scholarly journals Combinations of slow translating codon clusters can increase mRNA half-life

2018 ◽  
Author(s):  
Ajeet K. Sharma ◽  
Edward P. O’Brien
Keyword(s):  
Half Life ◽  
Opposite Effect ◽  
Synergistic Effects ◽  
Cellular Protein ◽  
Protein Levels ◽  
Dead Box ◽  
Naturally Occurring ◽  
Optimal Codon ◽  
Primary Determinant ◽  
Mrna Half Life

AbstractThe introduction of a single, non-optimal codon cluster into a transcript was found to cause ribosomes to queue upstream of it and decrease the transcript’s half-life through the action of the dead box protein Dhh1p, which interacts with stalled ribosomes and promotes transcript degradation. Naturally occurring transcripts often contain multiple slow codon clusters, but the influence of these combinations of clusters on a transcript’s half-life is unknown. Using a kinetic model that describes translation and mRNA degradation, we find that the introduction of a second slow codon cluster into a transcript near the 5′-end can have the opposite effect than that of the first cluster by increasing the mRNA’s half-life. We experimentally validate this finding by showing that S. cerevisiae transcripts that only have a slow codon cluster towards the 3′-end of the coding sequence have shorter half-lives than transcripts with non-optimal clusters at both 3′ and 5′ -ends. The origin of this increase in half-life arises from the 5′-end cluster suppressing the formation of ribosome queues upstream of the second cluster, thereby decreasing the opportunities for translation-dependent degradation machinery, such as the Dhh1p protein, to interact with stalled ribosomes. We also find in the model that in the presence of two slow codon clusters the cluster closest to 5′-end is the primary determinant of mRNA half-life. These results identify two of the rules governing the influence of slow codon clusters on mRNA half-life, demonstrate that multiple slow codon clusters can have synergistic effects, and indicate that codon usage bias can play a more nuanced role in controlling cellular protein levels than previously thought.

Combinations of slow-translating codon clusters can increase mRNA half-life in Saccharomyces cerevisiae

2021 ◽  
Vol 118 (51) ◽  
pp. e2026362118
Author(s):  
Ajeet K. Sharma ◽  
Johannes Venezian ◽  
Ayala Shiber ◽  
Günter Kramer ◽  
Bernd Bukau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quality Control ◽  
Half Life ◽  
Synergistic Effects ◽  
Cellular Protein ◽  
Start Codon ◽  
Translational Efficiency ◽  
Control Factors ◽  
The Impact ◽  
Mrna Half Life

The presence of a single cluster of nonoptimal codons was found to decrease a transcript’s half-life through the interaction of the ribosome-associated quality control machinery with stalled ribosomes in Saccharomyces cerevisiae. The impact of multiple nonoptimal codon clusters on a transcript’s half-life, however, is unknown. Using a kinetic model, we predict that inserting a second nonoptimal cluster near the 5′ end can lead to synergistic effects that increase a messenger RNA’s (mRNA’s) half-life in S. cerevisiae. Specifically, the 5′ end cluster suppresses the formation of ribosome queues, reducing the interaction of ribosome-associated quality control factors with stalled ribosomes. We experimentally validate this prediction by introducing two nonoptimal clusters into three different genes and find that their mRNA half-life increases up to fourfold. The model also predicts that in the presence of two clusters, the cluster closest to the 5′ end is the primary determinant of mRNA half-life. These results suggest the “translational ramp,” in which nonoptimal codons are located near the start codon and increase translational efficiency, may have the additional biological benefit of allowing downstream slow-codon clusters to be present without decreasing mRNA half-life. These results indicate that codon usage bias plays a more nuanced role in controlling cellular protein levels than previously thought.

scholarly journals OmpC regulation differs between ST131 and non-ST131 Escherichia coli clinical isolates and involves differential expression of the small RNA MicC

2020 ◽  
Vol 75 (5) ◽  
pp. 1151-1158
Author(s):  
Corey S Suelter ◽  
Nancy D Hanson
Keyword(s):  
Escherichia Coli ◽  
Half Life ◽  
Selective Advantage ◽  
Resistance Mechanisms ◽  
Northern Blotting ◽  
Rt Pcr ◽  
E Coli ◽  
Protein Levels ◽  
Porin Proteins ◽  
Mrna Half Life

Abstract Background Virulence genes and the expression of resistance mechanisms undoubtedly play a role in the successful spread of the pandemic clone Escherichia coli ST131. Porin down-regulation is a chromosomal mechanism associated with antibiotic resistance. Translation of porin proteins can be impacted by modifications in mRNA half-life and the interaction among small RNAs (sRNAs), the porin transcript and the sRNA chaperone Hfq. Modifications in the translatability of porin proteins could impact the fitness and therefore the success of E. coli ST131 isolates in the presence of antibiotic. Objectives To identify differences in the translatability of OmpC and OmpF porins for different STs of E. coli by comparing steady-state RNA levels, mRNA half-life, regulatory sRNA expression and protein production. Methods RNA expression was evaluated using real-time RT–PCR and OmpC mRNA half-life by northern blotting. OmpC, OmpF and Hfq protein levels were evaluated by immunoblotting. Results Differences between ST131 and non-ST131 isolates included: (i) the level of OmpC RNA and protein produced with mRNA expression higher for ST131 but OmpC protein levels lower compared with non-ST131 isolates; (ii) OmpC mRNA half-life (21–30 min for ST131 isolates compared with <2–23 min for non-ST131 isolates); and (iii) levels of the sRNA MicC (2- to 120-fold for ST131 isolates compared with −4- to 70-fold for non-ST131 isolates). Conclusions Mechanisms involved in the translatability of porin proteins differed among different STs of E. coli. These differences could provide a selective advantage to ST131 E. coli when confronted with an antibiotic-rich environment.

scholarly journals Interleukin 1β Regulation of the System xc− Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR

2015 ◽  
Vol 291 (4) ◽  
pp. 1643-1651 ◽  
Author(s):  
Jingxue Shi ◽  
Yan He ◽  
Sandra J. Hewett ◽  
James A. Hewett
Keyword(s):  
Half Life ◽  
Rna Binding ◽  
Critical Role ◽  
Primary Cultures ◽  
Dependent Manner ◽  
Protein Levels ◽  
Mouse Cortex ◽  
Primary Mouse ◽  
Rna Immunoprecipitation ◽  
Mrna Half Life

System xc− is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc− in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3′-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3′-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc− activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

Debris/Micrometeoroid Impacts and Synergistic Effects on Spacecraft Materials

MRS Bulletin ◽  
2010 ◽  
Vol 35 (1) ◽  
pp. 41-47 ◽  
Author(s):  
E. Grossman ◽  
I. Gouzman ◽  
R. Verker
Keyword(s):  
Space Debris ◽  
Synergistic Effects ◽  
Degradation Mechanism ◽  
Space Environment ◽  
Materials Properties ◽  
Gas Gun ◽  
Naturally Occurring ◽  
Space Activity ◽  
The Impact ◽  
Debris Impact

AbstractIn the last 40 years, the increased space activity created a new form of space environment of hypervelocity objects—space debris—that have no functional use. The space debris, together with naturally occurring ultrahigh velocity meteoroids, presents a significant hazard to spacecraft. Collision with space debris or meteoroids might result in disfunction of external units such as solar cells, affecting materials properties, contaminating optical devices, or destroying satellites. The collision normally results in the formation of additional debris, increasing the hazard for future missions. The hypervelocity debris effect is studied by retrieving materials from space or by using ground simulation facilities. Simulation facilities, which include the light gas gun and Laser Driven Flyer methods, are used for studying the materials degradation due to debris impact. The impact effect could be accelerated when occurring simultaneously with other space environment components, such as atomic oxygen, ultraviolet, or x-ray radiation. Understanding the degradation mechanism might help in developing materials that will withstand the increasing hazard from the space debris, allowing for longer space missions. The large increase in space debris population and the associated risk to space activity requires significant measures to mitigate this hazard. Most current efforts are being devoted to prevention of collisions by keeping track of the larger debris and avoiding formation of new debris.

Integration of in vitro neurotoxicity data with biokinetic modelling for the estimation of in vivo neurotoxicity

2007 ◽  
Vol 26 (4) ◽  
pp. 333-338 ◽  
Author(s):  
Anna Forsby ◽  
Bas Blaauboer
Keyword(s):  
Exposure Period ◽  
Cellular Protein ◽  
In Vitro Toxicity ◽  
Target Tissue ◽  
Receptor Function ◽  
Human Neuroblastoma ◽  
Protein Levels ◽  
Effective Doses

Risk assessment of neurotoxicity is mainly based on in vivo exposure, followed by tests on behaviour, physiology and pathology. In this study, an attempt to estimate lowest observed neurotoxic doses after single or repeated dose exposure was performed. Differentiated human neuroblastoma SH-SY5Y cells were exposed to acrylamide, lindane, parathion, paraoxon, phenytoin, diazepam or caffeine for 72 hours. The effects on protein synthesis and intracellular free Ca2+concentration were studied as physiological endpoints. Voltage operated Ca2 +channel function, acetylcholine receptor function and neurite degenerative effects were investigated as neurospecific endpoints for excitability, cholinergic signal transduction and axonopathy, respectively. The general cytotoxicity, determined as the total cellular protein levels after the 72 hours exposure period, was used for comparison to the specific endpoints and for estimation of acute lethality. The lowest concentration that induced 20% effect (EC 20) obtained for each compound, was used as a surrogate for the lowest neurotoxic level (LOEL) at the target site in vivo. The LOELs were integrated with data on adsorption, distribution, metabolism and excretion of the compounds in physiologically-based biokinetic (PBBK) models of the rat and the lowest observed effective doses (LOEDs) were estimated for the test compounds. A good correlation was observed between the estimated LOEDs and experimental LOEDs found in literature for rat for all test compounds, except for diazepam. However, when using in vitro data from the literature on diazepam's effect on gamma-amino butyric acid (GABA)A receptor function for the estimation of LOED, the correlation between the estimated and experimental LOEDs was improved from a 10 000-fold to a 10-fold difference. Our results indicate that it is possible to estimate LOEDs by integrating in vitro toxicity data as surrogates for lowest observed target tissue levels with PBBK models, provided that some knowledge about toxic mechanisms is known. Human & Experimental Toxicology (2007) 26, 333—338

Transcription and Translation of the Sex Message in the Smut Fungus Ustilago Violacea

1974 ◽  
Vol 16 (1) ◽  
pp. 49-62
Author(s):  
J. E. CUMMINS ◽  
A. W. DAY
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Information Exchange ◽  
Plasma Membranes ◽  
Cellular Protein ◽  
Mating Activity ◽  
Ustilago Violacea ◽  
Protein Levels ◽  
Amino Acid Turnover ◽  
Mating Conditions

In Ustilago violacea conjugation involves a period of courtship followed by the cooperative assembly of a conjugation tube between paired sporidia of opposite mating type. During courtship information is exchanged between the paired sporidia directing transcription and translation of a ‘sex message’. This information exchange is completed even though the plasma membranes and walls of the mating sporidia remain intact until after the sex message has been translated. Once translation is completed the copulatory organelle is assembled. Inhibitor studies show that translation of specific mRNA species (sex message) is essential during the 3-4 h period of courtship prior to assembly and that transcription of this sex message is completed 15-30 min prior to the completion of translation. Amino acid incorporation studies show that even though the cellular protein levels remain constant during courtship there is extensive amino acid turnover as the sporidia adjust to mating conditions. However, an enhancement of amino acid turnover by mating, in contrast to unmixed sporidia, is not detectable. Prototrophic strains continue to synthesize stable RNA during courtship while amino acid auxotrophs discontinue stable RNA synthesis immediately on transfer to mating conditions. Both prototrophs and auxotrophs show extensive RNA turnover during courtship but a specific enhancement of turnover due to mating activity is not detectable from the labelling pattern alone. A round of DNA replication may be completed during courtship but mating activity does not influence the gross pattern of DNA replication in either of the mating types.

scholarly journals The Middle Domain of Hsp90 Acts as a Discriminator between Different Types of Client Proteins

2006 ◽  
Vol 26 (22) ◽  
pp. 8385-8395 ◽  
Author(s):  
Patricija Hawle ◽  
Martin Siepmann ◽  
Anja Harst ◽  
Marco Siderius ◽  
H. Peter Reusch ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Fold Increase ◽  
Cellular Protein ◽  
Hydrolysis Rate ◽  
Cellular Activity ◽  
Protein Levels ◽  
Middle Domain ◽  
Client Proteins

ABSTRACT The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.

scholarly journals Induction of TAK (Cyclin T1/P-TEFb) in Purified Resting CD4+ T Lymphocytes by Combination of Cytokines

2001 ◽  
Vol 75 (23) ◽  
pp. 11336-11343 ◽  
Author(s):  
Romi Ghose ◽  
Li-Ying Liou ◽  
Christine H. Herrmann ◽  
Andrew P. Rice
Keyword(s):  
T Lymphocytes ◽  
Rna Polymerase Ii ◽  
Interleukin 2 ◽  
Cellular Protein ◽  
Peripheral Blood Lymphocytes ◽  
Tat Protein ◽  
Necrosis Factor Alpha ◽  
Cyclin T1 ◽  
Protein Levels ◽  
Hiv 1

ABSTRACT Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4+ T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4+ T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4+ T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4+ T lymphocytes harboring a latent provirus.

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally

2019 ◽  
Vol 476 (2) ◽  
pp. 333-352 ◽  
Author(s):  
Lisa Schmidtke ◽  
Katharina Schrick ◽  
Sabrina Saurin ◽  
Rudolf Käfer ◽  
Fabian Gather ◽  
...  
Keyword(s):  
Half Life ◽  
Rna Binding ◽  
Binding Protein ◽  
Regulatory Protein ◽  
Rna Binding Protein ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Type Iii ◽  
Type Iii Ifn ◽  
Mrna Half Life

Abstract Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3′-untranslated regions (3′-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3′-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP−/− mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo. Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.

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