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Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation  [PDF]
Johann W. Bauer, Clemens Brandl, Olaf Haubenreisser, Bjoern Wimmer, Manuela Weber, Thomas Karl, Alfred Klausegger, Michael Breitenbach, Helmut Hintner, Tobias von der Haar, Mick F. Tuite, Lore Breitenbach-Koller
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0067609
Abstract: Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.
Translation Initiation Factor AteIF(iso)4E Is Involved in Selective mRNA Translation in Arabidopsis Thaliana Seedlings  [PDF]
Ana Valeria Martínez-Silva, César Aguirre-Martínez, Carlos E. Flores-Tinoco, Naholi D. Alejandri-Ramírez, Tzvetanka D. Dinkova
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0031606
Abstract: One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5′end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions.
Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity  [PDF]
Amit Sharma,Alper Yilmaz,Kim Marsh,Alan Cochrane,Kathleen Boris-Lawrie
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002612
Abstract: Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
Sensitivity of mRNA Translation  [PDF]
Gilad Poker,Michael Margaliot,Tamir Tuller
Quantitative Biology , 2014,
Abstract: Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, exit, and elongation rates along the mRNA strand on the steady state protein translation rate. We focus on two special cases where exact closed-form expressions for the translation rate sensitivity can be derived. We discuss the ramifications of our results in the context of functional genomics, molecular evolution, and synthetic biology.
The many routes to regulating mRNA translation
Kristian E Baker, Jeff Coller
Genome Biology , 2006, DOI: 10.1186/gb-2006-7-12-332
Abstract: Regulation of gene expression at the level of mRNA translation is a fundamental mechanism for moderating cellular events. A recent meeting at Cold Spring Harbor Laboratory served to highlight the modes, mechanisms, and architecture of translational control and of the translational apparatus itself (a full list of presentations is available at http://meetings.cshl.edu/meetings/abstracts/2006transc_absstat.html webcite). This report concentrates on several prominent themes in the regulation of mRNA translation. Central to the topics discussed here is the observation that the translation of single specific mRNAs, subsets, or even a majority of the mRNAs in a cell, is controlled almost exclusively through a multitude of interactions that occur between RNA-binding proteins and regulatory elements embedded throughout the mRNA (Figure 1).Binding of proteins to regulatory elements in the 3' untranslated region (3' UTR) of mRNA can facilitate repression of mRNA translation, and evidence suggests that several mRNAs that can be regulated in this way are controlled via a common messenger ribonucleoprotein (mRNP) architecture. The core repression complex consists of proteins that mediate an interaction between the 3' UTR-binding proteins and an mRNA 5' cap-binding protein, either the translation initiation factor eIF4E or a mimic. This arrangement sequesters the mRNA 5' cap in a nonproductive closed-loop conformation and inhibits an obligatory step in translational initiation. An example of this mode of control was presented by Paul Lasko (McGill University, Montréal, Canada), who described the repression of two mRNAs, hunchback and caudal, that are important for axis formation in the Drosophila embryo. An important point to note is that although the recognition elements within the two mRNAs bind distinct proteins, the respective mRNP complexes both lead to repression of translation through an interaction with a unique mRNA cap-binding protein, 4EHP.Claudia Bagni (University of
The Dynamics of Supply and Demand in mRNA Translation  [PDF]
Chris A. Brackley ,M. Carmen Romano,Marco Thiel
PLOS Computational Biology , 2011, DOI: 10.1371/journal.pcbi.1002203
Abstract: We study the elongation stage of mRNA translation in eukaryotes and find that, in contrast to the assumptions of previous models, both the supply and the demand for tRNA resources are important for determining elongation rates. We find that increasing the initiation rate of translation can lead to the depletion of some species of aa-tRNA, which in turn can lead to slow codons and queueing. Particularly striking “competition” effects are observed in simulations of multiple species of mRNA which are reliant on the same pool of tRNA resources. These simulations are based on a recent model of elongation which we use to study the translation of mRNA sequences from the Saccharomyces cerevisiae genome. This model includes the dynamics of the use and recharging of amino acid tRNA complexes, and we show via Monte Carlo simulation that this has a dramatic effect on the protein production behaviour of the system.
Use of surface plasmon resonance for the measurement of low affinity binding interactions between HSP72 and measles virus nucleocapsid protein
Zhang Xinsheng,Oglesbee Michael
Biological Procedures Online , 2003, DOI: 10.1251/bpo59
Abstract: The 72 kDa heat shock protein (HSP72) is a molecular chaperone that binds native protein with low affinity. These interactions can alter function of the substrate, a property known as HSP-mediated activity control. In the present work, BIAcore instrumentation was used to monitor binding reactions between HSP72 and naturally occurring sequence variants of the measles virus (MV) nucleocapsid protein (N), a structural protein regulating transcription/replication of the viral genome. Binding reactions employed synthetic peptides mimicking a putative HSP72 binding motif of N. Sequences were identified that bound HSP72 with affinities comparable to well-characterized activity control reactions. These sequences, but not those binding with lesser affinity, supported HSP72 activity control of MV transcription/replication. BIAcore instrumentation thus provides an effective way to measure biologically relevant low affinity interactions with structural variants of viral proteins.
Semi-Markov models of mRNA-translation  [PDF]
Mieke Gorissen,Carlo Vanderzande
Quantitative Biology , 2011,
Abstract: Translation is the cellular process in which ribosomes make proteins from information encoded on messenger RNA (mRNA). We model translation with an exclusion process taking into account the experimentally determined, non-exponential, waiting time between steps of a ribosome. From numerical simulations using realistic parameter values, we determine the distribution P(E) of the number of proteins E produced by one mRNA. We find that for small E this distribution is not geometric. We present a simplified and analytically solvable semi-Markov model that relates P(E) to the distributions of the times to produce the first E proteins.
Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation  [PDF]
Claudia M. Casanova, Peter Sehr, Kerstin Putzker, Matthias W. Hentze, Beate Neumann, Kent E. Duncan, Christian Thoma
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045943
Abstract: Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.
Reduced stability of mRNA secondary structure near the translation-initiation site in dsDNA viruses
Tong Zhou, Claus O Wilke
BMC Evolutionary Biology , 2011, DOI: 10.1186/1471-2148-11-59
Abstract: Here, we surveyed the complete genomes of 650 dsDNA virus strains for signals of reduced stability of mRNA secondary structure near the start codon. Our analysis included viruses infecting eukaryotic, prokaryotic, and archaeic hosts. We found that many viruses showed evidence for reduced mRNA secondary-structure stability near the start codon. The effect was most pronounced in viruses infecting prokaryotes, but was also observed in viruses infecting eukaryotes and archaea. The reduction in stability generally increased with increasing genomic GC content. For bacteriophage, the reduction was correlated with a corresponding reduction of stability in the phage hosts.We conclude that reduced stability of the mRNA secondary structure near the start codon is a common feature for dsDNA viruses, likely driven by the same selective pressures that cause it in cellular organisms.Translation initiation is facilitated by specific nucleotide patterns near the start codon. Upstream of the start codon, sequence features such as the Shine-Dalgarno sequence (in prokaryotes) and the Kozak sequence (in eukaryotes) prime the ribosome to initiate translation [1-7]. Downstream of the start codon, various sequence features promote translation initiation. For example, in Escherichia coli, the codon AAA seems to enhance translation initiation [8]. More generally, translation initiation is enhanced if the mRNA downstream of the start codon is AT-rich and does not form a stable secondary structure [9-13].Experimental and computational work in E. coli showed that gene expression levels are correlated to the thermodynamic stability of mRNA secondary structure near the start codon—lower stability implied higher protein abundance [13]. Recent computational studies have shown that the secondary-structure stability of mRNA segments near the start codon is on average lower than expected [14,15]. Tuller et al. found that, in both E. coli and Saccharomyces cerevisiae, mRNA secondary-structure stability
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