity.

PubMed

Andrade, José M; Dos Santos, Ricardo F; Chelysheva, Irina; Ignatova, Zoya; Arraiano, Cecília M

2018-06-01

Ribosome biogenesis is a complex process involving multiple factors. Here, we show that the widely conserved RNA chaperone Hfq, which can regulate sRNA-mRNA basepairing, plays a critical role in rRNA processing and ribosome assembly in Escherichia coli Hfq binds the 17S rRNA precursor and facilitates its correct processing and folding to mature 16S rRNA Hfq assists ribosome assembly and associates with pre-30S particles but not with mature 30S subunits. Inactivation of Hfq strikingly decreases the pool of mature 70S ribosomes. The reduction in ribosome levels depends on residues located in the distal face of Hfq but not on residues found in the proximal and rim surfaces which govern interactions with the sRNAs. Our results indicate that Hfq-mediated regulation of ribosomes is independent of its function as sRNA-regulator. Furthermore, we observed that inactivation of Hfq compromises translation efficiency and fidelity, both features of aberrantly assembled ribosomes. Our work expands the functions of the Sm-like protein Hfq beyond its function in small RNA-mediated regulation and unveils a novel role of Hfq as crucial in ribosome biogenesis and translation. © 2018 The Authors.

Structure of a pentameric virion-associated fiber with a potential role in Orsay virus entry to host cells

PubMed Central

Yuan, Wang; Zhou, Ying; Wang, Tao; Demeler, Borries; Zhong, Weiwei; Tao, Yizhi J.

2017-01-01

Despite the wide use of Caenorhabditis elegans as a model organism, the first virus naturally infecting this organism was not discovered until six years ago. The Orsay virus and its related nematode viruses have a positive-sense RNA genome, encoding three proteins: CP, RdRP, and a novel δ protein that shares no homology with any other proteins. δ can be expressed either as a free δ or a CP-δ fusion protein by ribosomal frameshift, but the structure and function of both δ and CP-δ remain unknown. Using a combination of electron microscopy, X-ray crystallography, computational and biophysical analyses, here we show that the Orsay δ protein forms a ~420-Å long, pentameric fiber with an N-terminal α-helical bundle, a β-stranded filament in the middle, and a C-terminal head domain. The pentameric nature of the δ fiber has been independently confirmed by both mass spectrometry and analytical ultracentrifugation. Recombinant Orsay capsid containing CP-δ shows protruding long fibers with globular heads at the distal end. Mutant viruses with disrupted CP-δ fibers were generated by organism-based reverse genetics. These viruses were found to be either non-viable or with poor infectivity according to phenotypic and qRT-PCR analyses. Furthermore, addition of purified δ proteins to worm culture greatly reduced Orsay infectivity in a sequence-specific manner. Based on the structure resemblance between the Orsay CP-δ fiber and the fibers from reovirus and adenovirus, we propose that CP-δ functions as a cell attachment protein to mediate Orsay entry into worm intestine cells. PMID:28241071

Amalga-like virus infecting Antonospora locustae, a microsporidian pathogen of grasshoppers, plus related viruses associated with other arthropods.

PubMed

Pyle, Jesse D; Keeling, Patrick J; Nibert, Max L

2017-04-02

A previously reported Expressed Sequence Tag (EST) library from spores of microsporidian Antonospora locustae includes a number of clones with sequence similarities to plant amalgaviruses. Reexamining the sequence accessions from that library, we found additional such clones, contributing to a 3247-nt contig that approximates the length of an amalga-like virus genome. Using A. locustae spores stored from that previous study, and new ones obtained from the same source, we newly visualized the putative dsRNA genome of this virus and obtained amplicons yielding a 3387-nt complete genome sequence. Phylogenetic analyses suggested it as prototype strain of a new genus in family Amalgaviridae. The genome contains two partially overlapping long ORFs, with downstream ORF2 in the +1 frame relative to ORF1 and a proposed motif for +1 ribosomal frameshifting in the region of overlap. Subsequent database searches using the predicted fusion protein sequence of this new amalga-like virus identified related sequences in the transcriptome of a basal hexapod, the springtail species Tetrodontophora bielanensis. We speculate that this second new amalga-like virus (contig length, 3475 nt) likely also derived from a microsporidian, or related organism, which was associated with the springtail specimens at the time of sampling for transcriptome analysis. Other findings of interest include evidence that the ORF1 translation products of these two new amalga-like viruses contain a central region of predicted α-helical coiled coil, as recently reported for plant amalgaviruses, and transcriptome-based evidence for another new amalga-like virus in the transcriptome of another basal hexapod, the two-pronged bristletail species Campodea augens. Copyright © 2017 Elsevier B.V. All rights reserved.

Recurrent emergence of structural variants of LTR retrotransposon CsRn1 evolving novel expression strategy and their selective expansion in a carcinogenic liver fluke, Clonorchis sinensis.

PubMed

Kim, Seon-Hee; Kong, Yoon; Bae, Young-An

2017-06-01

Autonomous retrotransposons, in which replication and transcription are coupled, encode the essential gag and pol genes as a fusion or separate overlapping form(s) that are expressed in single transcripts regulated by a common upstream promoter. The element-specific expression strategies have driven development of relevant translational recoding mechanisms including ribosomal frameshifting to satisfy the protein stoichiometry critical for the assembly of infectious virus-like particles. Retrotransposons with different recoding strategies exhibit a mosaic distribution pattern across the diverse families of reverse transcribing elements, even though their respective distributions are substantially skewed towards certain family groups. However, only a few investigations to date have focused on the emergence of retrotransposons evolving novel expression strategy and causal genetic drivers of the structural variants. In this study, the bulk of genomic and transcribed sequences of a Ty3/gypsy-like CsRn1 retrotransposon in Clonorchis sinensis were analyzed for the comprehensive examination of its expression strategy. Our results demonstrated that structural variants with single open reading frame (ORF) have recurrently emerged from precedential CsRn1 copies encoding overlapping gag-pol ORFs by a single-nucleotide insertion in an upstream region of gag stop codon. In the parasite genome, some of the newly evolved variants appeared to undergo proliferative burst as active master lineages together with their ancestral copies. The genetic event was similarly observed in Opisthorchis viverrini, the closest neighbor of C. sinensis, whereas the resulting structural variants might have failed to overcome purifying selection and comprised minor remnant copies in the Opisthorchis genome. Copyright © 2017 Elsevier B.V. All rights reserved.

FunGene: the functional gene pipeline and repository.

PubMed

Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R

2013-01-01

Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.

Access to Ribosomal Protein Rpl25p by the Signal Recognition Particle Is Required for Efficient Cotranslational Translocation

PubMed Central

Dalley, Jane A.; Selkirk, Alexander

2008-01-01

Targeting of proteins to the endoplasmic reticulum (ER) occurs cotranslationally necessitating the interaction of the signal recognition particle (SRP) and the translocon with the ribosome. Biochemical and structural studies implicate ribosomal protein Rpl25p as a major ribosome interaction site for both these factors. Here we characterize an RPL25GFP fusion, which behaves as a dominant mutant leading to defects in co- but not posttranslational translocation in vivo. In these cells, ribosomes still interact with ER membrane and the translocon, but are defective in binding SRP. Overexpression of SRP can restore ribosome binding of SRP, but only partially rescues growth and translocation defects. Our results indicate that Rpl25p plays a critical role in the recruitment of SRP to the ribosome. PMID:18448667

Entrapping ribosomes for viral translation: tRNA mimicry as a molecular Trojan horse.

PubMed

Barends, Sharief; Bink, Hugo H J; van den Worm, Sjoerd H E; Pleij, Cornelis W A; Kraal, Barend

2003-01-10

Turnip yellow mosaic virus (TYMV) has a genomic plus-strand RNA with a 5' cap followed by overlapping and different reading frames for the movement protein and polyprotein, while the distal coat protein cistron is translated from a subgenomic RNA. The 3'-untranslated region harbors a tRNA-like structure (TLS) to which a valine moiety can be added and it is indispensable for virus viability. Here, we report about a surprising interaction between TYMV-RNA-programmed ribosomes and 3'-valylated TLS that yields polyprotein with the valine N terminally incorporated by a translation mechanism resistant to regular initiation inhibitors. Disruption of the TLS exclusively abolishes polyprotein synthesis, which can be restored by adding excess TLS in trans. Our observations imply a novel eukaryotic mechanism for internal initiation of mRNA translation.

Eaf1p Is Required for Recruitment of NuA4 in Targeting TFIID to the Promoters of the Ribosomal Protein Genes for Transcriptional Initiation In Vivo

PubMed Central

Uprety, Bhawana; Sen, Rwik

2015-01-01

NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a Δeaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions. PMID:26100014

[Immunochemistry of eukaryotic ribosomes].

PubMed

Lopaczyński, W; Gałasiński, W

1990-01-01

Immunochemical investigations of ribosomes should correlate with basic knowledge of the function, structure and activity of organelles in the cell processes. Our paper presents data of immunochemical methods used to determine the structure, function and differences of ribosomes. We present the usefulness of immunochemical methods to test human ribosomes, diagnosis and therapy of many diseases.

In vitro degradation of ribosomes.

PubMed

Mora, G; Rivas, A

1976-12-01

The cytoplasmic ribosomes from Euglena gracilis var. bacillaris are found to be of two types taking into consideration their stability "in vitro". In the group of unstable ribosomes the large subunit is degraded. The other group apparently does not suffer any degradation under the conditions described. However the RNAs extracted from both types of ribosomes are degraded during sucrose density gradients. The degradation of the largest RNA species has been reported previously, but no comment has been made about the stability of the ribosome itself.

Senescent changes in the ribosomes of animal cells in vivo and in vitro

NASA Technical Reports Server (NTRS)

Miquel, J.; Johnson, J. E., Jr.

1979-01-01

The paper examines RNA-ribosomal changes observed in protozoa and fixed postmitotic cells, as well as the characteristics of intermitotic cells. Attention is given to a discussion of the implications of the reported ribosomal changes as to the senescent deterioration of protein synthesis and physiological functions. A survey of the literature suggests that, while the data on ribosomal change in dividing cells both in vivo and in vitro are inconclusive, there is strong histological and biochemical evidence in favor of some degree of quantitative ribosomal loss in fixed postmitotic cells. Since these decreases in ribosomes are demonstrated in differential cells from nematodes, insects and mammals, they may represent a universal manifestation of cytoplasmic senescence in certain types of fixed postmitotic animal cells. The observed variability in ribosomal loss for cells belonging to the same type suggests that this involution phenomenon is rather related to the wear and tear suffered by a particular cell.

A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

PubMed Central

2009-01-01

Membrane protein integration occurs predominantly at the endoplasmic reticulum and is mediated by the translocon, which is formed by the Sec61p complex. The translocon binds to the ribosome at the polypeptide exit site such that integration occurs in a cotranslational manner. Ribosomal protein Rpl17 is positioned such that it contacts both the ribosome exit tunnel and the surface of the ribosome near the exit site, where it is intimately associated with the translocon. The presence of a trans-membrane (TM) segment inside the ribosomal exit tunnel leads to the recruitment of RAMP4 to the translocon at a site adjacent to Rpl17. This suggests a signaling function for Rpl17 such that it can recognize a TM segment inside the ribosome and triggers rearrangements of the translocon, priming it for subsequent TM segment integration. PMID:19468070

Translational Control of Hemoglobin Synthesis by an Initiation Factor Required for Recycling of Ribosomes and for their Binding to Messenger RNA

PubMed Central

Kaempfer, Raymond; Kaufman, Jennifer

1972-01-01

The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 37° requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes. These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor. PMID:4508325

Computational resources for ribosome profiling: from database to Web server and software.

PubMed

Wang, Hongwei; Wang, Yan; Xie, Zhi

2017-08-14

Ribosome profiling is emerging as a powerful technique that enables genome-wide investigation of in vivo translation at sub-codon resolution. The increasing application of ribosome profiling in recent years has achieved remarkable progress toward understanding the composition, regulation and mechanism of translation. This benefits from not only the awesome power of ribosome profiling but also an extensive range of computational resources available for ribosome profiling. At present, however, a comprehensive review on these resources is still lacking. Here, we survey the recent computational advances guided by ribosome profiling, with a focus on databases, Web servers and software tools for storing, visualizing and analyzing ribosome profiling data. This review is intended to provide experimental and computational biologists with a reference to make appropriate choices among existing resources for the question at hand. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Hypermethylation of 28S ribosomal RNA in β-thalassemia trait carriers.

PubMed

Sornjai, Wannapa; Lithanatudom, Pathrapol; Erales, Jenny; Joly, Philippe; Francina, Alain; Hacot, Sabine; Fucharoen, Suthat; Svasti, Saovaros; Diaz, Jean Jacques; Mertani, Hichem C; Smith, Duncan R

2017-01-01

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of β-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in β-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in β-thalassemia trait carriers. Copyright © 2016 Elsevier B.V. All rights reserved.

Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins.

PubMed

Li, Xiao-Ping; Tumer, Nilgun E

2017-04-11

Both ricin and Shiga holotoxins display no ribosomal activity in their native forms and need to be activated to inhibit translation in a cell-free translation inhibition assay. This is because the ribosome binding site of the ricin A chain (RTA) is blocked by the B subunit in ricin holotoxin. However, it is not clear why Shiga toxin 1 (Stx1) or Shiga toxin 2 (Stx2) holotoxin is not active in a cell-free system. Here, we compare the ribosome binding and depurination activity of Stx1 and Stx2 holotoxins with the A1 subunits of Stx1 and Stx2 using either the ribosome or a 10-mer RNA mimic of the sarcin/ricin loop as substrates. Our results demonstrate that the active sites of Stx1 and Stx2 holotoxins are blocked by the A2 chain and the B subunit, while the ribosome binding sites are exposed to the solvent. Unlike ricin, which is enzymatically active, but cannot interact with the ribosome, Stx1 and Stx2 holotoxins are enzymatically inactive but can interact with the ribosome.

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

PubMed Central

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

2015-01-01

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. PMID:26112308

Ribosomal stress induces L11- and p53-dependent apoptosis in mouse pluripotent stem cells.

PubMed

Morgado-Palacin, Lucia; Llanos, Susana; Serrano, Manuel

2012-02-01

Ribosome biogenesis is the most demanding energetic process in proliferating cells and it is emerging as a critical sensor of cellular homeostasis. Upon disturbance of ribosome biogenesis, specific free ribosomal proteins, most notably L11, bind and inhibit Mdm2, resulting in activation of the tumor suppressor p53. This pathway has been characterized in somatic and cancer cells, but its function in embryonic pluripotent cells has remained unexplored. Here, we show that treatment with low doses of Actinomycin D or depletion of ribosomal protein L37, two well-established inducers of ribosomal stress, activate p53 in an L11-dependent manner in mouse embryonic stem cells (ESCs) and in induced pluripotent stem cells (iPSCs). Activation of p53 results in transcriptional induction of p53 targets, including p21, Mdm2, Pidd, Puma, Noxa and Bax. Finally, ribosomal stress elicits L11- and p53-dependent apoptosis in ESCs/iPSCs. These results extend to pluripotent cells the functionality of the ribosomal stress pathway and we speculate that this could be a relevant cellular checkpoint during early embryogenesis.

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus.

PubMed

Bayas, Camille A; Wang, Jiarui; Lee, Marissa K; Schrader, Jared M; Shapiro, Lucy; Moerner, W E

2018-04-17

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single-particle tracking and superresolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in Escherichia coli , where RNase E colocalizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter , we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Importantly, RNase E cluster positions correlated with the subcellular location of chromosomal loci of two highly transcribed rRNA genes, suggesting that RNase E's function in rRNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter , with chromosomal readout serving as the template for this organization.

Selective Destruction Of Cells Infected With The Human Immunodeficiency Virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2006-03-28

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a varient of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

Selective destruction of cells infected with human immunodeficiency virus

DOEpatents

Keener, William K.; Ward, Thomas E.

2003-09-30

Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a variant of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements.

PubMed

Guimond, A; Moss, T

1999-02-01

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.

The primary structures of ribosomal proteins S14 and S16 from the archaebacterium Halobacterium marismortui. Comparison with eubacterial and eukaryotic ribosomal proteins.

PubMed

Kimura, J; Kimura, M

1987-09-05

The amino acid sequences of two ribosomal proteins, S14 and S16, from the archaebacterium Halobacterium marismortui have been determined. Sequence data were obtained by the manual and solid-phase sequencing of peptides derived from enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease as well as by chemical cleavage with cyanogen bromide. Proteins S14 and S16 contain 109 and 126 amino acid residues and have Mr values of 11,964 and 13,515, respectively. Comparison of the sequences with those of ribosomal proteins from other organisms demonstrates that S14 has a significant homology with the rat liver ribosomal protein S11 (36% identity) as well as with the Escherichia coli ribosomal protein S17 (37%), and that S16 is related to the yeast ribosomal protein YS22 (40%) and proteins S8 from E. coli (28%) and Bacillus stearothermophilus (30%). A comparison of the amino acid residues in the homologous regions of halophilic and nonhalophilic ribosomal proteins reveals that halophilic proteins have more glutamic acids, asparatic acids, prolines, and alanines, and less lysines, arginines, and isoleucines than their nonhalophilic counterparts. These amino acid substitutions probably contribute to the structural stability of halophilic ribosomal proteins.

Evidence for rRNA 2'-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes.

PubMed

Erales, Jenny; Marchand, Virginie; Panthu, Baptiste; Gillot, Sandra; Belin, Stéphane; Ghayad, Sandra E; Garcia, Maxime; Laforêts, Florian; Marcel, Virginie; Baudin-Baillieu, Agnès; Bertin, Pierre; Couté, Yohann; Adrait, Annie; Meyer, Mélanie; Therizols, Gabriel; Yusupov, Marat; Namy, Olivier; Ohlmann, Théophile; Motorin, Yuri; Catez, Frédéric; Diaz, Jean-Jacques

2017-12-05

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2'-O-methylation (2'-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2'-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2'-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2'-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2'-O-Me, we identified a repertoire of 2'-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2'-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2'-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2'-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Type 1 ribosome-inactivating proteins depurinate plant 25S rRNA without species specificity.

PubMed Central

Prestle, J; Schönfelder, M; Adam, G; Mundry, K W

1992-01-01

Four different type 1 ribosome-inactivating proteins (RIPs) with RNA N-glycosidase activity were tested for their ability to attack the large rRNA of plant ribosomes derived from tobacco plants, as well as from the plant species from which the particular RIP had been isolated. Incubation of tobacco ribosomes with RIPs isolated from either Phytolacca americana L. (pokeweed), Dianthus barbatus L. (carnation), Spinacia oleracea L. (spinach) or Chenopodium amaranthicolor Coste and Reyn. (chenopodium) rendered the 25S rRNA sensitive to aniline-catalyzed hydrolysis, generating a single rRNA-fragment of about 350 nucleotides. The same fragment was generated when rRNAs from pokeweed, carnation, spinach or chenopodium ribosomes were aniline-treated without any deliberate treatment of the ribosomes with the respective RIP. This indicated that ribosomes from all RIP-producing plants were already inactivated by their own RIPs during preparation. These results demonstrate that plant ribosomes are generally susceptible to RIP attack, including modification by their own RIPs. Direct sequencing of the newly generated fragments revealed that a single N-glycosidic bond at an adenosine residue within the highly conserved sequence 5'-AGUACGAGAGGA-3' was cleaved by all of the RIPs investigated, a situation also found in animal, yeast and Escherichia coli ribosomes. Images PMID:1620614

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

PubMed Central

Craig, Nessly; Goldstein, Lester

1969-01-01

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus). PMID:5765758

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity.

PubMed

Park, Sang-Wook; Stevens, Noah M; Vivanco, Jorge M

2002-12-01

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

PubMed

Body, A; Brownstein, B H

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.

Effects of Detergents on Ribosomal Precursor Subunits of Bacillus megaterium

PubMed Central

Body, Barbara A.; Brownstein, Bernard H.

1978-01-01

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction. PMID:412833

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing.

PubMed

Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L

2013-02-01

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

PubMed Central

Gamalinda, Michael; Jakovljevic, Jelena; Babiano, Reyes; Talkish, Jason; de la Cruz, Jesús; Woolford, John L.

2013-01-01

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2. PMID:23268442

Mutational analysis of FLASH and PTPN13 genes in colorectal carcinomas.

PubMed

Jeong, Eun Goo; Lee, Sung Hak; Yoo, Nam Jin; Lee, Sug Hyung

2008-01-01

The Fas-Fas ligand system is considered a major pathway for induction of apoptosis in cells and tissues. FLASH was identified as a pro-apoptotic protein that transmits apoptosis signal during Fas-mediated apoptosis. PTPN13 interacts with Fas and functions as both suppressor and inducer of Fas-mediated apoptosis. There are polyadenine tracts in both FLASH (A8 and A9 in exon 8) and PTPN13 (A8 in exon 7) genes that could be frameshift mutation targets in colorectal carcinomas. Because genes encoding proteins in Fas-mediated apoptosis frequently harbor somatic mutations in cancers, we explored the possibility as to whether mutations of FLASH and PTPN13 are a feature of colorectal carcinomas. We analysed human FLASH in exon 8 and PTPN13 in exon 7 for the detection of somatic mutations in 103 colorectal carcinomas by a polymerase chain reaction (PCR)- based single-strand conformation polymorphism (SSCP). We detected two mutations in FLASH gene, but none in PTPN13 gene. However, the two mutations were not frameshift (deletion or insertion) mutations in the polyadenine tracts of FLASH. The two mutations consisted of a deletion mutation (c.3734-3737delAGAA) and a missense mutation (c.3703A>C). These data indicate that frameshift mutation in the polyadenine tracts in both FLASH and PTPN13 genes is rare in colorectal carcinomas. Also, the data suggest that both FLASH and PTPN13 mutations in the polyadenine tracts may not have a crucial role in the pathogenesis of colorectal carcinomas.

VLITL is a major cross-β-sheet signal for fibrinogen Aα-chain frameshift variants

PubMed Central

Garnier, Cyrille; Briki, Fatma; Le Pogamp, Patrick; Dogan, Ahmet; Rioux-Leclercq, Nathalie; Goude, Renan; Beugnet, Caroline; Martin, Laurent; Delpech, Marc; Bridoux, Frank; Grateau, Gilles; Doucet, Jean

2017-01-01

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient’s kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for β-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-β-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis. PMID:29089309

Identification of a novel COL1A1 frameshift mutation, c.700delG, in a Chinese osteogenesis imperfecta family

PubMed Central

Wang, Xiran; Pei, Yu; Dou, Jingtao; Lu, Juming; Li, Jian; Lv, Zhaohui

2015-01-01

Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI. PMID:25983617

Mismatch repair deficiency commonly precedes adenoma formation in Lynch Syndrome-Associated colorectal tumorigenesis.

PubMed

Sekine, Shigeki; Mori, Taisuke; Ogawa, Reiko; Tanaka, Masahiro; Yoshida, Hiroshi; Taniguchi, Hirokazu; Nakajima, Takeshi; Sugano, Kokichi; Yoshida, Teruhiko; Kato, Mamoru; Furukawa, Eisaku; Ochiai, Atsushi; Hiraoka, Nobuyoshi

2017-08-01

Lynch syndrome is a cancer predisposition syndrome caused by germline mutations in mismatch repair (MMR) genes. MMR deficiency is a ubiquitous feature of Lynch syndrome-associated colorectal adenocarcinomas; however, it remains unclear when the MMR-deficient phenotype is acquired during tumorigenesis. To probe this issue, the present study examined genetic alterations and MMR statuses in Lynch syndrome-associated colorectal adenomas and adenocarcinomas, in comparison with sporadic adenomas. Among the Lynch syndrome-associated colorectal tumors, 68 of 86 adenomas (79%) and all adenocarcinomas were MMR-deficient, whereas all the sporadic adenomas were MMR-proficient, as determined by microsatellite instability testing and immunohistochemistry for MMR proteins. Sequencing analyses identified APC or CTNNB1 mutations in the majority of sporadic adenomas (58/84, 69%) and MMR-proficient Lynch syndrome-associated adenomas (13/18, 72%). However, MMR-deficient Lynch syndrome-associated adenomas had less APC or CTNNB1 mutations (25/68, 37%) and frequent frameshift RNF43 mutations involving mononucleotide repeats (45/68, 66%). Furthermore, frameshift mutations affecting repeat sequences constituted 14 of 26 APC mutations (54%) in MMR-deficient adenomas whereas these frameshift mutations were rare in MMR-proficient adenomas in patients with Lynch syndrome (1/12, 8%) and in sporadic adenomas (3/52, 6%). Lynch syndrome-associated adenocarcinomas exhibited mutation profiles similar to those of MMR-deficient adenomas. Considering that WNT pathway activation sufficiently drives colorectal adenoma formation, the distinct mutation profiles of WNT pathway genes in Lynch syndrome-associated adenomas suggest that MMR deficiency commonly precedes adenoma formation.

In vitro antibacterial activity of rifampicin in combination with imipenem, meropenem and doripenem against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

PubMed

Hu, Yi-Fan; Liu, Chang-Pan; Wang, Nai-Yu; Shih, Shou-Chuan

2016-08-24

Multidrug-resistant Pseudomonas aeruginosa has emerged as one of the most important healthcare-associated pathogens. Colistin is regarded as the last-resort antibiotic for multidrug-resistant Gram-negative bacteria, but is associated with high rates of acute kidney injury. The aim of this in vitro study is to search for an alternative treatment to colistin for multidrug-resistant P. aeruginosa infections. Multidrug and carbapenem-resistant P. aeruginosa isolates were collected between January 2009 and December 2012 at MacKay Memorial Hospital. Minimal inhibitory concentrations (MICs) were determined for various antibiotic combinations. Carbapenemase-producing genes including bla VIM, other β-lactamase genes and porin mutations were screened by PCR and sequencing. The efficacy of carbapenems (imipenem, meropenem, doripenem) with or without rifampicin was correlated with the type of porin mutation (frameshift mutation, premature stop codon mutation) in multidrug-resistant P. aeruginosa isolates without carbapenemase-producing genes. Of the 71 multidrug-resistant clinical P. aeruginosa isolates, only six harboured the bla VIM gene. Imipenem, meropenem and doripenem were significantly more effective (reduced fold-change of MICs) when combined with rifampicin in bla VIM-negative isolates, especially in isolates with porin frameshift mutation. Imipenem + rifampicin combination has a low MIC against multidrug-resistant P. aeruginosa, especially in isolates with porin frameshift mutation. The imipenem + rifampicin combination may provide an alternative treatment to colistin for multidrug -resistant P. aeruginosa infections, especially for patients with renal insufficiency.

The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

PubMed

Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

2009-07-13

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, R.C.

This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese's group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a groupmore » of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.« less

Automated insertion of sequences into a ribosomal RNA alignment: An application of computational linguistics in molecular biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, Ronald C.

This thesis involved the construction of (1) a grammar that incorporates knowledge on base invariancy and secondary structure in a molecule and (2) a parser engine that uses the grammar to position bases into the structural subunits of the molecule. These concepts were combined with a novel pinning technique to form a tool that semi-automates insertion of a new species into the alignment for the 16S rRNA molecule (a component of the ribosome) maintained by Dr. Carl Woese`s group at the University of Illinois at Urbana. The tool was tested on species extracted from the alignment and on a groupmore » of entirely new species. The results were very encouraging, and the tool should be substantial aid to the curators of the 16S alignment. The construction of the grammar was itself automated, allowing application of the tool to alignments for other molecules. The logic programming language Prolog was used to construct all programs involved. The computational linguistics approach used here was found to be a useful way to attach the problem of insertion into an alignment.« less

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

Revisiting the structures of several antibiotics bound to the bacterial ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bulkley, David; Innis, C. Axel; Blaha, Gregor

2010-10-08

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model.more » Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.« less

Revisiting the Structures of Several Antibiotics Bound to the Bacterial Ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

D Bulkley; C Innis; G Blaha

2011-12-31

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model.more » Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.« less

Ribosome flow model with positive feedback

PubMed Central

Margaliot, Michael; Tuller, Tamir

2013-01-01

Eukaryotic mRNAs usually form a circular structure; thus, ribosomes that terminatae translation at the 3′ end can diffuse with increased probability to the 5′ end of the transcript, initiating another cycle of translation. This phenomenon describes ribosomal flow with positive feedback—an increase in the flow of ribosomes terminating translating the open reading frame increases the ribosomal initiation rate. The aim of this paper is to model and rigorously analyse translation with feedback. We suggest a modified version of the ribosome flow model, called the ribosome flow model with input and output. In this model, the input is the initiation rate and the output is the translation rate. We analyse this model after closing the loop with a positive linear feedback. We show that the closed-loop system admits a unique globally asymptotically stable equilibrium point. From a biophysical point of view, this means that there exists a unique steady state of ribosome distributions along the mRNA, and thus a unique steady-state translation rate. The solution from any initial distribution will converge to this steady state. The steady-state distribution demonstrates a decrease in ribosome density along the coding sequence. For the case of constant elongation rates, we obtain expressions relating the model parameters to the equilibrium point. These results may perhaps be used to re-engineer the biological system in order to obtain a desired translation rate. PMID:23720534

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

PubMed

MacConnell, W P; Kaplan, N O

1982-05-25

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.

The translational landscape of Arabidopsis mitochondria.

PubMed

Planchard, Noelya; Bertin, Pierre; Quadrado, Martine; Dargel-Graffin, Céline; Hatin, Isabelle; Namy, Olivier; Mireau, Hakim

2018-06-05

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.

Structure of the ribosome post-recycling complex probed by chemical cross-linking and mass spectrometry

PubMed Central

Kiosze-Becker, Kristin; Ori, Alessandro; Gerovac, Milan; Heuer, André; Nürenberg-Goloub, Elina; Rashid, Umar Jan; Becker, Thomas; Beckmann, Roland; Beck, Martin; Tampé, Robert

2016-01-01

Ribosome recycling orchestrated by the ATP binding cassette (ABC) protein ABCE1 can be considered as the final—or the first—step within the cyclic process of protein synthesis, connecting translation termination and mRNA surveillance with re-initiation. An ATP-dependent tweezer-like motion of the nucleotide-binding domains in ABCE1 transfers mechanical energy to the ribosome and tears the ribosome subunits apart. The post-recycling complex (PRC) then re-initiates mRNA translation. Here, we probed the so far unknown architecture of the 1-MDa PRC (40S/30S·ABCE1) by chemical cross-linking and mass spectrometry (XL-MS). Our study reveals ABCE1 bound to the translational factor-binding (GTPase) site with multiple cross-link contacts of the helix–loop–helix motif to the S24e ribosomal protein. Cross-linking of the FeS cluster domain to the ribosomal protein S12 substantiates an extreme lever-arm movement of the FeS cluster domain during ribosome recycling. We were thus able to reconstitute and structurally analyse a key complex in the translational cycle, resembling the link between translation initiation and ribosome recycling. PMID:27824037

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Dyke, Natalya; Chanchorn, Ekkawit; Van Dyke, Michael W., E-mail: mvandyke@email.wcu.edu

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 processmore » 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.« less

Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

PubMed

Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P

2018-01-19

A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

Ribosome protection by antibiotic resistance ATP-binding cassette protein.

PubMed

Su, Weixin; Kumar, Veerendra; Ding, Yichen; Ero, Rya; Serra, Aida; Lee, Benjamin Sian Teck; Wong, Andrew See Weng; Shi, Jian; Sze, Siu Kwan; Yang, Liang; Gao, Yong-Gui

2018-05-15

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins. Copyright © 2018 the Author(s). Published by PNAS.

Crystallization of the two-domain N-terminal fragment of the archaeal ribosomal protein L10(P0) in complex with a specific fragment of 23S rRNA

NASA Astrophysics Data System (ADS)

Kravchenko, O. V.; Mitroshin, I. V.; Gabdulkhakov, A. G.; Nikonov, S. V.; Garber, M. B.

2011-07-01

Lateral L12-stalk (P1-stalk in Archaea, P1/P2-stalk in eukaryotes) is an obligatory morphological element of large ribosomal subunits in all organisms studied. This stalk is composed of the complex of ribosomal proteins L10(P0) and L12(P1) and interacts with 23S rRNA through the protein L10(P0). L12(P1)-stalk is involved in the formation of GTPase center of the ribosome and plays an important role in the ribosome interaction with translation factors. High mobility of this stalk puts obstacles in determination of its structure within the intact ribosome. Crystals of a two-domain N-terminal fragment of ribosomal protein L10(P0) from the archaeon Methanococcus jannaschii in complex with a specific fragment of rRNA from the same organism have been obtained. The crystals diffract X-rays at 3.2 Å resolution.

Modular Assembly of the Bacterial Large Ribosomal Subunit.

PubMed

Davis, Joseph H; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S; Lyumkis, Dmitry; Williamson, James R

2016-12-01

The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ∼4-5 Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be "re-routed" through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. Copyright © 2016 Elsevier Inc. All rights reserved.

Modular Assembly of the Bacterial Large Ribosomal Subunit

PubMed Central

Davis, Joseph H.; Tan, Yong Zi; Carragher, Bridget; Potter, Clinton S.; Lyumkis, Dmitry; Williamson, James R.

2016-01-01

SUMMARY The ribosome is a complex macromolecular machine and serves as an ideal system for understanding biological macromolecular assembly. Direct observation of ribosome assembly in vivo is difficult, as few intermediates have been isolated and thoroughly characterized. Herein, we deploy a genetic system to starve cells of an essential ribosomal protein, which results in the accumulation of assembly intermediates that are competent for maturation. Quantitative mass spectrometry and single-particle cryo-electron microscopy reveal 13 distinct intermediates, which were each resolved to ~4–5Å resolution and could be placed in an assembly pathway. We find that ribosome biogenesis is a parallel process, that blocks of structured rRNA and proteins assemble cooperatively, and that the entire process is dynamic and can be ‘re-routed’ through different pathways as needed. This work reveals the complex landscape of ribosome assembly in vivo and provides the requisite tools to characterize additional assembly pathways for ribosomes and other macromolecular machines. PMID:27912064

Targeting ricin to the ribosome.

PubMed

May, Kerrie L; Yan, Qing; Tumer, Nilgun E

2013-07-01

The plant toxin ricin is highly toxic for mammalian cells and is of concern for bioterrorism. Ricin belongs to a family of functionally related toxins, collectively referred to as ribosome inactivating proteins (RIPs), which disable ribosomes and halt protein synthesis. Currently there are no specific antidotes against ricin or related RIPs. The catalytic subunit of ricin is an N-glycosidase that depurinates a universally conserved adenine residue within the sarcin/ricin loop (SRL) of the 28S rRNA. This depurination activity inhibits translation and its biochemistry has been intensively studied. Yet, recent developments paint a more complex picture of toxicity, with ribosomal proteins and cellular signaling pathways contributing to the potency of ricin. In particular, several studies have now established the importance of the ribosomal stalk structure in facilitating the depurination activity and ribosome specificity of ricin and other RIPs. This review highlights recent developments defining toxin-ribosome interactions and examines the significance of these interactions for toxicity and therapeutic intervention. Copyright © 2013 Elsevier Ltd. All rights reserved.

The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Auerbach, Tamar; Mermershtain, Inbal; Davidovich, Chen

2010-04-26

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, themore » streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.« less

Eukaryotic ribosome display with in situ DNA recovery.

PubMed

He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jetzt, Amanda E.

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarlymore » to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. - Highlights: • Arginines 193 and 235 of RTA are critical for binding to the mammalian ribosome. • R193A/R235A has full catalytic activity on RNA but not on mammalian ribosomes. • R193A/R235A is less toxic than a mutant that targets the active site. • The toxin-ribosome interaction is a therapeutic target for ricin intoxication.« less

Chromatographic method for the isolation of active 40S and 60S subunits from rat liver polyribosomes.

PubMed

Kopacz-Jodczyk, T; Paszkiewicz-Gadek, A; Lopaczyński, W; Gałasiński, W

1984-04-06

A rapid and simple procedure for isolation of 40S and 60S ribosomal subunits by ion-exchange column chromatography is described. The dissociated ribosomes can be separated and non-ribosomal proteins and low-molecular-weight substances removed. An assessment by physicochemical and functional criteria showed that the ribosomal subunits obtained are active and sufficiently homogeneous.

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

A Listeria monocytogenes RNA helicase essential for growth and ribosomal maturation at low temperatures uses its C terminus for appropriate interaction with the ribosome.

PubMed

Netterling, Sakura; Vaitkevicius, Karolis; Nord, Stefan; Johansson, Jörgen

2012-08-01

Listeria monocytogenes, a Gram-positive food-borne human pathogen, is able to grow at temperatures close to 0°C and is thus of great concern for the food industry. In this work, we investigated the physiological role of one DExD-box RNA helicase in Listeria monocytogenes. The RNA helicase Lmo1722 was required for optimal growth at low temperatures, whereas it was dispensable at 37°C. A Δlmo1722 strain was less motile due to downregulation of the major subunit of the flagellum, FlaA, caused by decreased flaA expression. By ribosomal fractionation experiments, it was observed that Lmo1722 was mainly associated with the 50S subunit of the ribosome. Absence of Lmo1722 decreased the fraction of 50S ribosomal subunits and mature 70S ribosomes and affected the processing of the 23S precursor rRNA. The ribosomal profile could be restored to wild-type levels in a Δlmo1722 strain expressing Lmo1722. Interestingly, the C-terminal part of Lmo1722 was redundant for low-temperature growth, motility, 23S rRNA processing, and appropriate ribosomal maturation. However, Lmo1722 lacking the C terminus showed a reduced affinity for the 50S and 70S fractions, suggesting that the C terminus is important for proper guidance of Lmo1722 to the 50S subunit. Taken together, our results show that the Listeria RNA helicase Lmo1722 is essential for growth at low temperatures, motility, and rRNA processing and is important for ribosomal maturation, being associated mainly with the 50S subunit of the ribosome.

Ribosomal trafficking is reduced in Schwann cells following induction of myelination.

PubMed

Love, James M; Shah, Sameer B

2015-01-01

Local synthesis of proteins within the Schwann cell periphery is extremely important for efficient process extension and myelination, when cells undergo dramatic changes in polarity and geometry. Still, it is unclear how ribosomal distributions are developed and maintained within Schwann cell projections to sustain local translation. In this multi-disciplinary study, we expressed a plasmid encoding a fluorescently labeled ribosomal subunit (L4-GFP) in cultured primary rat Schwann cells. This enabled the generation of high-resolution, quantitative data on ribosomal distributions and trafficking dynamics within Schwann cells during early stages of myelination, induced by ascorbic acid treatment. Ribosomes were distributed throughout Schwann cell projections, with ~2-3 bright clusters along each projection. Clusters emerged within 1 day of culture and were maintained throughout early stages of myelination. Three days after induction of myelination, net ribosomal movement remained anterograde (directed away from the Schwann cell body), but ribosomal velocity decreased to about half the levels of the untreated group. Statistical and modeling analysis provided additional insight into key factors underlying ribosomal trafficking. Multiple regression analysis indicated that net transport at early time points was dependent on anterograde velocity, but shifted to dependence on anterograde duration at later time points. A simple, data-driven rate kinetics model suggested that the observed decrease in net ribosomal movement was primarily dictated by an increased conversion of anterograde particles to stationary particles, rather than changes in other directional parameters. These results reveal the strength of a combined experimental and theoretical approach in examining protein localization and transport, and provide evidence of an early establishment of ribosomal populations within Schwann cell projections with a reduction in trafficking following initiation of myelination.

A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death

PubMed Central

McCall, Ingrid C.; Perrot, Véronique; Weiss, Howard; Ovesepian, Armen; Baquero, Fernando

2017-01-01

ABSTRACT We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures. PMID:28174311

Ribonucleic Acid and Ribosomes of Bacillus stearothermophilus1

PubMed Central

Saunders, Grady F.; Campbell, L. Leon

1966-01-01

Saunders, Grady F. (University of Illinois, Urbana), and L. Leon Campbell. Ribonucleic acid and ribosomes of Bacillus stearothermophilus. J. Bacteriol. 91:332–339. 1966.—The ability of some thermophilic bacteria to grow at temperatures as high as 76 C emphasizes the remarkable thermal stability of their crucial macromolecules. An investigation of the ribonucleic acid (RNA) and ribosomes of Bacillus stearothermophilus was conducted. Washed log-phase cells were disrupted either by sonic treatment or by alumina grinding in 10−2m MgCl2–10−2m tris-(hydroxymethyl)aminomethane buffer, pH 7.4 (TM buffer). Ultracentrifugal analysis revealed peaks at 72.5S, 101S, and 135S, with the 101S peak being the most prominent. By lowering the Mg++ concentration to 10−3m, the ribosome preparation was dissociated to give 40S, 31S, and 54S peaks. These in turn were reassociated in the presence of 10−2m Mg++ to give the larger 73S and 135S particles. When heated in TM buffer, Escherichia coli ribosomes began a gradual dissociation at 58 C, and at 70 C underwent a large hyperchromic shift with a Tm at 72.8 C. In contrast, B. stearothermophilus ribosomes did not show a hyperchromic shift below 70 C; they had a Tm of 77.9 C. The thermal denaturation curves of the 4S, 16S, and 23S RNA from both organisms were virtually identical. The gross amino acid composition of B. stearothermophilus ribosomes showed no marked differences from that reported for E. coli ribosomes. These data suggest that the unusual thermal stability of B. stearothermophilus ribosomes may reflect either an unusual packing arrangement of the protein to the RNA or differences in the primary structure of the ribosomal proteins. Images PMID:5903099

Characterization of Founder Viruses in Very Early SIV Rectal Transmission

PubMed Central

Yuan, Zhe; Ma, Fangrui; Demers, Andrew J.; Wang, Dong; Xu, Jianqing; Lewis, Mark G.; Li, Qingsheng

2016-01-01

A better understanding of HIV-1 transmission is critical for developing preventative strategies. To that end, we analyzed 524 full-length env sequences of SIVmac251 at 6 and 10 days post intrarectal infection of rhesus macaques. There was no tissue compartmentalization of founder viruses across plasma, rectal and distal lymphatic tissues for most animals; however one animal has evidence of virus tissue compartmentalization. Despite identical viral inoculums, founder viruses were animal-specific, primarily derived from rare variants in the inoculum, and have a founder virus signature that can distinguish dominant founder variants from minor founder or untransmitted variants in the inoculum. Importantly, the sequences of post-transmission defective viruses were phylogenetically associated with competent viral variants in the inoculum and were mainly converted from competent viral variants by frameshift rather than APOBEC mediated mutations, suggesting the converting the transmitted viruses into defective viruses through frameshift mutation is an important component of rectal transmission bottleneck. PMID:28027479

Rare Compound Heterozygous Frameshift Mutations in ALMS1 Gene Identified Through Exome Sequencing in a Taiwanese Patient With Alström Syndrome.

PubMed

Tsai, Meng-Che; Yu, Hui-Wen; Liu, Tsunglin; Chou, Yen-Yin; Chiou, Yuan-Yow; Chen, Peng-Chieh

2018-01-01

Alström syndrome (AS) is a rare autosomal recessive disorder that shares clinical features with other ciliopathy-related diseases. Genetic mutation analysis is often required in making differential diagnosis but usually costly in time and effort using conventional Sanger sequencing. Herein we describe a Taiwanese patient presenting cone-rod dystrophy and early-onset obesity that progressed to diabetes mellitus with marked insulin resistance during adolescence. Whole exome sequencing of the patient's genomic DNA identified a novel frameshift mutation in exons 15 (c.10290_10291delTA, p.Lys3431Serfs * 10) and a rare mutation in 16 (c.10823_10824delAG, p.Arg3609Alafs * 6) of ALMS1 gene. The compound heterozygous mutations were predicted to render truncated proteins. This report highlighted the clinical utility of exome sequencing and extended the knowledge of mutation spectrum in AS patients.

Balancing Selection of a Frame-Shift Mutation in the MRC2 Gene Accounts for the Outbreak of the Crooked Tail Syndrome in Belgian Blue Cattle

PubMed Central

Li, Wanbo; Dive, Marc; Tamma, Nico; Michaux, Charles; Druet, Tom; Huijbers, Ivo J.; Isacke, Clare M.; Coppieters, Wouter; Georges, Michel; Charlier, Carole

2009-01-01

We herein describe the positional identification of a 2-bp deletion in the open reading frame of the MRC2 receptor causing the recessive Crooked Tail Syndrome in cattle. The resulting frame-shift reveals a premature stop codon that causes nonsense-mediated decay of the mutant messenger RNA, and the virtual absence of functional Endo180 protein in affected animals. Cases exhibit skeletal anomalies thought to result from impaired extracellular matrix remodeling during ossification, and as of yet unexplained muscular symptoms. We demonstrate that carrier status is very significantly associated with desired characteristics in the general population, including enhanced muscular development, and that the resulting heterozygote advantage caused a selective sweep which explains the unexpectedly high frequency (25%) of carriers in the Belgian Blue Cattle Breed. PMID:19779552

A novel NDUFS4 frameshift mutation causes Leigh disease in the Hutterite population.

PubMed

Lamont, Ryan E; Beaulieu, Chandree L; Bernier, Francois P; Sparkes, Rebecca; Innes, A Micheil; Jackel-Cram, Candice; Ober, Carole; Parboosingh, Jillian S; Lemire, Edmond G

2017-03-01

Leigh disease is a progressive, infantile-onset, neurodegenerative disorder characterized by feeding difficulties, failure to thrive, hypotonia, seizures, and central respiratory compromise. Metabolic and neuroimaging investigations typically identify abnormalities consistent with a disorder of mitochondrial energy metabolism. Mutations in more than 35 genes affecting the mitochondrial respiratory chain encoded from both the nuclear and mitochondrial genomes have been associated with Leigh disease. The clinical presentations of five individuals of Hutterite descent with Leigh disease are described herein. An identity-by-descent mapping and candidate gene approach was used to identify a novel homozygous c.393dupA frameshift mutation in the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (NDUFS4) gene. The carrier frequency of this mutation was estimated in >1,300 Hutterite individuals to be 1 in 27. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harada, Y.; Li, H.; Li, Hua

2009-04-28

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of {approx}1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings,more » we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.« less

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

PubMed Central

Harada, Yoichiro; Li, Hua; Li, Huilin; Lennarz, William J.

2009-01-01

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes. PMID:19365066

Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

PubMed Central

Ochs, Kerstin; Rust, René C.; Niepmann, Michael

1999-01-01

Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. PMID:10438840

Ribosomes are optimized for autocatalytic production

NASA Astrophysics Data System (ADS)

Reuveni, Shlomi; Ehrenberg, Måns; Paulsson, Johan

2017-07-01

Many fine-scale features of ribosomes have been explained in terms of function, revealing a molecular machine that is optimized for error-correction, speed and control. Here we demonstrate mathematically that many less well understood, larger-scale features of ribosomes—such as why a few ribosomal RNA molecules dominate the mass and why the ribosomal protein content is divided into 55-80 small, similarly sized segments—speed up their autocatalytic production.

Comparison of preribosomal RNA processing pathways in yeast, plant and human cells - focus on coordinated action of endo- and exoribonucleases.

PubMed

Tomecki, Rafal; Sikorski, Pawel J; Zakrzewska-Placzek, Monika

2017-07-01

Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo- and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans. © 2017 Federation of European Biochemical Societies.

Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation.

PubMed

Artieri, Carlo G; Fraser, Hunter B

2014-12-01

The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

[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.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

PubMed

Hussmann, Jeffrey A; Patchett, Stephanie; Johnson, Arlen; Sawyer, Sara; Press, William H

2015-12-01

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast

PubMed Central

Hussmann, Jeffrey A.; Patchett, Stephanie; Johnson, Arlen; Sawyer, Sara; Press, William H.

2015-01-01

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics. PMID:26656907

Clinical Investigation Program.

DTIC Science & Technology

1985-10-01

Patients with Nasopharyngeal Carcinoma (NPC) and Determination of the Effect of IFN on Epstein - Barr Virus (EBV)-related Immunological Markers (0) (P...with Nasopharyngeal Carcinoma (NPC) and Determination of the Effect of IFN on Epstein - Barr virus (EBV)-related Immunological Markers Principal...Ribosomal Vaccine (0) (PR) (P) 12A/84 Lieberman, M. M. Relationship of the Immune 26 Response to the Heat Sensitivity of the Moloney Virus -Induced YAC

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

PubMed

Fuchs, Gabriele; Petrov, Alexey N; Marceau, Caleb D; Popov, Lauren M; Chen, Jin; O'Leary, Seán E; Wang, Richard; Carette, Jan E; Sarnow, Peter; Puglisi, Joseph D

2015-01-13

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Multicentric prevalence study of anti P ribosomal autoantibodies in juvenile onset systemic lupus erythematosus compared with adult onset systemic lupus erythematosus.

PubMed

Pisoni, Cecilia N; Muñoz, Sebastián Andrés; Carrizo, Carolina; Cosatti, Micaela; Álvarez, Analía; Dubinsky, Diana; Bresan, Eleonora; Russo, Ricardo; Borgia, Ezequiel; García, Mercedes; Sansinanea, Pierina; Basta, María Cristina; D'Amico, Maria Agustina; Barreira, Juan Carlos; Lancioni, Eliana; Soriano, Enrique; Cunto, Carmen de; Beron, Ana; Eimon, Alicia

2015-01-01

To investigate the prevalence and associations with clinical manifestations of anti- P ribosomal antibodies in patients with juvenile-onset and adult-onset systemic lupus erythematosus (SLE). Clinical and serological data of 30 patients with juvenile-onset SLE (age at onset younger than 16 years old) were compared with data of 92 patients with adult-onset SLE. Symptoms occurring during the entire disease course were considered. Anti- P ribosomal antibodies were tested by ELISA. Anti- P ribosomal antibodies were found significantly more often in pediatric-onset SLE patients (26.7% vs. 6.5%; OR=5.21 [CI95%=1.6-16.5], p=0.003). Alopecia (OR=10.11, CI 95%=1.25-97) and skin rash (non discoid) (OR=4.1, CI 95%=1.25-13.89) were significantly associated with anti- P ribosomal antibodies. Anti-ribosomal P antibodies are more often found in patients with juvenile SLE. Alopecia and skin rash were the only clinical manifestations associated to anti-ribosomal P antibodies. Copyright © 2013 Elsevier España, S.L.U. All rights reserved.

The 5S RNP Couples p53 Homeostasis to Ribosome Biogenesis and Nucleolar Stress

PubMed Central

Sloan, Katherine E.; Bohnsack, Markus T.; Watkins, Nicholas J.

2013-01-01

Summary Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14ARF, a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production. PMID:24120868

Mechanisms of ribosome stalling by SecM at multiple elongation steps

PubMed Central

Zhang, Jun; Pan, Xijiang; Yan, Kaige; Sun, Shan; Gao, Ning; Sui, Sen-Fang

2015-01-01

Regulation of translating ribosomes is a major component of gene expression control network. In Escherichia coli, ribosome stalling by the C-terminal arrest sequence of SecM regulates the SecA-dependent secretion pathway. Previous studies reported many residues of SecM peptide and ribosome exit tunnel are critical for stalling. However, the underlying molecular mechanism is still not clear at the atomic level. Here, we present two cryo-EM structures of the SecM-stalled ribosomes at 3.3–3.7 Å resolution, which reveal two different stalling mechanisms at distinct elongation steps of the translation cycle: one is due to the inactivation of ribosomal peptidyl-transferase center which inhibits peptide bond formation with the incoming prolyl-tRNA; the other is the prolonged residence of the peptidyl-RNA at the hybrid A/P site which inhibits the full-scale tRNA translocation. These results demonstrate an elegant control of translation cycle by regulatory peptides through a continuous, dynamic reshaping of the functional center of the ribosome. DOI: http://dx.doi.org/10.7554/eLife.09684.001 PMID:26670735

Sir2a regulates rDNA transcription and multiplication rate in the human malaria parasite Plasmodium falciparum

PubMed Central

Mancio-Silva, Liliana; Lopez-Rubio, Jose Juan; Claes, Aurélie; Scherf, Artur

2013-01-01

The Plasmodium falciparum histone deacetylase Sir2a localizes at telomeric regions where it contributes to epigenetic silencing of clonally variant virulence genes. Apart from telomeres, PfSir2a also accumulates in the nucleolus, which harbours the developmentally regulated ribosomal RNA genes. Here we investigate the nucleolar function of PfSir2a and demonstrate that PfSir2a fine-tunes ribosomal RNA gene transcription. Using a parasite line in which PfSir2a has been disrupted, we observe that histones near the transcription start sites of all ribosomal RNA genes are hyperacetylated and that transcription of ribosomal RNA genes is upregulated. Complementation of the PfSir2a-disrupted parasites restores the ribosomal RNA levels, whereas PfSir2a overexpression in wild-type parasites decreases ribosomal RNA synthesis. Furthermore, we observe that PfSir2a modulation of ribosomal RNA synthesis is linked to an altered number of daughter merozoites and the parasite multiplication rate. These findings provide new insights into an epigenetic mechanism that controls malaria parasite proliferation and virulence. PMID:23443558

Identification of MICA alleles with a long Leu-repeat in the transmembrane region and no cytoplasmic tail due to a frameshift-deletion in exon 4.

PubMed

Obuchi, N; Takahashi, M; Nouchi, T; Satoh, M; Arimura, T; Ueda, K; Akai, J; Ota, M; Naruse, T; Inoko, H; Numano, F; Kimura, A

2001-06-01

MHC class I chain-related gene A (MICA) is located close to HLA-B gene and expressed in epithelial cells. The MICA gene is reported to be highly polymorphic as are the classical class I genes. To further assess the polymorphism in the MICA gene, we analyzed a total of 60 HLA-homozygous cells for the sequences spanning exons 2-6. In the analysis, four new MICA alleles were identified and six variations were recognized in exon 6. MICA*017, which was identified in three HLA-B57 homozygous cells (DBB, DEM and WIN), differed from MICA*002 in exon 3 and had a guanine deletion at the 3' end of exon 4. MICA*015 identified in an HLA-B45 homozygous cell (OMW) also had the same deletion that causes a frameshift mutation resulting in complete change of the transmembrane region and premature termination in the cytoplasmic tail; these alleles have a long hydrophobic leucine-rich region instead of the alanine repeat in the transmembrane region and terminate at the second position in the cytoplasmic domain. The frameshift deletion was found only in HLA-B45- or -B57-positive panels tested, suggesting a strong linkage disequilibrium between the deletion and B45 or B57. MICA*048, which was different in exon 5 from MICA*008, was identified in an HLA-B61 homozygous cell (TA21), while MICA*00901 identified in HLA-B51 homozygous cells (LUY and KT2) was distinguished from MICA*009 by exon 6.

Differentiation of the Ribosomal Protein Compositions in the Genus Escherichia and Its Related Bacteria

PubMed Central

Osawa, Syozo; Itoh, Takuzi; Otaka, Eiko

1971-01-01

Compositions of the ribosomal proteins of 60 bacterial strains belonging to the genus Escherichia and its related genera were examined by use of a column of carboxymethyl cellulose. The ribosomes were classified into seven groups and were further differentiated into several types (subgroups) according to their protein compositions. It was shown that ribosomal protein composition is a useful characteristic for studies of bacterial taxonomy. PMID:5563866

Cis-regulatory RNA elements that regulate specialized ribosome activity.

PubMed

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.

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

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

PubMed

Su, Ting; Cheng, Jingdong; Sohmen, Daniel; Hedman, Rickard; Berninghausen, Otto; von Heijne, Gunnar; Wilson, Daniel N; Beckmann, Roland

2017-05-30

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.

Recent studies implicate the nucleolus as the major site of nuclear translation.

PubMed

McLeod, Tina; Abdullahi, Akilu; Li, Min; Brogna, Saverio

2014-08-01

The nucleolus is the most prominent morphological feature within the nucleus of eukaryotic cells and is best known for its role in ribosome biogenesis. It forms around highly transcribed ribosomal RNA gene repeats which yield precursor rRNAs that are co-transcriptionally processed, folded and, while still within the nucleolus, associate with most of the ribosomal proteins. The nucleolus is therefore often thought of as a factory for making ribosomal subunits, which are exported as inactive precursors to the cytoplasm where late maturation makes them capable of mRNA binding and translation initiation. However, recent studies have shown substantial evidence for the presence of functional, translation competent ribosomal subunits within the nucleus, particularly in the nucleolus. These observations raise the intriguing possibility that the nucleolus, as well as being a ribosome factory, is also an important nuclear protein-synthesis plant.

The AAA-ATPase NVL2 is a component of pre-ribosomal particles that interacts with the DExD/H-box RNA helicase DOB1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagahama, Masami; Yamazoe, Takeshi; Hara, Yoshimitsu

2006-08-04

Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase,more » whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone.« less

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

PubMed

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R; Raikhel, Natasha V

2015-01-06

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

Ribosomes: Ribozymes that Survived Evolution Pressures but Is Paralyzed by Tiny Antibiotics

NASA Astrophysics Data System (ADS)

Yonath, Ada

An impressive number of crystal structures of ribosomes, the universal cellular machines that translate the genetic code into proteins, emerged during the last decade. The determination of ribosome high resolution structure, which was widely considered formidable, led to novel insights into the ribosomal function, namely, fidelity, catalytic mechanism, and polymerize activities. They also led to suggestions concerning its origin and shed light on the action, selectivity and synergism of ribosomal antibiotics; illuminated mechanisms acquiring bacterial resistance and provided structural information for drug improvement and design. These studies required the pioneering and implementation of advanced technologies, which directly influenced the remarkable increase of the number of structures deposited in the Protein Data Bank.

Differences in the ribosomes prepared from lactating and non-lactating bovine mammary gland

PubMed Central

Herrington, M. D.; Hawtrey, A. O.

1971-01-01

1. Ribosomes prepared from bovine lactating mammary gland are able to synthesize protein, whereas similar preparations from non-lactating glands are not. Washing the ribosome suspensions through a medium containing 0.5m-ammonium chloride enhanced their ability to incorporate phenylalanine into polyphenylalanine. 2. Ribosomes isolated from non-lactating bovine mammary gland, in contrast with those from rat liver and lactating mammary gland, contained significant amounts of extraneous nucleases. These enzymes could be removed by washing with a medium A buffer containing 0.5m-ammonium chloride. 3. Only those ribosomes from functionally active tissues were able to bind polyuridylic acid and phenylalanyl-tRNA. PMID:5165653

New Fluorescent Macrolide Derivatives for Studying Interactions of Antibiotics and Their Analogs with the Ribosomal Exit Tunnel.

PubMed

Tereshchenkov, A G; Shishkina, A V; Karpenko, V V; Chertkov, V A; Konevega, A L; Kasatsky, P S; Bogdanov, A A; Sumbatyan, N V

2016-10-01

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

The Unexplored Mechanisms and Regulatory Functions of Ribosomal Translocation

NASA Astrophysics Data System (ADS)

Alejo, Jose Luis

In every cell, protein synthesis is carried out by the ribosome, a complex macromolecular RNA-protein assembly. Decades of structural and kinetic studies have increased our understanding of ribosome initiation, decoding, translocation and termination. Yet, the underlying mechanism of these fundamental processes has yet to be fully delineated. Hence, the molecular basis of regulation remains obscure. Here, single-molecule fluorescence methods are applied to decipher the mechanism and regulatory roles of the multi-step process of directional substrate translocation on the ribosome that accompanies every round of protein synthesis. In Chapter 1, single-molecule fluorescence resonance energy transfer (smFRET) is introduced as a tool for studying bacterial ribosome translocation. Chapter 2 details the experimental methods. In Chapter 3, the elongation factor G(EF-G)-catalyzed movement of substrates through the ribosome is examined from several perspectives or signals reporting on various degrees of freedom of ribosome dynamics. Two ribosomal states interconvert in the presence of EF-G(GDP), displaying novel head domain motions, until relocking takes place. In Chapter 4, in order to test if the mentioned fluctuations leading to relocking are correlated to the engagement of the P-site by the peptidyl-tRNA, the translocation of miscoded tRNAs is studied. Severe defects in the relocking stages of translocation reveal the correlation between this new stage of translocation and P-site tRNA engagement.

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.

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

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

Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome

PubMed Central

Arenz, Stefan; Graf, Michael; Nguyen, Fabian; Huter, Paul; Polikanov, Yury S.; Blanchard, Scott C.; Wilson, Daniel N.

2016-01-01

The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis. PMID:27330110

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

PubMed Central

Lindström, Mikael S.

2012-01-01

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site. PMID:23285058

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome.

PubMed

Jetzt, Amanda E; Li, Xiao-Ping; Tumer, Nilgun E; Cohick, Wendie S

2016-11-01

Ricin is a potent ribotoxin that is considered a bioterror threat due to its ease of isolation and possibility of aerosolization. In yeast, mutation of arginine residues away from the active site results in a ricin toxin A chain (RTA) variant that is unable to bind the ribosome and exhibits reduced cytotoxicity. The goal of the present work was to determine if these residues contribute to ribosome binding and cytotoxicity of RTA in mammalian cells. The RTA mutant R193A/R235A did not interact with mammalian ribosomes, while a G212E variant with a point mutation near its active site bound ribosomes similarly to wild-type (WT) RTA. R193A/R235A retained full catalytic activity on naked RNA but had reduced activity on mammalian ribosomes. To determine the effect of this mutant in intact cells, pre R193A/R235A containing a signal sequence directing it to the endoplasmic reticulum and mature R193A/R235A that directly targeted cytosolic ribosomes were each expressed. Depurination and protein synthesis inhibition were reduced by both pre- and mature R193A/R235A relative to WT. Protein synthesis inhibition was reduced to a greater extent by R193A/R235A than by G212E. Pre R193A/R235A caused a greater reduction in caspase activation and loss of mitochondrial membrane potential than G212E relative to WT RTA. These findings indicate that an RTA variant with reduced ribosome binding is less toxic than a variant with less catalytic activity but normal ribosome binding activity. The toxin-ribosome interaction represents a novel target for the development of therapeutics to prevent or treat ricin intoxication. Copyright © 2016 Elsevier Inc. All rights reserved.

The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes.

PubMed

Schäfer, Thorsten; Strauss, Daniela; Petfalski, Elisabeth; Tollervey, David; Hurt, Ed

2003-03-17

Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.

Structure of ratcheted ribosomes with tRNAs in hybrid states

PubMed Central

Julián, Patricia; Konevega, Andrey L.; Scheres, Sjors H. W.; Lázaro, Melisa; Gil, David; Wintermeyer, Wolfgang; Rodnina, Marina V.; Valle, Mikel

2008-01-01

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G. PMID:18971332

Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries.

PubMed

Lamichhane, Tek N; Abeydeera, N Dinuka; Duc, Anne-Cécile E; Cunningham, Philip R; Chow, Christine S

2011-01-28

Ribosomal RNA is the catalytic portion of ribosomes, and undergoes a variety of conformational changes during translation. Structural changes in ribosomal RNA can be facilitated by the presence of modified nucleotides. Helix 31 of bacterial 16S ribosomal RNA harbors two modified nucleotides, m²G966 and m⁵C967, that are highly conserved among bacteria, though the degree and nature of the modifications in this region are different in eukaryotes. Contacts between helix 31 and the P-site tRNA, initiation factors, and ribosomal proteins highlight the importance of this region in translation. In this work, a heptapeptide M13 phage-display library was screened for ligands that target the wild-type, naturally modified bacterial helix 31. Several peptides, including TYLPWPA, CVRPFAL, TLWDLIP, FVRPFPL, ATPLWLK, and DIRTQRE, were found to be prevalent after several rounds of screening. Several of the peptides exhibited moderate affinity (in the high nM to low µM range) to modified helix 31 in biophysical assays, including surface plasmon resonance (SPR), and were also shown to bind 30S ribosomal subunits. These peptides also inhibited protein synthesis in cell-free translation assays.

Selective ribosome profiling as a tool to study the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

PubMed Central

Becker, Annemarie H.; Oh, Eugene; Weissman, Jonathan S.; Kramer, Günter; Bukau, Bernd

2014-01-01

A plethora of factors is involved in the maturation of newly synthesized proteins, including chaperones, membrane targeting factors, and enzymes. Many factors act cotranslationally through association with ribosome-nascent chain complexes (RNCs), but their target specificities and modes of action remain poorly understood. We developed selective ribosome profiling (SeRP) to identify substrate pools and points of RNC engagement of these factors. SeRP is based on sequencing mRNA fragments covered by translating ribosomes (general ribosome profiling, RP), combined with a procedure to selectively isolate RNCs whose nascent polypeptides are associated with the factor of interest. Factor–RNC interactions are stabilized by crosslinking, the resulting factor–RNC adducts are then nuclease-treated to generate monosomes, and affinity-purified. The ribosome-extracted mRNA footprints are converted to DNA libraries for deep sequencing. The protocol is specified for general RP and SeRP in bacteria. It was first applied to the chaperone trigger factor and is readily adaptable to other cotranslationally acting factors, including eukaryotic factors. Factor–RNC purification and sequencing library preparation takes 7–8 days, sequencing and data analysis can be completed in 5–6 days. PMID:24136347

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

PubMed

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

2016-10-13

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.

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.

The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress.

PubMed

Sloan, Katherine E; Bohnsack, Markus T; Watkins, Nicholas J

2013-10-17

Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, I. Non-Mendelian Mutations*

PubMed Central

Gillham, Nicholas W.; Boynton, John E.; Burkholder, Barbara

1970-01-01

Uniparentally inherited mutations to antibiotic resistance and dependence in Chlamydomonas reinhardi exhibit an altered chloroplast ribosome phenotype. Genetic studies demonstrate an absolute correlation between the drug resistance or dependence and the ribosome phenotype in two such mutants. Images PMID:5289000

LOCAL TRANSLATION. Comment on "Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling".

PubMed

Reid, David W; Nicchitta, Christopher V

2015-06-12

Jan et al. (Research Articles, 7 November 2014, p. 716) propose that ribosomes translating secretome messenger RNAs (mRNAs) traffic from the cytosol to the endoplasmic reticulum (ER) upon emergence of the signal peptide and return to the cytosol after termination. An accounting of controls demonstrates that mRNAs initiate translation on ER-bound ribosomes and that ribosomes are retained on the ER through many cycles of translation. Copyright © 2015, American Association for the Advancement of Science.

Ribosomal RNA and ribosomal proteins in corynebacteria.

PubMed

Martín, Juan F; Barreiro, Carlos; González-Lavado, Eva; Barriuso, Mónica

2003-09-04

Ribosomal RNAs (rRNAs) (16S, 23S, 5S) encoded by the rrn operons and ribosomal proteins play a very important role in the formation of ribosomes and in the control of translation. Five copies of the rrn operon were reported by hybridization studies in Brevibacterium (Corynebacterium) lactofermentum but the genome sequence of Corynebacterium glutamicum provided evidence for six rrn copies. All six copies of the C. glutamicum 16S rRNA have a size of 1523 bp and each of the six copies of the 5S contain 120 bp whereas size differences are found between the six copies of the 23S rRNA. The anti-Shine-Dalgarno sequence at the 3'-end of the 16S rRNA was 5'-CCUCCUUUC-3'. Each rrn operon is transcribed as a large precursor rRNA (pre-rRNA) that is processed by RNaseIII and other RNases at specific cleavage boxes that have been identified in the C. glutamicum pre-rRNA. A secondary structure of the C. glutamicum 16S rRNA is proposed. The 16S rRNA sequence has been used as a molecular evolution clock allowing the deduction of a phylogenetic tree of all Corynebacterium species. In C. glutamicum, there are 11 ribosomal protein gene clusters encoding 42 ribosomal proteins. The organization of some of the ribosomal protein gene cluster is identical to that of Escherichia coli whereas in other clusters the organization of the genes is rather different. Some specific ribosomal protein genes are located in a different cluster in C. glutamicum when compared with E. coli, indicating that the control of expression of these genes is different in E. coli and C. glutamicum.

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

PubMed Central

Stage-Zimmermann, Tracy; Schmidt, Ute; Silver, Pamela A.

2000-01-01

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. PMID:11071906

Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bae, Hee Kyong; Shinozuka, Junko; Islam, Zahidul

2009-06-01

Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultantmore » fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.« less

Eukaryotic 5S rRNA biogenesis

PubMed Central

Ciganda, Martin; Williams, Noreen

2012-01-01

The ribosome is a large complex containing both protein and RNA which must be assembled in a precise manner to allow proper functioning in the critical role of protein synthesis. 5S rRNA is the smallest of the RNA components of the ribosome, and although it has been studied for decades, we still do not have a clear understanding of its function within the complex ribosome machine. It is the only RNA species that binds ribosomal proteins prior to its assembly into the ribosome. Its transport into the nucleolus requires this interaction. Here we present an overview of some of the key findings concerning the structure and function of 5S rRNA and how its association with specific proteins impacts its localization and function. PMID:21957041

Recent structural studies on Dom34/aPelota and Hbs1/aEF1α: important factors for solving general problems of ribosomal stall in translation

PubMed Central

Kobayashi, Kan; Ishitani, Ryuichiro; Nureki, Osamu

2013-01-01

In the translation process, translating ribosomes usually move on an mRNA until they reach the stop codon. However, when ribosomes translate an aberrant mRNA, they stall. Then, ribosomes are rescued from the aberrant mRNA, and the aberrant mRNA is subsequently degraded. In eukaryotes, Pelota (Dom34 in yeast) and Hbs1 are responsible for solving general problems of ribosomal stall in translation. In archaea, aPelota and aEF1α, homologous to Pelota and Hbs1, respectively, are considered to be involved in that process. In recent years, great progress has been made in determining structures of Dom34/aPelota and Hbs1/aEF1α. In this review, we focus on the functional roles of Dom34/aPelota and Hbs1/aEF1α in ribosome rescue, based on recent structural studies of them. We will also present questions to be answered by future work. PMID:27493551

Direct measurement of the mechanical work during translocation by the ribosome

DOE PAGES

Liu, Tingting; Kaplan, Ariel; Alexander, Lisa; ...

2014-08-11

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functionsmore » as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano–chemical cycle.« less

The N-terminal sequence of ribosomal protein L10 from the archaebacterium Halobacterium marismortui and its relationship to eubacterial protein L6 and other ribosomal proteins.

PubMed

Dijk, J; van den Broek, R; Nasiulas, G; Beck, A; Reinhardt, R; Wittmann-Liebold, B

1987-08-01

The amino-terminal sequence of ribosomal protein L10 from Halobacterium marismortui has been determined up to residue 54, using both a liquid- and a gas-phase sequenator. The two sequences are in good agreement. The protein is clearly homologous to protein HcuL10 from the related strain Halobacterium cutirubrum. Furthermore, a weaker but distinct homology to ribosomal protein L6 from Escherichia coli and Bacillus stearothermophilus can be detected. In addition to 7 identical amino acids in the first 36 residues in all four sequences a number of conservative replacements occurs, of mainly hydrophobic amino acids. In this common region the pattern of conserved amino acids suggests the presence of a beta-alpha fold as it occurs in ribosomal proteins L12 and L30. Furthermore, several potential cases of homology to other ribosomal components of the three ur-kingdoms have been found.

Macrolide antibiotic interaction and resistance on the bacterial ribosome.

PubMed

Poehlsgaard, Jacob; Douthwaite, Stephen

2003-02-01

Our understanding of the fine structure of many antibiotic target sites has reached a new level of enlightenment in the last couple of years due to the advent, by X-ray crystallography, of high-resolution structures of the bacterial ribosome. Many classes of clinically useful antibiotics bind to the ribosome to inhibit bacterial protein synthesis. Macrolide, lincosamide and streptogramin B (MLSB) antibiotics form one of the largest groups, and bind to the same site on the 50S ribosomal subunit. Here, we review the molecular details of the ribosomal MLSB site to put into perspective the main points from a wealth of biochemical and genetic data that have been collected over several decades. The information is now available to understand, at atomic resolution, how macrolide antibiotics interact with their ribosomal target, how the target is altered to confer resistance, and in which directions we need to look if we are to rationally design better drugs to overcome the extant resistance mechanisms.

A model for the study of ligand binding to the ribosomal RNA helix h44

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dibrov, Sergey M.; Parsons, Jerod; Hermann, Thomas

2010-09-02

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structuralmore » model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region.« less

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling

PubMed Central

Su, Ting; Cheng, Jingdong; Sohmen, Daniel; Hedman, Rickard; Berninghausen, Otto; von Heijne, Gunnar; Wilson, Daniel N; Beckmann, Roland

2017-01-01

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50–55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome. DOI: http://dx.doi.org/10.7554/eLife.25642.001 PMID:28556777

Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids

NASA Astrophysics Data System (ADS)

Rogers, Joseph M.; Kwon, Sunbum; Dawson, Simon J.; Mandal, Pradeep K.; Suga, Hiroaki; Huc, Ivan

2018-03-01

Translation, the mRNA-templated synthesis of peptides by the ribosome, can be manipulated to incorporate variants of the 20 cognate amino acids. Such approaches for expanding the range of chemical entities that can be produced by the ribosome may accelerate the discovery of molecules that can perform functions for which poorly folded, short peptidic sequences are ill suited. Here, we show that the ribosome tolerates some artificial helical aromatic oligomers, so-called foldamers. Using a flexible tRNA-acylation ribozyme—flexizyme—foldamers were attached to tRNA, and the resulting acylated tRNAs were delivered to the ribosome to initiate the synthesis of non-cyclic and cyclic foldamer-peptide hybrid molecules. Passing through the ribosome exit tunnel requires the foldamers to unfold. Yet foldamers encode sufficient folding information to influence the peptide structure once translation is completed. We also show that in cyclic hybrids, the foldamer portion can fold into a helix and force the peptide segment to adopt a constrained and stretched conformation.

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

The Phosphorylation of Ribosomal Protein in Lemna minor

PubMed Central

Trewavas, A.

1973-01-01

Sterile cultures of Lemna minor have been labeled with 32P1, and the ribosomal proteins have been examined for radioactivity. In relatively short term labeling a radioactive protein was found which ran as a single component in both urea/acetic acid and sodium lauryl sulfate gel electrophoresis. Acid hydrolysis of the labeled protein permitted the isolation of serine phosphate. After labeling to equilibrium with 32P1, calculation indicated only 0.6 to 0.75 atom of this protein phosphorus per ribosome. The phosphorylated protein is found in both polysomes and “derived” monomers and appears to be located in the ribosomal small subunit. Its apparent molecular weight is 42,000. Addition of growth-inhibiting concentrations of abscisic acid does not alter the apparent degree of labeling of this protein in 5 hours, but after 24 hours of treatment the total protein phosphorus was reduced from 0.75 atom of phosphorus per ribosome to 0.36 atom of phosphorus per ribosome. PMID:16658405

Ribosomes slide on lysine-encoding homopolymeric A stretches

PubMed Central

Koutmou, Kristin S; Schuller, Anthony P; Brunelle, Julie L; Radhakrishnan, Aditya; Djuranovic, Sergej; Green, Rachel

2015-01-01

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome ‘sliding’ represents an unexpected type of ribosome movement possible during translation. DOI: http://dx.doi.org/10.7554/eLife.05534.001 PMID:25695637

The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged Escherichia coli ribosomes.

PubMed

Dey, Sandip; Biswas, Chiranjit; Sengupta, Jayati

2018-06-21

The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Escherichia coli Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that E. coli HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo-electron microscopy structure of the 50S-HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival. © 2018 Dey et al.

Fluctuations in protein synthesis from a single RNA template: stochastic kinetics of ribosomes.

PubMed

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.

Cell illustrator 4.0: a computational platform for systems biology.

PubMed

Nagasaki, Masao; Saito, Ayumu; Jeong, Euna; Li, Chen; Kojima, Kaname; Ikeda, Emi; Miyano, Satoru

2011-01-01

Cell Illustrator is a software platform for Systems Biology that uses the concept of Petri net for modeling and simulating biopathways. It is intended for biological scientists working at bench. The latest version of Cell Illustrator 4.0 uses Java Web Start technology and is enhanced with new capabilities, including: automatic graph grid layout algorithms using ontology information; tools using Cell System Markup Language (CSML) 3.0 and Cell System Ontology 3.0; parameter search module; high-performance simulation module; CSML database management system; conversion from CSML model to programming languages (FORTRAN, C, C++, Java, Python and Perl); import from SBML, CellML, and BioPAX; and, export to SVG and HTML. Cell Illustrator employs an extension of hybrid Petri net in an object-oriented style so that biopathway models can include objects such as DNA sequence, molecular density, 3D localization information, transcription with frame-shift, translation with codon table, as well as biochemical reactions.

Cell Illustrator 4.0: a computational platform for systems biology.

PubMed

Nagasaki, Masao; Saito, Ayumu; Jeong, Euna; Li, Chen; Kojima, Kaname; Ikeda, Emi; Miyano, Satoru

2010-01-01

Cell Illustrator is a software platform for Systems Biology that uses the concept of Petri net for modeling and simulating biopathways. It is intended for biological scientists working at bench. The latest version of Cell Illustrator 4.0 uses Java Web Start technology and is enhanced with new capabilities, including: automatic graph grid layout algorithms using ontology information; tools using Cell System Markup Language (CSML) 3.0 and Cell System Ontology 3.0; parameter search module; high-performance simulation module; CSML database management system; conversion from CSML model to programming languages (FORTRAN, C, C++, Java, Python and Perl); import from SBML, CellML, and BioPAX; and, export to SVG and HTML. Cell Illustrator employs an extension of hybrid Petri net in an object-oriented style so that biopathway models can include objects such as DNA sequence, molecular density, 3D localization information, transcription with frame-shift, translation with codon table, as well as biochemical reactions.

Evolutionary and dispersal history of Triatoma infestans, main vector of Chagas disease, by chromosomal markers.

PubMed

Panzera, Francisco; Ferreiro, María J; Pita, Sebastián; Calleros, Lucía; Pérez, Ruben; Basmadjián, Yester; Guevara, Yenny; Brenière, Simone Frédérique; Panzera, Yanina

2014-10-01

Chagas disease, one of the most important vector-borne diseases in the Americas, is caused by Trypanosoma cruzi and transmitted to humans by insects of the subfamily Triatominae. An effective control of this disease depends on elimination of vectors through spraying with insecticides. Genetic research can help insect control programs by identifying and characterizing vector populations. In southern Latin America, Triatoma infestans is the main vector and presents two distinct lineages, known as Andean and non-Andean chromosomal groups, that are highly differentiated by the amount of heterochromatin and genome size. Analyses with nuclear and mitochondrial sequences are not conclusive about resolving the origin and spread of T. infestans. The present paper includes the analyses of karyotypes, heterochromatin distribution and chromosomal mapping of the major ribosomal cluster (45S rDNA) to specimens throughout the distribution range of this species, including pyrethroid-resistant populations. A total of 417 specimens from seven different countries were analyzed. We show an unusual wide rDNA variability related to number and chromosomal position of the ribosomal genes, never before reported in species with holocentric chromosomes. Considering the chromosomal groups previously described, the ribosomal patterns are associated with a particular geographic distribution. Our results reveal that the differentiation process between both T. infestans chromosomal groups has involved significant genomic reorganization of essential coding sequences, besides the changes in heterochromatin and genomic size previously reported. The chromosomal markers also allowed us to detect the existence of a hybrid zone occupied by individuals derived from crosses between both chromosomal groups. Our genetic studies support the hypothesis of an Andean origin for T. infestans, and suggest that pyrethroid-resistant populations from the Argentinean-Bolivian border are most likely the result of recent secondary contact between both lineages. We suggest that vector control programs should make a greater effort in the entomological surveillance of those regions with both chromosomal groups to avoid rapid emergence of resistant individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

Kirromycin, an Inhibitor of Protein Biosynthesis that Acts on Elongation Factor Tu

PubMed Central

Wolf, Heinz; Chinali, Gianni; Parmeggiani, Andrea

1974-01-01

Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNAPhe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNAPhe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNAPhe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same Km for GTP as the one dependent on Phe-tRNAPhe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNAPhe, but not tRNAPhe or Ac-Phe-tRNAPhe, stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components. PMID:4373734

Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana.

PubMed

Palm, Denise; Simm, Stefan; Darm, Katrin; Weis, Benjamin L; Ruprecht, Maike; Schleiff, Enrico; Scharf, Christian

2016-01-01

Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast.

Proteome distribution between nucleoplasm and nucleolus and its relation to ribosome biogenesis in Arabidopsis thaliana

PubMed Central

Palm, Denise; Simm, Stefan; Darm, Katrin; Weis, Benjamin L.; Ruprecht, Maike; Schleiff, Enrico; Scharf, Christian

2016-01-01

ABSTRACT Ribosome biogenesis is an essential process initiated in the nucleolus. In eukaryotes, multiple ribosome biogenesis factors (RBFs) can be found in the nucleolus, the nucleus and in the cytoplasm. They act in processing, folding and modification of the pre-ribosomal (r)RNAs, incorporation of ribosomal proteins (RPs), export of pre-ribosomal particles to the cytoplasm, and quality control mechanisms. Ribosome biogenesis is best established for Saccharomyces cerevisiae. Plant ortholog assignment to yeast RBFs revealed the absence of about 30% of the yeast RBFs in plants. In turn, few plant specific proteins have been identified by biochemical experiments to act in plant ribosome biogenesis. Nevertheless, a complete inventory of plant RBFs has not been established yet. We analyzed the proteome of the nucleus and nucleolus of Arabidopsis thaliana and the post-translational modifications of these proteins. We identified 1602 proteins in the nucleolar and 2544 proteins in the nuclear fraction with an overlap of 1429 proteins. For a randomly selected set of proteins identified by the proteomic approach we confirmed the localization inferred from the proteomics data by the localization of GFP fusion proteins. We assigned the identified proteins to various complexes and functions and found about 519 plant proteins that have a potential to act as a RBFs, but which have not been experimentally characterized yet. Last, we compared the distribution of RBFs and RPs in the various fractions with the distribution established for yeast. PMID:26980300

Induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway delays the initiation but fails to eradicate established murine acute myeloid leukemia.

PubMed

Jaako, P; Ugale, A; Wahlestedt, M; Velasco-Hernandez, T; Cammenga, J; Lindström, M S; Bryder, D

2017-01-01

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

The Ribosome: The Cell's Protein-Synthesizing Machine and How Antibiotics Disrupt It

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venki Ramakrishnan

Determining the structure of the ribosome has made it possible for Ramakrishnan and his colleagues to image antibiotics bound to the ribosome, leading to a better understanding of their action, which could help in the development of novel drugs. In his ta

The Ribosome: The Cell's Protein-Synthesizing Machine and How Antibiotics Disrupt It

ScienceCinema

Venki Ramakrishnan

2017-12-09

Determining the structure of the ribosome has made it possible for Ramakrishnan and his colleagues to image antibiotics bound to the ribosome, leading to a better understanding of their action, which could help in the development of novel drugs. In his ta

Improve homology search sensitivity of PacBio data by correcting frameshifts.

PubMed

Du, Nan; Sun, Yanni

2016-09-01

Single-molecule, real-time sequencing (SMRT) developed by Pacific BioSciences produces longer reads than secondary generation sequencing technologies such as Illumina. The long read length enables PacBio sequencing to close gaps in genome assembly, reveal structural variations, and identify gene isoforms with higher accuracy in transcriptomic sequencing. However, PacBio data has high sequencing error rate and most of the errors are insertion or deletion errors. During alignment-based homology search, insertion or deletion errors in genes will cause frameshifts and may only lead to marginal alignment scores and short alignments. As a result, it is hard to distinguish true alignments from random alignments and the ambiguity will incur errors in structural and functional annotation. Existing frameshift correction tools are designed for data with much lower error rate and are not optimized for PacBio data. As an increasing number of groups are using SMRT, there is an urgent need for dedicated homology search tools for PacBio data. In this work, we introduce Frame-Pro, a profile homology search tool for PacBio reads. Our tool corrects sequencing errors and also outputs the profile alignments of the corrected sequences against characterized protein families. We applied our tool to both simulated and real PacBio data. The results showed that our method enables more sensitive homology search, especially for PacBio data sets of low sequencing coverage. In addition, we can correct more errors when comparing with a popular error correction tool that does not rely on hybrid sequencing. The source code is freely available at https://sourceforge.net/projects/frame-pro/ yannisun@msu.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Mutations in CTC1, Encoding the CTS Telomere Maintenance Complex Component 1, Cause Cerebroretinal Microangiopathy with Calcifications and Cysts

PubMed Central

Polvi, Anne; Linnankivi, Tarja; Kivelä, Tero; Herva, Riitta; Keating, James P.; Mäkitie, Outi; Pareyson, Davide; Vainionpää, Leena; Lahtinen, Jenni; Hovatta, Iiris; Pihko, Helena; Lehesjoki, Anna-Elina

2012-01-01

Cerebroretinal microangiopathy with calcifications and cysts (CRMCC) is a rare multisystem disorder characterized by extensive intracranial calcifications and cysts, leukoencephalopathy, and retinal vascular abnormalities. Additional features include poor growth, skeletal and hematological abnormalities, and recurrent gastrointestinal bleedings. Autosomal-recessive inheritance has been postulated. The pathogenesis of CRMCC is unknown, but its phenotype has key similarities with Revesz syndrome, which is caused by mutations in TINF2, a gene encoding a member of the telomere protecting shelterin complex. After a whole-exome sequencing approach in four unrelated individuals with CRMCC, we observed four recessively inherited compound heterozygous mutations in CTC1, which encodes the CTS telomere maintenance complex component 1. Sanger sequencing revealed seven more compound heterozygous mutations in eight more unrelated affected individuals. Two individuals who displayed late-onset cerebral findings, a normal fundus appearance, and no systemic findings did not have CTC1 mutations, implying that systemic findings are an important indication for CTC1 sequencing. Of the 11 mutations identified, four were missense, one was nonsense, two resulted in in-frame amino acid deletions, and four were short frameshift-creating deletions. All but two affected individuals were compound heterozygous for a missense mutation and a frameshift or nonsense mutation. No individuals with two frameshift or nonsense mutations were identified, which implies that severe disturbance of CTC1 function from both alleles might not be compatible with survival. Our preliminary functional experiments did not show evidence of severely affected telomere integrity in the affected individuals. Therefore, determining the underlying pathomechanisms associated with deficient CTC1 function will require further studies. PMID:22387016

Mutations in REEP6 Cause Autosomal-Recessive Retinitis Pigmentosa.

PubMed

Arno, Gavin; Agrawal, Smriti A; Eblimit, Aiden; Bellingham, James; Xu, Mingchu; Wang, Feng; Chakarova, Christina; Parfitt, David A; Lane, Amelia; Burgoyne, Thomas; Hull, Sarah; Carss, Keren J; Fiorentino, Alessia; Hayes, Matthew J; Munro, Peter M; Nicols, Ralph; Pontikos, Nikolas; Holder, Graham E; Asomugha, Chinwe; Raymond, F Lucy; Moore, Anthony T; Plagnol, Vincent; Michaelides, Michel; Hardcastle, Alison J; Li, Yumei; Cukras, Catherine; Webster, Andrew R; Cheetham, Michael E; Chen, Rui

2016-12-01

Retinitis pigmentosa (RP) is the most frequent form of inherited retinal dystrophy. RP is genetically heterogeneous and the genes identified to date encode proteins involved in a wide range of functional pathways, including photoreceptor development, phototransduction, the retinoid cycle, cilia, and outer segment development. Here we report the identification of biallelic mutations in Receptor Expression Enhancer Protein 6 (REEP6) in seven individuals with autosomal-recessive RP from five unrelated families. REEP6 is a member of the REEP/Yop1 family of proteins that influence the structure of the endoplasmic reticulum but is relatively unstudied. The six variants identified include three frameshift variants, two missense variants, and a genomic rearrangement that disrupts exon 1. Human 3D organoid optic cups were used to investigate REEP6 expression and confirmed the expression of a retina-specific isoform REEP6.1, which is specifically affected by one of the frameshift mutations. Expression of the two missense variants (c.383C>T [p.Pro128Leu] and c.404T>C [p.Leu135Pro]) and the REEP6.1 frameshift mutant in cultured cells suggest that these changes destabilize the protein. Furthermore, CRISPR-Cas9-mediated gene editing was used to produce Reep6 knock-in mice with the p.Leu135Pro RP-associated variant identified in one RP-affected individual. The homozygous knock-in mice mimic the clinical phenotypes of RP, including progressive photoreceptor degeneration and dysfunction of the rod photoreceptors. Therefore, our study implicates REEP6 in retinal homeostasis and highlights a pathway previously uncharacterized in retinal dystrophy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A Frameshift Mutation in KIT is Associated with  White Spotting in the Arabian Camel.

PubMed

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F; Brooks, Samantha

2017-03-09

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five kITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation.

A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel

PubMed Central

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F.; Brooks, Samantha

2017-01-01

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five KITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation. PMID:28282952

Molecular mechanisms of transformation of C3H/10T1/2 C1 8 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds.

PubMed Central

Landolph, J R

1994-01-01

Carcinogenic arsenic, nickel, and chromium compounds induced morphological and neoplastic transformation but no mutation to ouabain resistance in 10T1/2 mouse embryo cells; lead chromate also did not induce mutation to ouabain or 6-thioguanine resistance in Chinese hamster ovary cells. The mechanism of metal-induced morphological transformation was likely not due to the specific base substitution mutations measured in ouabain resistance mutation assays, and for lead chromate, likely not due to this type of base substitution mutation or to frameshift mutations. Preliminary data indicate increases in steady-state levels of c-myc RNA in arsenic-, nickel-, and chromium-transformed cell lines. We also showed that carcinogenic nickel, chromium, and arsenic compounds and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) induced stable anchorage independence (Al) in diploid human fibroblasts (DHF) but no focus formation or immortality. Nickel subsulfide and lead chromate induced Al but not mutation to 6-thioguanine resistance. The mechanism of induction of Al by metal salts in DHF was likely not by the type of base substitution or frameshift mutations measured in these assays. MNNG induced Al, mutation to 6-thioguanine resistance, and mutation to ouabain resistance, and might induce Al by base substitution or frameshift mutations. Dexamethasone, aspirin, and salicylic acid inhibited nickel subsulfide, MNNG, and 12-O-tetrade-canoylphorbol-13-acetate (TPA)-induced Al in DHF, suggesting that arachidonic acid metabolism and oxygen radical generation play a role in induction of Al. We propose that nickel compounds stimulate arachidonic acid metabolism, consequent oxygen radical generation, and oxygen radical attack upon DNA.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1. PMID:7843085

Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency.

PubMed

Olsen, Rikke K J; Koňaříková, Eliška; Giancaspero, Teresa A; Mosegaard, Signe; Boczonadi, Veronika; Mataković, Lavinija; Veauville-Merllié, Alice; Terrile, Caterina; Schwarzmayr, Thomas; Haack, Tobias B; Auranen, Mari; Leone, Piero; Galluccio, Michele; Imbard, Apolline; Gutierrez-Rios, Purificacion; Palmfeldt, Johan; Graf, Elisabeth; Vianey-Saban, Christine; Oppenheim, Marcus; Schiff, Manuel; Pichard, Samia; Rigal, Odile; Pyle, Angela; Chinnery, Patrick F; Konstantopoulou, Vassiliki; Möslinger, Dorothea; Feichtinger, René G; Talim, Beril; Topaloglu, Haluk; Coskun, Turgay; Gucer, Safak; Botta, Annalisa; Pegoraro, Elena; Malena, Adriana; Vergani, Lodovica; Mazzà, Daniela; Zollino, Marcella; Ghezzi, Daniele; Acquaviva, Cecile; Tyni, Tiina; Boneh, Avihu; Meitinger, Thomas; Strom, Tim M; Gregersen, Niels; Mayr, Johannes A; Horvath, Rita; Barile, Maria; Prokisch, Holger

2016-06-02

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Clinical features of X linked juvenile retinoschisis in Chinese families associated with novel mutations in the RS1 gene.

PubMed

Li, Xiaoxin; Ma, Xiang; Tao, Yong

2007-06-07

To describe the clinical phenotype of X linked juvenile retinoschisis (XLRS) in 12 Chinese families with 11 different mutations in the XLRS1 (RS1) gene. Complete ophthalmic examinations were carried out in 29 affected males (12 probands), 38 heterozygous females carriers, and 100 controls. The coding regions of the RS1 gene that encodes retinoschisin were amplified by polymerase chain reaction and directly sequenced. Of the 29 male participants, 28 (96.6%) displayed typical foveal schisis. Eleven different RS1 mutations were identified in 12 families; four of these mutations, two frameshift mutations (26 del T of exon 1 and 488 del G of exon 5), and two missense mutations (Asp145His and Arg156Gly) of exon 5, had not been previously described. One non-disease-related polymorphism (NSP): 576C to T (Pro192Pro) change was also newly reported herein. We compared genotypes and observed more severe clinical features in families with the following mutations: frameshift mutation (26 del T) of exon 1, the splice donor site mutation (IVS1+2T to C),or Arg102Gln, Arg209His, and Arg213Gln mutations. Severe XLRS phenotypes are associated with the frameshift mutation 26 del T, splice donor site mutation (IVS1+2T to C), and Arg102Gln, Asp145His, Arg209His, and Arg213Gln mutations. The wide variability in the phenotype in Chinese patients with XLRS and different mutations in the RS1 gene is described. Identification of mutations in the RS1 gene and expanded information on clinical manifestations will facilitate early diagnosis, appropriate early therapy, and genetic counseling regarding the prognosis of XLRS.

Clinical features of X linked juvenile retinoschisis in Chinese families associated with novel mutations in the RS1 gene

PubMed Central

Ma, Xiang; Tao, Yong

2007-01-01

Purpose To describe the clinical phenotype of X linked juvenile retinoschisis (XLRS) in 12 Chinese families with 11 different mutations in the XLRS1 (RS1) gene. Methods Complete ophthalmic examinations were carried out in 29 affected males (12 probands), 38 heterozygous females carriers, and 100 controls. The coding regions of the RS1 gene that encodes retinoschisin were amplified by polymerase chain reaction and directly sequenced. Results Of the 29 male participants, 28 (96.6%) displayed typical foveal schisis. Eleven different RS1 mutations were identified in 12 families; four of these mutations, two frameshift mutations (26 del T of exon 1 and 488 del G of exon 5), and two missense mutations (Asp145His and Arg156Gly) of exon 5, had not been previously described. One non-disease-related polymorphism (NSP): 576C to T (Pro192Pro) change was also newly reported herein. We compared genotypes and observed more severe clinical features in families with the following mutations: frameshift mutation (26 del T) of exon 1, the splice donor site mutation (IVS1+2T to C),or Arg102Gln, Arg209His, and Arg213Gln mutations. Conclusions Severe XLRS phenotypes are associated with the frameshift mutation 26 del T, splice donor site mutation (IVS1+2T to C), and Arg102Gln, Asp145His, Arg209His, and Arg213Gln mutations. The wide variability in the phenotype in Chinese patients with XLRS and different mutations in the RS1 gene is described. Identification of mutations in the RS1 gene and expanded information on clinical manifestations will facilitate early diagnosis, appropriate early therapy, and genetic counseling regarding the prognosis of XLRS. PMID:17615541

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma irradiation

DTIC Science & Technology

2011-10-01

release of L11 from the nucleolus to the nucleoplasm, where it binds to c-Myc protein, and to the cytoplasm, where it binds to c-myc mRNA. We also found...rRNA and ribosomal proteins (RPs), rRNA processing, and the as- sembly of the mature ribosome subunits in the nucleolus fol- lowed by their transport...from the nucleolus or from intact ribosomes to suppress MDM2 (68). However, whether L11 suppresses c-Myc in response to ribosomal stress is not known

Evolution of ribosomal proteins in Enterobacteriaceae.

PubMed Central

Hori, H; Osawa, S

1978-01-01

The evolution of ribosomal proteins of about 70 bacterial strains belonging to the family Enterobacteriaceae has been studied by use of previously reported data (S. Osawa, T. Itoh, and E. Otaka, J. Bacteriol. 107:168-178, 1971) and those obtained in this paper. The proximity of the bacteria was quantified by co-chromatographing the differentially labeled ribosomal proteins from two strains on a column of carboxymethyl cellulose in various combinations. The were then classified into 12 groups (=species?) according to their ribosomal protein compositions and were placed in a phylogenic tree. PMID:346556

4′-O-substitutions determine selectivity of aminoglycoside antibiotics

PubMed Central

Perez-Fernandez, Déborah; Shcherbakov, Dmitri; Matt, Tanja; Leong, Ng Chyan; Kudyba, Iwona; Duscha, Stefan; Boukari, Heithem; Patak, Rashmi; Dubbaka, Srinivas Reddy; Lang, Kathrin; Meyer, Martin; Akbergenov, Rashid; Freihofer, Pietro; Vaddi, Swapna; Thommes, Pia; Ramakrishnan, V.; Vasella, Andrea; Böttger, Erik C.

2014-01-01

Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here we present a series of 4′,6′-O-acetal and 4′-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug-binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4′-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets. PMID:24473108

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

Purification, characterization and crystallization of the human 80S ribosome

PubMed Central

Khatter, Heena; Myasnikov, Alexander G.; Mastio, Leslie; Billas, Isabelle M. L.; Birck, Catherine; Stella, Stefano; Klaholz, Bruno P.

2014-01-01

Ribosomes are key macromolecular protein synthesis machineries in the cell. Human ribosomes have so far not been studied to atomic resolution because of their particularly complex structure as compared with other eukaryotic or prokaryotic ribosomes, and they are difficult to prepare to high homogeneity, which is a key requisite for high-resolution structural work. We established a purification protocol for human 80S ribosomes isolated from HeLa cells that allows obtaining large quantities of homogenous samples as characterized by biophysical methods using analytical ultracentrifugation and multiangle laser light scattering. Samples prepared under different conditions were characterized by direct single particle imaging using cryo electron microscopy, which helped optimizing the preparation protocol. From a small data set, a 3D reconstruction at subnanometric resolution was obtained showing all prominent structural features of the human ribosome, and revealing a salt concentration dependence of the presence of the exit site tRNA, which we show is critical for obtaining crystals. With these well-characterized samples first human 80S ribosome crystals were obtained from several crystallization conditions in capillaries and sitting drops, which diffract to 26 Å resolution at cryo temperatures and for which the crystallographic parameters were determined, paving the way for future high-resolution work. PMID:24452798

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex.

PubMed

Xie, Ping

2015-10-09

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by "hungry" codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel.

Dwell-Time Distribution, Long Pausing and Arrest of Single-Ribosome Translation through the mRNA Duplex

PubMed Central

Xie, Ping

2015-01-01

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by “hungry” codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel. PMID:26473825

Defective ribosome assembly in Shwachman-Diamond syndrome.

PubMed

Wong, Chi C; Traynor, David; Basse, Nicolas; Kay, Robert R; Warren, Alan J

2011-10-20

Shwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

PubMed

Tan, Thomas C J; Knight, John; Sbarrato, Thomas; Dudek, Kate; Willis, Anne E; Zamoyska, Rose

2017-07-25

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.

PubMed

Sharma, Divya; Cukras, Anthony R; Rogers, Elizabeth J; Southworth, Daniel R; Green, Rachel

2007-12-07

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

tRNA-derived short RNAs bind to Saccharomyces cerevisiae ribosomes in a stress-dependent manner and inhibit protein synthesis in vitro

PubMed Central

Kasprzyk, Marta; Twardowski, Tomasz

2016-01-01

Recently, a number of ribosome-associated non-coding RNAs (rancRNAs) have been discovered in all three domains of life. In our previous studies, we have described several types of rancRNAs in Saccharomyces cerevisiae, derived from many cellular RNAs, including mRNAs, rRNAs, tRNAs and snoRNAs. Here, we present the evidence that the tRNA fragments from simple eukaryotic organism S. cerevisiae directly bind to the ribosomes. Interestingly, rancRNA-tRFs in yeast are derived from both, 5′- and 3′-part of the tRNAs and both types of tRFs associate with the ribosomes in vitro. The location of tRFs within the ribosomes is distinct from classical A- and P-tRNA binding sites. Moreover, 3′-tRFs bind to the distinct site than 5′-tRFs. These interactions are stress dependent and as a consequence, provoke regulation of protein biosynthesis. We observe strong correlation between tRF binding to the ribosomes and inhibition of protein biosynthesis in particular environmental conditions. These results implicate the existence of an ancient and conserved mechanism of translation regulation with the involvement of ribosome-associating tRNA-derived fragments. PMID:27609601

RNase activity in erythroid cell lysates.

PubMed

Burka, E R

1969-09-01

The characteristics of degradation of reticulocyte ribonucleic acid (RNA) and ribosomes were studied in a whole erythroid cell lysate system. The process followed Michaelis-Menten kinetics, and indicated that RNA degradation in the erythroid cell is mediated by an enzyme previously isolated from reticulocyte hemolysates. Erythroid cell RNase activity had a temperature optimum of 50 degrees C, a pH optimum of 7.0, was not energy dependent, was heat labile at physiologic pH, and was inhibited by Mg(++), Ca(++), and exposure to bentonite and deoxycholate. Free sulfhydryl groups were not essential for RNase activity. Of the substrates occurring naturally within the erythroid cell, isolated ribosomal RNA was most susceptible to the action of the enzyme, intact ribosomes least susceptible, and transfer RNA intermediate between them. Natural substrates were degraded completely to nucleotides in cell lysates. Competitive inhibition studies indicate that one enzyme system is capable of degrading both RNA and ribosomes, although the existence of more than one enzyme has not been excluded. Erythroid cell lysates quickly broke down polyribosomes into single ribosomes. The more rapid degradation of ribosomes, as compared with transfer RNA, which occurs in vivo, as opposed to findings in vitro, suggests that there is a special intracellular mechanism responsible for ribosome degradation in the maturing erythroid cell.

Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome.

PubMed

Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping; Tumer, Nilgun E

2016-12-01

The A1 subunits of Shiga toxin 1 (Stx1A1) and Shiga toxin 2 (Stx2A1) interact with the conserved C termini of ribosomal-stalk P-proteins to remove a specific adenine from the sarcin/ricin loop. We previously showed that Stx2A1 has higher affinity for the ribosome and higher catalytic activity than Stx1A1. To determine if conserved arginines at the distal face of the active site contribute to the higher affinity of Stx2A1 for the ribosome, we mutated Arg172, Arg176, and Arg179 in both toxins. We show that Arg172 and Arg176 are more important than Arg179 for the depurination activity and toxicity of Stx1A1 and Stx2A1. Mutation of a single arginine reduced the depurination activity of Stx1A1 more than that of Stx2A1. In contrast, mutation of at least two arginines was necessary to reduce depurination by Stx2A1 to a level similar to that of Stx1A1. R176A and R172A/R176A mutations eliminated interaction of Stx1A1 and Stx2A1 with ribosomes and with the stalk, while mutation of Arg170 at the active site reduced the binding affinity of Stx1A1 and Stx2A1 for the ribosome, but not for the stalk. These results demonstrate that conserved arginines at the distal face of the active site are critical for interactions of Stx1A1 and Stx2A1 with the stalk, while a conserved arginine at the active site is critical for non-stalk-specific interactions with the ribosome. Arginine mutations at either site reduced ribosome interactions of Stx1A1 and Stx2A1 similarly, indicating that conserved arginines are critical for ribosome interactions but do not contribute to the higher affinity of Stx2A1 for the ribosome. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

PubMed Central

Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook

2016-01-01

ABSTRACT The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S. Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits. PMID:27297882

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

PubMed Central

Barna, Maria; Pusic, Aya; Zollo, Ornella; Costa, Maria; Kondrashov, Nadya; Rego, Eduardo; Rao, Pulivarthi H; Ruggero, Davide

2008-01-01

The Myc oncogene regulates the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III, and rDNA1,2. An outstanding question is whether and how increasing the cellular protein synthesis capacity can affect the multi-step process leading to cancer. We utilized ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ–Myc/+ transgenic mice to normal levels and show that in this context Myc's oncogenic potential is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a novel paradigm that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation employed to regulate the expression of selective mRNAs. We show that an aberrant increase in cap-dependent translation downstream Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (p58-PITSLRE)3-5, which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ–Myc/+ mice. When accurate translational control is re-established in Eμ–Myc/+ mice, genome instability is suppressed. Our findings reveal how perturbations in translational control provide a highly specific outcome on gene expression, genome stability, and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level. PMID:19011615

Whole-exome analysis of foetal autopsy tissue reveals a frameshift mutation in OBSL1, consistent with a diagnosis of 3-M Syndrome.

PubMed

Marshall, Christian R; Farrell, Sandra A; Cushing, Donna; Paton, Tara; Stockley, Tracy L; Stavropoulos, Dimitri J; Ray, Peter N; Szego, Michael; Lau, Lynette; Pereira, Sergio L; Cohn, Ronald D; Wintle, Richard F; Abuzenadah, Adel M; Abu-Elmagd, Muhammad; Scherer, Stephen W

2015-01-01

We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a recurrent set of clinical symptoms.

A Novel Frameshift Mutation of the USH2A Gene in a Korean Patient with Usher Syndrome Type II.

PubMed

Boo, Sung Hyun; Song, Min-Jung; Kim, Hee-Jin; Cho, Yang-Sun; Chu, Hosuk; Ko, Moon-Hee; Chung, Won-Ho; Kim, Jong-Won; Hong, Sung Hwa

2013-03-01

Usher syndrome type II (USH2) is the most common form of Usher syndrome, characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa. It has been shown that mutations in the USH2A gene are responsible for USH2. The authors herein describe a 34-year-old Korean woman with the typical clinical manifestation of USH2; she had bilateral hearing disturbance and progressive visual deterioration, without vestibular dysfunction. Molecular genetic study of the USH2A gene revealed a novel frameshift mutation (c.2310delA; Glu771LysfsX17). She was heterozygous for this mutation, and no other mutation was found in USH2A, suggesting the possibility of an intronic or large genomic rearrangement mutation. To the best of our knowledge, this is the first report of a genetically confirmed case of USH2 in Korea. More investigations are needed to delineate genotype-phenotype correlations and ethnicity-specific genetic background of Usher syndrome.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosatelli, M.C.; Faa, V.; Sardu, R.

This study reports the molecular characterization of [beta]-thalassemia in the Sardinian population. Three thousand [beta]-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common [beta]-thalassemia mutations in the Mediterranean at-risk populations. The mutation which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. The authors reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the [beta]-thalassemia chromosomes. The other two relatively commonmore » mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study they have detected a novel [beta]-thalassemia mutation, i.e., a frameshift at codon 1, in three [beta]-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system.« less

Frameshift mutation in the APOA5 gene causing hypertriglyceridemia in a Pakistani family: Management and considerations for cardiovascular risk.

PubMed

Thériault, Sébastien; Don-Wauchope, Andrew; Chong, Michael; Lali, Ricky; Morrison, Katherine M; Paré, Guillaume

2016-01-01

We report a novel homozygous apolipoprotein A5 (APOA5) frameshift mutation (c.G425del-C, p.Arg143AlafsTer57) identified in a 12-year-old boy of Pakistani origin with severe hypertriglyceridemia (up to 35 mmol/L) and type V hyperlipoproteinemia. The patient did not respond to fibrate therapy, but his condition improved under a very low fat diet, although compliance was suboptimal. Heterozygous status was detected in both parents (consanguineous union) and one sibling, all showing moderate hypertriglyceridemia (between 5 and 10 mmol/L). There was a significant family history of premature cardiovascular disease. The index case was also diagnosed with a coronary artery anomaly. Considering the recently reported association of rare mutations in APOA5 with the risk of early myocardial infarction, we discuss the implications of these findings for the young man and his family. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

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.

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan, Haitian; Hahm, Joseph; Diggs, Stephen

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

[Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

PubMed

Yang, Qing-Li; Shen, Ji-Qing; Jiang, Zhi-Hua; Yang, Yi-Chao; Li, Hong-Mei; Chen, Ying-Dan; Zhou, Xiao-Nong

2014-06-01

To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

Structural and Functional Analysis of BipA, a Regulator of Virulence in Enteropathogenic Escherichia coli

DOE PAGES

Fan, Haitian; Hahm, Joseph; Diggs, Stephen; ...

2015-07-10

The translational GTPase BipA regulates the expression of virulence and pathogenicity factors in several eubacteria. BipA-dependent expression of virulence factors occurs under starvation conditions, such as encountered during infection of a host. Under these conditions, BipA associates with the small ribosomal subunit. BipA also has a second function to promote the efficiency of late steps in biogenesis of large ribosomal subunits at low temperatures, presumably while bound to the ribosome. During starvation, the cellular concentration of stress alarmone guanosine-3', 5'-bis pyrophosphate (ppGpp) is increased. This increase allows ppGpp to bind to BipA and switch its binding specificity from ribosomes tomore » small ribosomal subunits. A conformational change of BipA upon ppGpp binding could explain the ppGpp regulation of the binding specificity of BipA. Here, we present the structures of the full-length BipA from Escherichia coli in apo, GDP-, and ppGpp-bound forms. The crystal structure and small-angle x-ray scattering data of the protein with bound nucleotides, together with a thermodynamic analysis of the binding of GDP and of ppGpp to BipA, indicate that the ppGpp-bound form of BipA adopts the structure of the GDP form. This suggests furthermore, that the switch in binding preference only occurs when both ppGpp and the small ribosomal subunit are present. Finally, this molecular mechanism would allow BipA to interact with both the ribosome and the small ribosomal subunit during stress response.« less

Proteopedia Entry: The Large Ribosomal Subunit of "Haloarcula Marismortui"

ERIC Educational Resources Information Center

Decatur, Wayne A.

2010-01-01

This article presents a "Proteopedia" page that shows the refined version of the structure of the "Haloarcula" large ribosomal subunit as solved by the laboratories of Thomas Steitz and Peter Moore. The landmark structure is of great impact as it is the first atomic-resolution structure of the highly conserved ribosomal subunit which harbors…

Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA.

PubMed

Hochstatter, Julia; Hölzel, Michael; Rohrmoser, Michaela; Schermelleh, Lothar; Leonhardt, Heinrich; Keough, Rebecca; Gonda, Thomas J; Imhof, Axel; Eick, Dirk; Längst, Gernot; Németh, Attila

2012-07-13

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing

PubMed Central

Sloan, Katherine E.; Mattijssen, Sandy; Lebaron, Simon; Tollervey, David; Pruijn, Ger J.M.

2013-01-01

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference–mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells. PMID:23439679

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.

Thermus Thermophilus as a Model System for the Study of Ribosomal Antibiotic Resistance

NASA Astrophysics Data System (ADS)

Gregory, Steven T.

2018-03-01

Ribosomes are the intracellular ribonucleoprotein machines responsible for the translation of mRNA sequence into protein sequence. As an essential cell component, the ribosome is the target of numerous antibiotics that bind to critical functional sites to impair protein synthesis. Mutations causing resistance to antibiotics arise in antibiotic binding sites, and an understanding of the basis of resistance will be an essential component of efforts to develop new antibiotics by rational drug design. We have identified a number of antibiotic-resistance mutations in ribosomal genes of the thermophilic bacterium Thermus thermophilus. This species offers two primary advantages for examining the structural basis of antibiotic-resistance, in particular, its potential for genetic manipulation and the suitability of its ribosomes for analysis by X-ray crystallography. Mutations we have identified in this organism are in many instances identical to those found in other bacterial species, including important pathogens, a result of the extreme conservation of ribosome functional sites. Here I summarize the advantages of this organism as a model system to study antibiotic-resistance mechanisms at the molecular level.

Mpp10 represents a platform for the interaction of multiple factors within the 90S pre-ribosome

PubMed Central

Kharde, Satyavati; Ahmed, Yasar Luqman; Stier, Gunter; Kunze, Ruth; Sinning, Irmgard

2017-01-01

In eukaryotes, ribosome assembly is a highly complex process that involves more than 200 assembly factors that ensure the folding, modification and processing of the different rRNA species as well as the timely association of ribosomal proteins. One of these factors, Mpp10 associates with Imp3 and Imp4 to form a complex that is essential for the normal production of the 18S rRNA. Here we report the crystal structure of a complex between Imp4 and a short helical element of Mpp10 to a resolution of 1.88 Å. Furthermore, we extend the interaction network of Mpp10 and characterize two novel interactions. Mpp10 is able to bind the ribosome biogenesis factor Utp3/Sas10 through two conserved motifs in its N-terminal region. In addition, Mpp10 interacts with the ribosomal protein S5/uS7 using a short stretch within an acidic loop region. Thus, our findings reveal that Mpp10 provides a platform for the simultaneous interaction with multiple proteins in the 90S pre-ribosome. PMID:28813493

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway.

PubMed

Llanos, Susana; Serrano, Manuel

2010-10-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely, UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage.

The double life of the ribosome: When its protein folding activity supports prion propagation.

PubMed

Voisset, Cécile; Blondel, Marc; Jones, Gary W; Friocourt, Gaëlle; Stahl, Guillaume; Chédin, Stéphane; Béringue, Vincent; Gillet, Reynald

2017-03-04

It is no longer necessary to demonstrate that ribosome is the central machinery of protein synthesis. But it is less known that it is also key player of the protein folding process through another conserved function: the protein folding activity of the ribosome (PFAR). This ribozyme activity, discovered more than 2 decades ago, depends upon the domain V of the large rRNA within the large subunit of the ribosome. Surprisingly, we discovered that anti-prion compounds are also potent PFAR inhibitors, highlighting an unexpected link between PFAR and prion propagation. In this review, we discuss the ancestral origin of PFAR in the light of the ancient RNA world hypothesis. We also consider how this ribosomal activity fits into the landscape of cellular protein chaperones involved in the appearance and propagation of prions and other amyloids in mammals. Finally, we examine how drugs targeting the protein folding activity of the ribosome could be active against mammalian prion and other protein aggregation-based diseases, making PFAR a promising therapeutic target for various human protein misfolding diseases.

Temporal programming of chloroplast and cytoplasmic ribosomal RNA transcription in the synchronous cell cycle of Chlamydomonas reinhardtii

PubMed Central

1977-01-01

Approximately 90% of the Chlamydomonas reinhardtii chloroplast and cytoplasmic rRNAs was transcribed in the nuclear G1 phase, which occurred during the light period under an alternating light-dark synchronization regime of 12 h each. The remaining 10% of chloroplast and cytoplasmic rRNAs was transcribed from its respective DNAs in the dark period, in the midst of an apparent turnover of a transcription appeared to be prokaryotic in sophistication. The transcription was not interrupted during chloroplast DNA synthesis which occurred during the light period. However, transcription of the nuclear DNA was repressed severely during the nuclear S phase in the dark period. The patterns of incorporation of 32P into chloroplast and cytoplasmic rRNA species in the cell cycle were similar to those of the actual rRNA synthesis as measured optically. However, the quantity of 32P incorporation per unit amount of rRNA synthesized varied considerably during the cell cycle, increasing in all rRNA's during the dark period. 32P incorporation data obtained from continuous and pulse 32P-labeling experiments also revealed a turnover of a small amount of both cytoplasmic and chloroplast rRNAs at the end of the S phase. The 32P incorporation into cytoplasmic and chloroplast rRNAs was well matched temporally with the 32P incorporation into their corresponding ribosomes, indicating that the newly synthesized rRNA molecules are utilized without delay throughout the cell cycle in the assembly of ribosomes. PMID:833204

SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

PubMed

Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

2012-07-15

In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

DOE PAGES

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.; ...

2014-12-22

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

Amino acid sequences of ribosomal proteins S11 from Bacillus stearothermophilus and S19 from Halobacterium marismortui. Comparison of the ribosomal protein S11 family.

PubMed

Kimura, M; Kimura, J; Hatakeyama, T

1988-11-21

The complete amino acid sequences of ribosomal proteins S11 from the Gram-positive eubacterium Bacillus stearothermophilus and of S19 from the archaebacterium Halobacterium marismortui have been determined. A search for homologous sequences of these proteins revealed that they belong to the ribosomal protein S11 family. Homologous proteins have previously been sequenced from Escherichia coli as well as from chloroplast, yeast and mammalian ribosomes. A pairwise comparison of the amino acid sequences showed that Bacillus protein S11 shares 68% identical residues with S11 from Escherichia coli and a slightly lower homology (52%) with the homologous chloroplast protein. The halophilic protein S19 is more related to the eukaryotic (45-49%) than to the eubacterial counterparts (35%).

The primary structures of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Hatakeyama, T; Kimura, M

1988-11-21

The complete amino acid sequences of ribosomal proteins L16, L23 and L33 from the archaebacterium Halobacterium marismortui were determined. The sequences were established by manual sequencing of peptides produced with several proteases as well as by cleavage with dilute HCl. Proteins L16, L23 and L33 consist of 119, 154 and 69 amino acid residues, and their molecular masses are 13,538, 16,812 and 7620 Da, respectively. The comparison of their sequences with those of ribosomal proteins from other organisms revealed that L23 and L33 are related to eubacterial ribosomal proteins from Escherichia coli and Bacillus stearothermophilus, while protein L16 was found to be homologous to a eukaryotic ribosomal protein from yeast. These results provide information about the special phylogenetic position of archaebacteria.

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

PubMed Central

Umaer, Khan; Ciganda, Martin

2014-01-01

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention. PMID:24706018

TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

PubMed

Ghosh, Abhishek; Rideout, Elizabeth J; Grewal, Savraj S

2014-10-01

The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

TIF-IA-Dependent Regulation of Ribosome Synthesis in Drosophila Muscle Is Required to Maintain Systemic Insulin Signaling and Larval Growth

PubMed Central

Ghosh, Abhishek; Rideout, Elizabeth J.; Grewal, Savraj S.

2014-01-01

The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis—a limiting step in ribosome biogenesis—via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2—a secreted factor that binds and inhibits dILP activity—from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis. PMID:25356674

A new system for naming ribosomal proteins

PubMed Central

Ban, Nenad; Beckmann, Roland; Cate, Jamie HD; Dinman, Jonathan D; Dragon, François; Ellis, Steven R; Lafontaine, Denis LJ; 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 MT; Steitz, Thomas A; Tchorzewski, Marek; Tollervey, David; Warren, Alan J; Williamson, James R; Wilson, Daniel; Yonath, Ada; Yusupov, Marat

2015-01-01

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. PMID:24524803

Origins of the plant chloroplasts and mitochondria based on comparisons of 5S ribosomal RNAs

NASA Technical Reports Server (NTRS)

Delihas, N.; Fox, G. E.

1987-01-01

In this paper, we provide macromolecular comparisons utilizing the 5S ribosomal RNA structure to suggest extant bacteria that are the likely descendants of chloroplast and mitochondria endosymbionts. The genetic stability and near universality of the 5S ribosomal gene allows for a useful means to study ancient evolutionary changes by macromolecular comparisons. The value in current and future ribosomal RNA comparisons is in fine tuning the assignment of ancestors to the organelles and in establishing extant species likely to be descendants of bacteria involved in presumed multiple endosymbiotic events.

Towards the Batch Synthesis of Long DNA

DTIC Science & Technology

2002-10-01

mishybridizations which arise because of frame-shifting (in the special case of pairs of batch ssDNAs [as opposed to semi-ligated “DNA Frankensteins ...of DNA in solution, despite the possible influences of steric constraints, applied electric potential, etc. 78 Although Howorka et al. do not

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

PubMed Central

Koch, Miriam; Willi, Jessica; Pradère, Ugo; Hall, Jonathan

2017-01-01

Abstract The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation. PMID:28369621

Nuclear export of the small ribosomal subunit requires the Ran–GTPase cycle and certain nucleoporins

PubMed Central

Moy, Terence I.; Silver, Pamela A.

1999-01-01

After their assembly in the nucleolus, ribosomal subunits are exported from the nucleus to the cytoplasm. After export, the 20S rRNA in the small ribosomal subunit is cleaved to yield 18S rRNA and the small 5′ ITS1 fragment. The 5′ ITS1 RNA is normally degraded by the cytoplasmic Xrn1 exonuclease, but in strains lacking XRN1, the 5′ ITS1 fragment accumulates in the cytoplasm. Using the cytoplasmic localization of the 5′ ITS1 fragment as an indicator for the export of the small ribosomal subunit, we have identified genes that are required for ribosome export. Ribosome export is dependent on the Ran–GTPase as mutations in Ran or its regulators caused 5′ ITS1 to accumulate in the nucleoplasm. Mutations in the genes encoding the nucleoporin Nup82 and in the NES exporter Xpo1/Crm1 also caused the nucleoplasmic accumulation of 5′ ITS1. Mutants in a subset of nucleoporins and in the nuclear transport factors Srp1, Kap95, Pse1, Cse1, and Mtr10 accumulate the 5′ ITS1 in the nucleolus and affect ribosome assembly. In contrast, we did not detect nuclear accumulation of 5′ ITS1 in 28 yeast strains that have mutations in other genes affecting nuclear trafficking. PMID:10465789

Miscoding-induced stalling of substrate translocation on the bacterial ribosome.

PubMed

Alejo, Jose L; Blanchard, Scott C

2017-10-10

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G-catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

Miscoding-induced stalling of substrate translocation on the bacterial ribosome

PubMed Central

Alejo, Jose L.; Blanchard, Scott C.

2017-01-01

Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors. PMID:28973849

Metabolism of ribosomal proteins microinjected into the oocytes of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsurugi, K.; Motizuki, M.; Mitsui, K.

1988-01-01

When the total proteins from Xenopus laevis 60 S ribosomal subunits (TP60) were /sup 3/H-labeled in vitro and injected back into X. laevis oocytes, most /sup 3/H-TP60 are integrated into the cytoplasmic 60 S subunits via the nucleus during 16 h of incubation. In the oocytes whose rRNA synthesis is inhibited, /sup 3/H-TP60 are rapidly degraded with a half-life of 2-3 h. This degradation ceased as soon as rRNA synthesis was resumed, suggesting that ribosomal proteins unassociated with nascent rRNA are unstable in the oocytes. The degradation of /sup 3/H-TP60 in the absence of RNA synthesis was inhibited by iodoacetamide,more » a cysteine protease inhibitor, resulting in the accumulation of /sup 3/H-TP60 in the nucleus reaching about a threefold concentration in the cytoplasm. Considering the results with enucleated oocytes, we suggest that the X. laevis nucleus has a limited capacity to accumulate ribosomal proteins in an active manner but that those ribosomal proteins accumulated in excess over rRNA synthesis are degraded by a cysteine protease in the nucleus. By contrast, ribosomal proteins from Escherichia coli only equilibrate between the nucleus and the cytoplasm and are degraded by serine protease(s) in the cytoplasm without being integrated in the form of ribosomes in the nucleus.« less

Telomere and ribosomal DNA repeats are chromosomal targets of the bloom syndrome DNA helicase

PubMed Central

Schawalder, James; Paric, Enesa; Neff, Norma F

2003-01-01

Background Bloom syndrome is one of the most cancer-predisposing disorders and is characterized by genomic instability and a high frequency of sister chromatid exchange. The disorder is caused by loss of function of a 3' to 5' RecQ DNA helicase, BLM. The exact role of BLM in maintaining genomic integrity is not known but the helicase has been found to associate with several DNA repair complexes and some DNA replication foci. Results Chromatin immunoprecipitation of BLM complexes recovered telomere and ribosomal DNA repeats. The N-terminus of BLM, required for NB localization, is the same as the telomere association domain of BLM. The C-terminus is required for ribosomal DNA localization. BLM localizes primarily to the non-transcribed spacer region of the ribosomal DNA repeat where replication forks initiate. Bloom syndrome cells expressing the deletion alleles lacking the ribosomal DNA and telomere association domains have altered cell cycle populations with increased S or G2/M cells relative to normal. Conclusion These results identify telomere and ribosomal DNA repeated sequence elements as chromosomal targets for the BLM DNA helicase during the S/G2 phase of the cell cycle. BLM is localized in nuclear bodies when it associates with telomeric repeats in both telomerase positive and negative cells. The BLM DNA helicase participates in genomic stability at ribosomal DNA repeats and telomeres. PMID:14577841

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients

PubMed Central

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H.; Mills, Jason A.; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M.; Podsakoff, Gregory M.; Gadue, Paul; French, Deborah L.; Mason, Philip J.; Bessler, Monica

2013-01-01

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the “safe harbor” AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells. PMID:23744582

Ribosomal and hematopoietic defects in induced pluripotent stem cells derived from Diamond Blackfan anemia patients.

PubMed

Garçon, Loïc; Ge, Jingping; Manjunath, Shwetha H; Mills, Jason A; Apicella, Marisa; Parikh, Shefali; Sullivan, Lisa M; Podsakoff, Gregory M; Gadue, Paul; French, Deborah L; Mason, Philip J; Bessler, Monica; Weiss, Mitchell J

2013-08-08

Diamond Blackfan anemia (DBA) is a congenital disorder with erythroid (Ery) hypoplasia and tissue morphogenic abnormalities. Most DBA cases are caused by heterozygous null mutations in genes encoding ribosomal proteins. Understanding how haploinsufficiency of these ubiquitous proteins causes DBA is hampered by limited availability of tissues from affected patients. We generated induced pluripotent stem cells (iPSCs) from fibroblasts of DBA patients carrying mutations in RPS19 and RPL5. Compared with controls, DBA fibroblasts formed iPSCs inefficiently, although we obtained 1 stable clone from each fibroblast line. RPS19-mutated iPSCs exhibited defects in 40S (small) ribosomal subunit assembly and production of 18S ribosomal RNA (rRNA). Upon induced differentiation, the mutant clone exhibited globally impaired hematopoiesis, with the Ery lineage affected most profoundly. RPL5-mutated iPSCs exhibited defective 60S (large) ribosomal subunit assembly, accumulation of 12S pre-rRNA, and impaired erythropoiesis. In both mutant iPSC lines, genetic correction of ribosomal protein deficiency via complementary DNA transfer into the "safe harbor" AAVS1 locus alleviated abnormalities in ribosome biogenesis and hematopoiesis. Our studies show that pathological features of DBA are recapitulated by iPSCs, provide a renewable source of cells to model various tissue defects, and demonstrate proof of principle for genetic correction strategies in patient stem cells.

Development of a tissue-specific ribosome profiling approach in Drosophila enables genome-wide evaluation of translational adaptations

PubMed Central

2017-01-01

Recent advances in next-generation sequencing approaches have revolutionized our understanding of transcriptional expression in diverse systems. However, measurements of transcription do not necessarily reflect gene translation, the process of ultimate importance in understanding cellular function. To circumvent this limitation, biochemical tagging of ribosome subunits to isolate ribosome-associated mRNA has been developed. However, this approach, called TRAP, lacks quantitative resolution compared to a superior technology, ribosome profiling. Here, we report the development of an optimized ribosome profiling approach in Drosophila. We first demonstrate successful ribosome profiling from a specific tissue, larval muscle, with enhanced resolution compared to conventional TRAP approaches. We next validate the ability of this technology to define genome-wide translational regulation. This technology is leveraged to test the relative contributions of transcriptional and translational mechanisms in the postsynaptic muscle that orchestrate the retrograde control of presynaptic function at the neuromuscular junction. Surprisingly, we find no evidence that significant changes in the transcription or translation of specific genes are necessary to enable retrograde homeostatic signaling, implying that post-translational mechanisms ultimately gate instructive retrograde communication. Finally, we show that a global increase in translation induces adaptive responses in both transcription and translation of protein chaperones and degradation factors to promote cellular proteostasis. Together, this development and validation of tissue-specific ribosome profiling enables sensitive and specific analysis of translation in Drosophila. PMID:29194454

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

PubMed Central

Dembowski, Jill A.; Kuo, Benjamin; Woolford, John L.

2013-01-01

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA3 pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps. PMID:23788678

Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection

PubMed Central

Au, Hilda H.; Cornilescu, Gabriel; Mouzakis, Kathryn D.; Ren, Qian; Burke, Jordan E.; Lee, Seonghoon; Butcher, Samuel E.; Jan, Eric

2015-01-01

The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirus Israeli acute paralysis virus (IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame. PMID:26554019

sORFs.org: a repository of small ORFs identified by ribosome profiling

PubMed Central

Olexiouk, Volodimir; Crappé, Jeroen; Verbruggen, Steven; Verhegen, Kenneth; Martens, Lennart; Menschaert, Gerben

2016-01-01

With the advent of ribosome profiling, a next generation sequencing technique providing a “snap-shot’’ of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these ‘sORFs’, indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects. PMID:26527729

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

PubMed

Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

2017-06-29

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

PubMed Central

Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M.; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

2016-01-01

Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

LOOP IIId of the HCV IRES is essential for the structural rearrangement of the 40S-HCV IRES complex

PubMed Central

Angulo, Jenniffer; Ulryck, Nathalie; Deforges, Jules; Chamond, Nathalie; Lopez-Lastra, Marcelo; Masquida, Benoît; Sargueil, Bruno

2016-01-01

As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a ‘kissing complex’ between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation. PMID:26626152

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

PubMed Central

Jha, Sujata; Rollins, Madeline G.; Fuchs, Gabriele; Procter, Dean J.; Hall, Elizabeth A.; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N.; Walsh, Derek

2017-01-01

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements1. However, beyond differential subunit expression during development2,3, evidence for regulated ribosome specification within individual cells has remained elusive1. Here, we report that a poxvirus kinase phosphorylates serine/threonine residues in the small ribosomal subunit protein, Receptor for Activated C Kinase (RACK1) that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs whose 5’ untranslated regions (UTRs) contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analysis revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, where these leaders act as translational enhancers for poorly understood reasons. Phosphomimetics and inter-species chimeras demonstrated that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase4 confer a translational advantage. Our findings uncover ribosome customization through a novel trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie the enigmatic poxvirus polyA-leaders4. PMID:28636603

The Ribosomal RNA is a Useful Marker to Visualize Rhizobia Interacting with Legume Plants

ERIC Educational Resources Information Center

Rinaudi, Luciana; Isola, Maria C.; Giordano, Walter

2004-01-01

Symbiosis between rhizobia and leguminous plants leads to the formation of nitrogen-fixing root nodules. In the present article, we recommend the use of the ribosomal RNA (rRNA) isolated from legume nodules in an experimental class with the purpose of introducing students to the structure of eukaryotic and prokaryotic ribosomes and of…

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.

Arginine residues on the opposite side of the active site stimulate the catalysis of ribosome depurination by ricin A chain by interacting with the P-protein stalk.

PubMed

Li, Xiao-Ping; Kahn, Peter C; Kahn, Jennifer Nielsen; Grela, Przemyslaw; Tumer, Nilgun E

2013-10-18

Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K(m) values and catalytic rates (k(cat)) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K(m) and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.

The Functional Role of eL19 and eB12 Intersubunit Bridge in the Eukaryotic Ribosome.

PubMed

Kisly, Ivan; Gulay, Suna P; Mäeorg, Uno; Dinman, Jonathan D; Remme, Jaanus; Tamm, Tiina

2016-05-22

During translation, the two eukaryotic ribosomal subunits remain associated through 17 intersubunit bridges, five of which are eukaryote specific. These are mainly localized to the peripheral regions and are believed to stabilize the structure of the ribosome. The functional importance of these bridges remains largely unknown. Here, the essentiality of the eukaryote-specific bridge eB12 has been investigated. The main component of this bridge is ribosomal protein eL19 that is composed of an N-terminal globular domain, a middle region, and a long C-terminal α-helix. The analysis of deletion mutants demonstrated that the globular domain and middle region of eL19 are essential for cell viability, most likely functioning in ribosome assembly. The eB12 bridge, formed by contacts between the C-terminal α-helix of eL19 and 18S rRNA in concert with additional stabilizing interactions involving either eS7 or uS17, is dispensable for viability. Nevertheless, eL19 mutants impaired in eB12 bridge formation displayed slow growth phenotypes, altered sensitivity/resistance to translational inhibitors, and enhanced hyperosmotic stress tolerance. Biochemical analyses determined that the eB12 bridge contributes to the stability of ribosome subunit interactions in vitro. 60S subunits containing eL19 variants defective in eB12 bridge formation failed to form 80S ribosomes regardless of Mg(2+) concentration. The reassociation of 40S and mutant 60S subunits was markedly improved in the presence of deacetylated tRNA, emphasizing the importance of tRNAs during the subunit association. We propose that the eB12 bridge plays an important role in subunit joining and in optimizing ribosome functionality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-wide assessment of differential translations with ribosome profiling data.

PubMed

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-04-04

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations.

Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation.

PubMed

Wasserman, Michael R; Alejo, Jose L; Altman, Roger B; Blanchard, Scott C

2016-04-01

Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Multi-perspective smFRET reveals rate-determining late intermediates of ribosomal translocation

PubMed Central

Wasserman, Michael R.; Alejo, Jose L.; Altman, Roger B.; Blanchard, Scott C.

2016-01-01

Directional translocation of the ribosome through the messenger RNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of transfer and messenger RNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we have tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations reveal direct evidence of structurally and kinetically distinct, late intermediates during substrate movement, whose resolution is rate-determining to the translocation mechanism. These steps involve intra-molecular events within the EFG(GDP)-bound ribosome, including exaggerated, reversible fluctuations of the small subunit head domain, which ultimately facilitate peptidyl-tRNA’s movement into its final post-translocation position. PMID:26926435

Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex.

PubMed

Cukras, Anthony R; Southworth, Daniel R; Brunelle, Julie L; Culver, Gloria M; Green, Rachel

2003-08-01

Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment.

PubMed

Gomez, James E; Kaufmann-Malaga, Benjamin B; Wivagg, Carl N; Kim, Peter B; Silvis, Melanie R; Renedo, Nikolai; Ioerger, Thomas R; Ahmad, Rushdy; Livny, Jonathan; Fishbein, Skye; Sacchettini, James C; Carr, Steven A; Hung, Deborah T

2017-02-21

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

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

HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

PubMed

Vizoso-Vázquez, A; Barreiro-Alonso, A; González-Siso, M I; Rodríguez-Belmonte, E; Lamas-Maceiras, M; Cerdán, M E

2018-04-30

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

PubMed Central

Smirnov, Alexandre; Entelis, Nina; Martin, Robert P.; Tarassov, Ivan

2011-01-01

5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes. PMID:21685364

The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif.

PubMed

Chan, Y L; Paz, V; Olvera, J; Wool, I G

1993-04-30

The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939. Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene. The mRNA for the protein is about 500 nucleotides in length. Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37. We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37.

Drug-Sensing by the Ribosome Induces Translational Arrest via Active Site Perturbation

PubMed Central

Arenz, Stefan; Meydan, Sezen; Starosta, Agata L.; Berninghausen, Otto; Beckmann, Roland; Vázquez-Laslop, Nora; Wilson, Daniel N.

2014-01-01

SUMMARY During protein synthesis, nascent polypeptide chains within the ribosomal tunnel can act in cis to induce ribosome stalling and regulate expression of downstream genes. The Staphylococcus aureus ErmCL leader peptide induces stalling in the presence of clinically important macrolide antibiotics, such as erythromycin, leading to the induction of the downstream macrolide resistance methyltransferase ErmC. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmCL-stalled ribosome at 3.9 Å resolution. The structure reveals how the ErmCL nascent chain directly senses the presence of the tunnel-bound drug and thereby induces allosteric conformational rearrangements at the peptidyltransferase center (PTC) of the ribosome. ErmCL-induced perturbations of the PTC prevent stable binding and accommodation of the aminoacyl-tRNA at the A-site leading to inhibition of peptide bond formation and translation arrest. PMID:25306253

Ribosome rearrangements at the onset of translational bypassing

PubMed Central

Agirrezabala, Xabier; Samatova, Ekaterina; Klimova, Mariia; Zamora, Miguel; Gil-Carton, David; Rodnina, Marina V.; Valle, Mikel

2017-01-01

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNAGly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap. PMID:28630923

Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae.

PubMed

Groth-Malonek, Milena; Wahrmund, Ute; Polsakiewicz, Monika; Knoop, Volker

2007-04-01

Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid liverworts.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parham, James F.; Macey, J. Robert; Papenfuss, Theodore J.

As part of an ongoing project to generate a mitochondrial database for terrestrial tortoises based on museum specimens, the complete mitochondrial genome sequences of 10 species and a {approx}14 kb sequence from an eleventh species are reported. The sampling of the present study emphasizes Mediterranean tortoises (genus Testudo and their close relatives). Our new sequences are aligned, along with those of two testudinoid turtles from GenBank, Chrysemys picta and Mauremys reevesii, yielding an alignment of 14,858 positions, of which 3,238 are parsimony informative. We develop a phylogenetic taxonomy for Testudo and related species based on well-supported, diagnosable clades. Several well-supportedmore » nodes are recovered, including the monophyly of a restricted Testudo, T. kleinmanni + T. marginata (the Chersus clade), and the placement of the enigmatic African pancake tortoise (Malacochersustornieri) within the predominantly Palearctic greater Testudo group (Testudona tax. nov.). Despite the large amount of sequence reported, there is low statistical support for some nodes within Testudona and Sowe do not propose names for those groups. A preliminary and conservative estimation of divergence times implies a late Miocene diversification for the testudonan clade (6-12 million years ago), matching their first appearance in the fossil record. The multi-continental distribution of testudonan turtles can be explained by the establishment of permanent connections between Europe, Africa, and Asia at this time. The arrival of testudonan turtles to Africa occurred after one or more initial tortoise invasions gave rise to the diverse (>25 species) 'Geochelone complex.'Two unusual genomic features are reported for the mtDNA of one tortoise, M. tornieri: (1) nad4 has a shift of reading frame that we suggest is resolved by translational frameshifting of the mRNA on the ribosome during protein synthesis and (2) there are two copies of the control region and trnF, with the latter having experienced multiple nucleotide substitutions in a pattern suggesting that each is being maintained by selection.« less

Vertical Transmission of the Retrotransposable Elements R1 and R2 during the Evolution of the Drosophila Melanogaster Species Subgroup

PubMed Central

Eickbush, D. G.; Eickbush, T. H.

1995-01-01

R1 and R2 are non-long-terminal repeat retrotransposable elements that insert into specific sequences of insect 28S ribosomal RNA genes. These elements have been extensively described in Drosophila melanogaster. To determine whether these elements have been horizontally or vertically transmitted, we characterized R1 and R2 elements from the seven other members of the melanogaster species subgroup by genomic blotting and nucleotide sequencing. Each species was found to have homogeneous families of R1 and R2 elements with the exception of erecta and orena, which have no R2 elements. The DNA sequences of multiple R1 and R2 copies from each species indicated nucleotide divergence within each species averaged only 0.48% for R1 and 0.35% for R2, well below the level of divergence among the species. Most copies of R1 and R2 (40 of 47) sequenced from the seven species were potentially functional, as indicated by the absence of premature termination codons or translational frameshifts that would destroy the open reading frame of the element. The sequence relationships of both the R1 and R2 elements from the various members of the melanogaster subgroup closely followed that of the species phylogeny, suggesting that R1 and R2 have been stably maintained by vertical transmission since the origin of this species subgroup 17-20 million years ago. The remarkable stability of R1 and R2, compared to what has been suggested for transposable elements that insert at multiple locations in these same species, may be due to their unique specificity for sites in the rRNA gene locus. Under low copy number conditions, when it is essential for any mobile element to transpose, the insertion specificities of R1 and R2 ensure uniform developmentally regulated target sites that can be occupied with little or no detrimental effect on the host. PMID:7713424

Global translational impacts of the loss of the tRNA modification t6A in yeast.

PubMed

Thiaville, Patrick C; Legendre, Rachel; Rojas-Benítez, Diego; Baudin-Baillieu, Agnès; Hatin, Isabelle; Chalancon, Guilhem; Glavic, Alvaro; Namy, Olivier; de Crécy-Lagard, Valérie

2016-01-01

The universal tRNA modification t 6 A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t 6 A 37 leads to severe growth defects in baker's yeast, phenotypes similar to those caused by defects in mcm 5 s 2 U 34 synthesis. Mutants in mcm 5 s 2 U 34 can be suppressed by overexpression of tRNA Lys UUU , but we show t 6 A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t 6 A and mcm 5 s 2 U are not determinants for each other's formation. Our results suggest that t 6 A deficiency, like mcm 5 s 2 U deficiency, leads to protein folding defects, and show that the absence of t 6 A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t 6 A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t 6 A absence were examined by ribosome profiling. Interestingly, the absence of t 6 A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t 6 A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I 34 :C 3 pairs while increasing the elongation rate of rare tRNAs and G 34 :U 3 pairs. This work reveals that the consequences of t 6 A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.

Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania.

PubMed

Padmanabhan, P K; Samant, M; Cloutier, S; Simard, M J; Papadopoulou, B

2012-12-01

Few natural antisense (as) RNAs have been reported as yet in the unicellular protozoan Leishmania. Here, we describe that Leishmania produces natural asRNAs complementary to all ribosomal RNA (rRNA) species. Interestingly, we show that drug-induced apoptosis-like programmed cell death triggers fragmentation of asRNA complementary to the large subunit gamma (LSU-γ) rRNA, one of the six 28S rRNA processed fragments in Leishmania. Heat and oxidative stress also induce fragmentation of asrRNA, but to a lesser extent. Extensive asrRNA cleavage correlates with rRNA breakdown and translation inhibition. Indeed, overexpression of asLSU-γ rRNA accelerates rRNA degradation upon induction of apoptosis. In addition, we provide mechanistic insight into the regulation of apoptosis-induced asrRNA fragmentation by a 67 kDa ATP-dependent RNA helicase of the DEAD-box subfamily. This helicase binds both sense (s)LSU-γ and asLSU-γ rRNAs, and appears to have a key role in protecting rRNA from degradation by preventing asrRNA cleavage and thus cell death. Remarkably, the asrRNA fragmentation process operates not only in trypanosomatid protozoa but also in mammals. Our findings uncover a novel mechanism of regulation involving asrRNA fragmentation and rRNA breakdown, that is triggered by apoptosis and conditions of reduced translation under stress, and seems to be evolutionary conserved.

Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria.

PubMed

Glotz, C; Müssig, J; Gewitz, H S; Makowski, I; Arad, T; Yonath, A; Wittmann, H G

1987-11-01

Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E. coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols. A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution. In addition, from one crystal form large crystals could be grown in X-ray capillaries. In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, II. A New Mendelian Mutation*

PubMed Central

Boynton, John E.; Gillham, Nicholas W.; Burkholder, Barbara

1970-01-01

A new mutation of Chlamydomonas reinhardi, cr-1, is characterized. The mutation exhibits Mendelian inheritance and affects the sedimentation velocity and formation of intact chloroplast ribosomes. The mutant grows reasonably well when supplied with sodium acetate as a carbon source, but poorly when forced to grow photosynthetically using carbon dioxide. Since the mutant cr-1 accumulates large subunits of the chloroplast ribosome, we postulate that it is blocked in the formation of the small subunit. A tentative model explaining the behavior of the several mutants in Chlamydomonas now known to have altered chloroplast ribosomal phenotypes is presented. Images PMID:16591885

Ribosome profiling reveals the what, when, where and how of protein synthesis.

PubMed

Brar, Gloria A; Weissman, Jonathan S

2015-11-01

Ribosome profiling, which involves the deep sequencing of ribosome-protected mRNA fragments, is a powerful tool for globally monitoring translation in vivo. The method has facilitated discovery of the regulation of gene expression underlying diverse and complex biological processes, of important aspects of the mechanism of protein synthesis, and even of new proteins, by providing a systematic approach for experimental annotation of coding regions. Here, we introduce the methodology of ribosome profiling and discuss examples in which this approach has been a key factor in guiding biological discovery, including its prominent role in identifying thousands of novel translated short open reading frames and alternative translation products.

Fluctuations and synchrony of RNA synthesis in nucleoli.

PubMed

Pliss, Artem; Kuzmin, Andrey N; Kachynski, Aliaksandr V; Baev, Alexander; Berezney, Ronald; Prasad, Paras N

2015-06-01

Ribosomal RNA (rRNA) sequences are synthesized at exceptionally high rates and, together with ribosomal proteins (r-proteins), are utilized as building blocks for the assembly of pre-ribosomal particles. Although it is widely acknowledged that tight regulation and coordination of rRNA and r-protein production are fundamentally important for the maintenance of cellular homeostasis, still little is known about the real-time kinetics of the ribosome component synthesis in individual cells. In this communication we introduce a label-free MicroRaman spectrometric approach for monitoring rRNA synthesis in live cultured cells. Remarkably high and rapid fluctuations of rRNA production rates were revealed by this technique. Strikingly, the changes in the rRNA output were synchronous for ribosomal genes located in separate nucleoli of the same cell. Our findings call for the development of new concepts to elucidate the coordination of ribosomal components production. In this regard, numerical modeling further demonstrated that the production of rRNA and r-proteins can be coordinated, regardless of the fluctuations in rRNA synthesis. Overall, our quantitative data reveal a spectacular interplay of inherently stochastic rates of RNA synthesis and the coordination of gene expression.

Focal accumulation of preribosomes outside the nucleolus during metaphase–anaphase in budding yeast

PubMed Central

Moriggi, Giulia; Gaspar, Sonia G.; Nieto, Blanca; Bustelo, Xosé R.

2017-01-01

Saccharomyces cerevisiae contains one nucleolus that remains intact in the mother-cell side of the nucleus throughout most of mitosis. Based on this, it is assumed that the bulk of ribosome production during cell division occurs in the mother cell. Here, we show that the ribosome synthesis machinery localizes not only in the nucleolus but also at a center that is present in the bud side of the nucleus after the initiation of mitosis. This center can be visualized by live microscopy as a punctate body located in close proximity to the nuclear envelope and opposite to the nucleolus. It contains ribosomal DNA (rDNA) and precursors of both 40S and 60S ribosomal subunits. Proteins that actively participate in ribosome synthesis, but not functionally defective variants, accumulate in that site. The formation of this body occurs in the metaphase-to-anaphase transition when discrete regions of rDNA occasionally exit the nucleolus and move into the bud. Collectively, our data unveil the existence of a previously unknown mechanism for preribosome accumulation at the nuclear periphery in budding yeast. We propose that this might be a strategy to expedite the delivery of ribosomes to the growing bud. PMID:28588079

Oxidative stress damages rRNA inside the ribosome and differentially affects the catalytic center

PubMed Central

Willi, Jessica; Küpfer, Pascal; Evéquoz, Damien; Fernandez, Guillermo; Polacek, Norbert

2018-01-01

Abstract Intracellular levels of reactive oxygen species (ROS) increase as a consequence of oxidative stress and represent a major source of damage to biomolecules. Due to its high cellular abundance RNA is more frequently the target for oxidative damage than DNA. Nevertheless the functional consequences of damage on stable RNA are poorly understood. Using a genome-wide approach, based on 8-oxo-guanosine immunoprecipitation, we present evidence that the most abundant non-coding RNA in a cell, the ribosomal RNA (rRNA), is target for oxidative nucleobase damage by ROS. Subjecting ribosomes to oxidative stress, we demonstrate that oxidized 23S rRNA inhibits the ribosome during protein biosynthesis. Placing single oxidized nucleobases at specific position within the ribosome's catalytic center by atomic mutagenesis resulted in markedly different functional outcomes. While some active site nucleobases tolerated oxidative damage well, oxidation at others had detrimental effects on protein synthesis by inhibiting different sub-steps of the ribosomal elongation cycle. Our data provide molecular insight into the biological consequences of RNA oxidation in one of the most central cellular enzymes and reveal mechanistic insight on the role of individual active site nucleobases during translation. PMID:29309687

Depletion of ribosomal protein L37 occurs in response to DNA damage and activates p53 through the L11/MDM2 pathway

PubMed Central

Llanos, Susana; Serrano, Manuel

2013-01-01

Perturbation of ribosomal biogenesis has recently emerged as a relevant p53-activating pathway. This pathway can be initiated by depletion of certain ribosomal proteins, which is followed by the binding and inhibition of MDM2 by a different subset of ribosomal proteins that includes L11. Here, we report that depletion of L37 leads to cell cycle arrest in a L11- and p53-dependent manner. DNA damage can initiate ribosomal stress, although little is known about the mechanisms involved. We have found that some genotoxic insults, namely UV light and cisplatin, lead to proteasomal degradation of L37 in the nucleoplasm and to the ensuing L11-dependent stabilization of p53. Moreover, ectopic L37 overexpression can attenuate the DNA damage response mediated by p53. These results support the concept that DNA damage-induced proteasomal degradation of L37 constitutes a mechanistic link between DNA damage and the ribosomal stress pathway, and is a relevant contributing signaling pathway for the activation of p53 in response to DNA damage. PMID:20935493

Pseudoknot and translational control in the expression of the S15 ribosomal protein.

PubMed

Bénard, L; Philippe, C; Ehresmann, B; Ehresmann, C; Portier, C

1996-01-01

Translational autocontrol of the expression of the ribosomal protein S15 proceeds through the transitory formation of a pseudoknot. A synopsis of the known data is used to propose a molecular model of the mechanism involved and for the role of the pseudoknot. This latter structure is able to recruit 30S ribosomal subunits to initiate translation, but also to bind S15 and to stop translation by trapping the ribosome on its loading site. Information on the S15 protein recognition of the messenger RNA site was deduced from mutational analyses and chemical probing. A comparison of this messenger site with the S15 ribosomal binding site was conducted by analysing hydroxyl radical footprintings of these two sites. The existence of two subsites in 16S RNA suggests that the ribosomal protein S15 might present either two different binding sites or at least one common subsite. Clues for the presence of a common site between the messenger and 16S RNA are given which cannot rule out that recognition specificity is linked to a few other determinants. Whether these determinants are different or not remains an open question.

Translational profiling of B cells infected with the Epstein-Barr virus reveals 5' leader ribosome recruitment through upstream open reading frames.

PubMed

Bencun, Maja; Klinke, Olaf; Hotz-Wagenblatt, Agnes; Klaus, Severina; Tsai, Ming-Han; Poirey, Remy; Delecluse, Henri-Jacques

2018-04-06

The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. We have used ribosome profiling to characterize viral translation in infected cells and map new translation initiation sites. We show here that EBV transcripts are translated with highly variable efficiency, owing to variable transcription and translation rates, variable ribosome recruitment to the leader region and coverage by monosomes versus polysomes. Some transcripts were hardly translated, others mainly carried monosomes, showed ribosome accumulation in leader regions and most likely represent non-coding RNAs. A similar process was visible for a subset of lytic genes including the key transactivators BZLF1 and BRLF1 in cells infected with weakly replicating EBV strains. This suggests that ribosome trapping, particularly in the leader region, represents a new checkpoint for the repression of lytic replication. We could identify 25 upstream open reading frames (uORFs) located upstream of coding transcripts that displayed 5' leader ribosome trapping, six of which were located in the leader region shared by many latent transcripts. These uORFs repressed viral translation and are likely to play an important role in the regulation of EBV translation.

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

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.

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

The amino terminal end determines the stability and assembling capacity of eukaryotic ribosomal stalk proteins P1 and P2.

PubMed

Camargo, Hendricka; Nusspaumer, Gretel; Abia, David; Briceño, Verónica; Remacha, Miguel; Ballesta, Juan P G

2011-05-01

The eukaryotic ribosomal proteins P1 and P2 bind to protein P0 through their N-terminal domain to form the essential ribosomal stalk. A mutational analysis points to amino acids at positions 2 and 3 as determinants for the drastic difference of Saccharomyces cerevisiae P1 and P2 half-life, and suggest different degradation mechanisms for each protein type. Moreover, the capacity to form P1/P2 heterodimers is drastically affected by mutations in the P2β four initial amino acids, while these mutations have no effect on P1β. Binding of P2β and, to a lesser extent, P1β to the ribosome is also seriously affected showing the high relevance of the amino acids in the first turn of the NTD α-helix 1 for the stalk assembly. The negative effect of some mutations on ribosome binding can be reversed by the presence of the second P1/P2 couple in the ribosome, indicating a stabilizing structural influence between the two heterodimers. Unexpectedly, some mutations totally abolish heterodimer formation but allow significant ribosome binding and, therefore, a previous P1 and P2 association seems not to be an absolute requirement for stalk assembly. Homology modeling of the protein complexes suggests that the mutated residues can affect the overall protein conformation. © The Author(s) 2011. Published by Oxford University Press.

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain.

PubMed

Koch, Miriam; Willi, Jessica; Pradère, Ugo; Hall, Jonathan; Polacek, Norbert

2017-06-20

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson-Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

PubMed Central

Ahmed, Tofayel; Shi, Jian

2017-01-01

Abstract Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c. PMID:28582576

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

PubMed

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-05-18

The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits.

Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

PubMed Central

Espinar-Marchena, Francisco; Rodríguez-Galán, Olga; Fernández-Fernández, José; Linnemann, Jan; de la Cruz, Jesús

2018-01-01

Abstract The contribution of most ribosomal proteins to ribosome synthesis has been quite well analysed in Saccharomyces cerevisiae. However, few yeast ribosomal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre-60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA2 and 27SA3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S particles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S particle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans-acting factors required for 27SB pre-rRNA processing but accumulate practically all factors required for processing of 27SA3 pre-rRNA. We have also analysed the functional interaction between the eukaryote-specific carboxy-terminal extensions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alterations in mature 60S subunits. PMID:29788267

sORFs.org: a repository of small ORFs identified by ribosome profiling.

PubMed

Olexiouk, Volodimir; Crappé, Jeroen; Verbruggen, Steven; Verhegen, Kenneth; Martens, Lennart; Menschaert, Gerben

2016-01-04

With the advent of ribosome profiling, a next generation sequencing technique providing a "snap-shot'' of translated mRNA in a cell, many short open reading frames (sORFs) with ribosomal activity were identified. Follow-up studies revealed the existence of functional peptides, so-called micropeptides, translated from these 'sORFs', indicating a new class of bio-active peptides. Over the last few years, several micropeptides exhibiting important cellular functions were discovered. However, ribosome occupancy does not necessarily imply an actual function of the translated peptide, leading to the development of various tools assessing the coding potential of sORFs. Here, we introduce sORFs.org (http://www.sorfs.org), a novel database for sORFs identified using ribosome profiling. Starting from ribosome profiling, sORFs.org identifies sORFs, incorporates state-of-the-art tools and metrics and stores results in a public database. Two query interfaces are provided, a default one enabling quick lookup of sORFs and a BioMart interface providing advanced query and export possibilities. At present, sORFs.org harbors 263 354 sORFs that demonstrate ribosome occupancy, originating from three different cell lines: HCT116 (human), E14_mESC (mouse) and S2 (fruit fly). sORFs.org aims to provide an extensive sORFs database accessible to researchers with limited bioinformatics knowledge, thus enabling easy integration into personal projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Genome Mining for Ribosomally Synthesized Natural Products

PubMed Central

Velásquez, Juan E.; van der Donk, Wilfred

2011-01-01

In recent years, the number of known peptide natural products that are synthesized via the ribosomal pathway has rapidly grown. Taking advantage of sequence homology among genes encoding precursor peptides or biosynthetic proteins, in silico mining of genomes combined with molecular biology approaches has guided the discovery of a large number of new ribosomal natural products, including lantipeptides, cyanobactins, linear thiazole/oxazole-containing peptides, microviridins, lasso peptides, amatoxins, cyclotides, and conopeptides. In this review, we describe the strategies used for the identification of these ribosomally-synthesized and posttranslationally modified peptides (RiPPs) and the structures of newly identified compounds. The increasing number of chemical entities and their remarkable structural and functional diversity may lead to novel pharmaceutical applications. PMID:21095156

The linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility

PubMed Central

Bashan, Anat; Yonath, Ada

2009-01-01

Crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to their large size, complex structure, inherent flexibility and high conformational variability. For the case of the small ribosomal subunit, which caused extreme difficulties, post crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. This cluster played a dual role in ribosomal crystallography: providing anomalous phasing power and dramatically increased the resolution, by stabilization of a selected functional conformation. Thus, four out of the fourteen clusters that bind to each of the crystallized small subunits are attached to a specific ribosomal protein in a fashion that may control a significant component of the subunit internal flexibility, by “gluing” symmetrical related subunits. Here we highlight basic issues in the relationship between metal ions and macromolecules and present common traits controlling in the interactions between polymetalates and various macromolecules, which may be extended towards the exploitation of polymetalates for therapeutical treatment. PMID:19915655

Genome-wide assessment of differential translations with ribosome profiling data

PubMed Central

Xiao, Zhengtao; Zou, Qin; Liu, Yu; Yang, Xuerui

2016-01-01

The closely regulated process of mRNA translation is crucial for precise control of protein abundance and quality. Ribosome profiling, a combination of ribosome foot-printing and RNA deep sequencing, has been used in a large variety of studies to quantify genome-wide mRNA translation. Here, we developed Xtail, an analysis pipeline tailored for ribosome profiling data that comprehensively and accurately identifies differentially translated genes in pairwise comparisons. Applied on simulated and real datasets, Xtail exhibits high sensitivity with minimal false-positive rates, outperforming existing methods in the accuracy of quantifying differential translations. With published ribosome profiling datasets, Xtail does not only reveal differentially translated genes that make biological sense, but also uncovers new events of differential translation in human cancer cells on mTOR signalling perturbation and in human primary macrophages on interferon gamma (IFN-γ) treatment. This demonstrates the value of Xtail in providing novel insights into the molecular mechanisms that involve translational dysregulations. PMID:27041671

The ribosome as an optimal decoder: a lesson in molecular recognition.

PubMed

Savir, Yonatan; Tlusty, Tsvi

2013-04-11

The ribosome is a complex molecular machine that, in order to synthesize proteins, has to decode mRNAs by pairing their codons with matching tRNAs. Decoding is a major determinant of fitness and requires accurate and fast selection of correct tRNAs among many similar competitors. However, it is unclear whether the modern ribosome, and in particular its large conformational changes during decoding, are the outcome of adaptation to its task as a decoder or the result of other constraints. Here, we derive the energy landscape that provides optimal discrimination between competing substrates and thereby optimal tRNA decoding. We show that the measured landscape of the prokaryotic ribosome is sculpted in this way. This model suggests that conformational changes of the ribosome and tRNA during decoding are means to obtain an optimal decoder. Our analysis puts forward a generic mechanism that may be utilized broadly by molecular recognition systems. Copyright © 2013 Elsevier Inc. All rights reserved.

Downregulation of ribosome biogenesis during early forebrain development

PubMed Central

Chau, Kevin F; Shannon, Morgan L; Fame, Ryann M; Fonseca, Erin; Mullan, Hillary; Johnson, Matthew B; Sendamarai, Anoop K; Springel, Mark W; Laurent, Benoit

2018-01-01

Forebrain precursor cells are dynamic during early brain development, yet the underlying molecular changes remain elusive. We observed major differences in transcriptional signatures of precursor cells from mouse forebrain at embryonic days E8.5 vs. E10.5 (before vs. after neural tube closure). Genes encoding protein biosynthetic machinery were strongly downregulated at E10.5. This was matched by decreases in ribosome biogenesis and protein synthesis, together with age-related changes in proteomic content of the adjacent fluids. Notably, c-MYC expression and mTOR pathway signaling were also decreased at E10.5, providing potential drivers for the effects on ribosome biogenesis and protein synthesis. Interference with c-MYC at E8.5 prematurely decreased ribosome biogenesis, while persistent c-MYC expression in cortical progenitors increased transcription of protein biosynthetic machinery and enhanced ribosome biogenesis, as well as enhanced progenitor proliferation leading to subsequent macrocephaly. These findings indicate large, coordinated changes in molecular machinery of forebrain precursors during early brain development. PMID:29745900

A Plant 5S Ribosomal RNA Mimic Regulates Alternative Splicing of Transcription Factor IIIA Pre-mRNAs

PubMed Central

Hammond, Ming C.; Wachter, Andreas; Breaker, Ronald R.

2009-01-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with striking resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs (pre-mRNAs) from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. Since the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing. PMID:19377483

Structural insights into cell cycle control by essential GTPase Era.

PubMed

Ji, Xinhua

Era (Escherichia coli Ras-like protein), essential for bacterial cell viability, is composed of an N-terminal GTPase domain and a C-terminal KH domain. In bacteria, it is required for the processing of 16S ribosomal RNA (rRNA) and maturation of 30S (small) ribosomal subunit. Era recognizes 10 nucleotides ( 1530 GAUCACCUCC 1539 ) near the 3' end of 16S rRNA and interacts with helix 45 (h45, nucleotides 1506-1529). GTP binding enables Era to bind RNA, RNA binding stimulates Era's GTP-hydrolyzing activity, and GTP hydrolysis releases Era from matured 30S ribosomal subunit. As such, Era controls cell growth rate via regulating the maturation of the 30S ribosomal subunit. Ribosomes manufacture proteins in all living organisms. The GAUCA sequence and h45 are highly conserved in all three kingdoms of life. Homologues of Era are present in eukaryotic cells. Hence, the mechanism of bacterial Era action also sheds light on the cell cycle control of eukaryotes.

The Structure of the Anti-tuberculosis Antibiotics Viomycin and Capreomycin Bound to the 70S Ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanley, R.; Blaha, G; Grodzicki, R

2010-01-01

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

The structures of the anti-tuberculosis antibiotics viomycin and capreomycin bound to the 70S ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanley, Robin E.; Blaha, Gregor; Grodzicki, Robert L.

2010-05-03

Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-{angstrom} resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in themore » pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.« less

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

PubMed Central

Amanatiadou, Elsa P.; Papadopoulos, Giorgio L.; Strouboulis, John; Vizirianakis, Ioannis S.

2015-01-01

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

Assembly and disassembly of the nucleolus during the cell cycle.

PubMed

Hernandez-Verdun, Danièle

2011-01-01

The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs.

PubMed

Hammond, Ming C; Wachter, Andreas; Breaker, Ronald R

2009-05-01

Transcription factor IIIA (TFIIIA) is required for eukaryotic synthesis of 5S ribosomal RNA by RNA polymerase III. Here we report the discovery of a structured RNA element with clear resemblance to 5S rRNA that is conserved within TFIIIA precursor mRNAs from diverse plant lineages. TFIIIA protein expression is controlled by alternative splicing of the exon containing the plant 5S rRNA mimic (P5SM). P5SM triggers exon skipping upon binding of ribosomal protein L5, a natural partner of 5S rRNA, which demonstrates the functional adaptation of its structural mimicry. As the exon-skipped splice product encodes full-length TFIIIA protein, these results reveal a ribosomal protein-mRNA interaction that is involved in 5S rRNA synthesis and has implications for cross-coordination of ribosomal components. This study also provides insight into the origin and function of a newfound class of structured RNA that regulates alternative splicing.

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.

Molecular basis for erythromycin-dependent ribosome-stalling during translation of the ErmBL leader peptide

PubMed Central

Arenz, Stefan; Ramu, Haripriya; Gupta, Pulkit; Berninghausen, Otto; Beckmann, Roland; Vázquez-Laslop, Nora; Mankin, Alexander S.; Wilson, Daniel N.

2014-01-01

In bacteria, ribosome-stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here, we present a cryo-electron microscopy (EM) structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase center (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural bases for understanding how the drug and the nascent peptide cooperate to inhibit peptide-bond formation and induce translation arrest. PMID:24662426

Structural basis for 16S ribosomal RNA cleavage by the cytotoxic domain of colicin E3.

PubMed

Ng, C Leong; Lang, Kathrin; Meenan, Nicola Ag; Sharma, Amit; Kelley, Ann C; Kleanthous, Colin; Ramakrishnan, V

2010-10-01

The toxin colicin E3 targets the 30S subunit of bacterial ribosomes and cleaves a phosphodiester bond in the decoding center. We present the crystal structure of the 70S ribosome in complex with the cytotoxic domain of colicin E3 (E3-rRNase). The structure reveals how the rRNase domain of colicin binds to the A site of the decoding center in the 70S ribosome and cleaves the 16S ribosomal RNA (rRNA) between A1493 and G1494. The cleavage mechanism involves the concerted action of conserved residues Glu62 and His58 of the cytotoxic domain of colicin E3. These residues activate the 16S rRNA for 2' OH-induced hydrolysis. Conformational changes observed for E3-rRNase, 16S rRNA and helix 69 of 23S rRNA suggest that a dynamic binding platform is required for colicin E3 binding and function.

The Use of Plant-Derived Ribosome Inactivating Proteins in Immunotoxin Development: Past, Present and Future Generations

PubMed Central

Rust, Aleksander; Partridge, Lynda J.; Davletov, Bazbek

2017-01-01

Ribosome inactivating proteins (RIPs) form a class of toxins that was identified over a century ago. They continue to fascinate scientists and the public due to their very high activity and long-term stability which might find useful applications in the therapeutic killing of unwanted cells but can also be used in acts of terror. We will focus our review on the canonical plant-derived RIPs which display ribosomal RNA N-glycosidase activity and irreversibly inhibit protein synthesis by cleaving the 28S ribosomal RNA of the large 60S subunit of eukaryotic ribosomes. We will place particular emphasis on therapeutic applications and the generation of immunotoxins by coupling antibodies to RIPs in an attempt to target specific cells. Several generations of immunotoxins have been developed and we will review their optimisation as well as their use and limitations in pre-clinical and clinical trials. Finally, we endeavour to provide a perspective on potential future developments for the therapeutic use of immunotoxins. PMID:29076988

Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution

NASA Astrophysics Data System (ADS)

Quade, Nick; Boehringer, Daniel; Leibundgut, Marc; van den Heuvel, Joop; Ban, Nenad

2015-07-01

Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.

Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome

NASA Astrophysics Data System (ADS)

Adio, Sarah; Senyushkina, Tamara; Peske, Frank; Fischer, Niels; Wintermeyer, Wolfgang; Rodnina, Marina V.

2015-06-01

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

Analysis of immune responses in genital tracts of mice immunised with purified ribosomal fractions of Neisseria gonorrhoeae.

PubMed Central

Kita, E; Kashiba, S

1984-01-01

Immunisation of ddY mice with the purified ribosomal fraction of Neisseria gonorrhoeae was found to protect against intravaginal challenge with homologous organisms. This protection correlated with the presence of bactericidal antibody to purified ribosomal fraction in serum as well as in vaginal secretions. Analysis of the vaginal fluids from control mice and those immunised with purified ribosomal fraction showed that the enhanced elimination of gonococci in immune mice might be because of an early response of leucocytes generated by the reaction mediated by antibody and complement. Absorption studies showed that there was at least one major protective antigen in purified ribosomal fraction, other than cell surface substances such as lipopolysaccharide, outer membrane proteins, and pili. Bactericidal assays mediated by antibody and complement showed that matched samples of serum and vaginal fluid from immune mice had comparable gonococcidal activity, which was augmented by the effect of progesterone. Although delayed hypersensitivity was produced in immune mice that were resistant to N gonorrhoeae, the exact role of cellular immunity could not be clarified in this study. These results suggest that antibody to purified ribosomal fraction plays a major part in protection against gonococcal infection in the genital tract, and that such protection may entail both cellular immunity and hormonal changes. PMID:6430462

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

PubMed

Hatakeyama, T; Kimura, M

1988-03-15

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jie; Lancaster, Laura; Trakhanov, Sergei

2012-03-26

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 {angstrom} crystal structure of the RF3 {center_dot} GDPNP {center_dot} ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7{sup o} rotation of the body and 14{sup o} rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. Wemore » suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.« less

Cytoplasmic long noncoding RNAs are frequently bound to and degraded at ribosomes in human cells

PubMed Central

Carlevaro-Fita, Joana; Rahim, Anisa; Guigó, Roderic; Vardy, Leah A.; Johnson, Rory

2016-01-01

Recent footprinting studies have made the surprising observation that long noncoding RNAs (lncRNAs) physically interact with ribosomes. However, these findings remain controversial, and the overall proportion of cytoplasmic lncRNAs involved is unknown. Here we make a global, absolute estimate of the cytoplasmic and ribosome-associated population of stringently filtered lncRNAs in a human cell line using polysome profiling coupled to spike-in normalized microarray analysis. Fifty-four percent of expressed lncRNAs are detected in the cytoplasm. The majority of these (70%) have >50% of their cytoplasmic copies associated with polysomal fractions. These interactions are lost upon disruption of ribosomes by puromycin. Polysomal lncRNAs are distinguished by a number of 5′ mRNA-like features, including capping and 5′UTR length. On the other hand, nonpolysomal “free cytoplasmic” lncRNAs have more conserved promoters and a wider range of expression across cell types. Exons of polysomal lncRNAs are depleted of endogenous retroviral insertions, suggesting a role for repetitive elements in lncRNA localization. Finally, we show that blocking of ribosomal elongation results in stabilization of many associated lncRNAs. Together these findings suggest that the ribosome is the default destination for the majority of cytoplasmic long noncoding RNAs and may play a role in their degradation. PMID:27090285