scholarly journals Author response: Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

2020 ◽  
Author(s):  
Szymon Juszkiewicz ◽  
Greg Slodkowicz ◽  
Zhewang Lin ◽  
Paula Freire-Pritchett ◽  
Sew-Yeu Peak-Chew ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Feedback Inhibition ◽  
Author Response ◽  
Cis Acting

scholarly journals Ribosome collisions trigger cis-acting feedback inhibition of translation initiation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Szymon Juszkiewicz ◽  
Greg Slodkowicz ◽  
Zhewang Lin ◽  
Paula Freire-Pritchett ◽  
Sew-Yeu Peak-Chew ◽  
...  
Keyword(s):  
Quality Control ◽  
Translation Initiation ◽  
Quantitative Proteomics ◽  
Feedback Inhibition ◽  
The Other ◽  
Cis Acting ◽  
Ribosome Density ◽  
In Cis

Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognised by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 stabilises GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway’s output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products.

scholarly journals A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF

2020 ◽  
Vol 21 (18) ◽  
pp. 6974
Author(s):  
Niki Vassilaki ◽  
Efseveia Frakolaki ◽  
Katerina I. Kalliampakou ◽  
Panagiotis Sakellariou ◽  
Ioly Kotta-Loizou ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Translation Initiation ◽  
Site Directed Mutagenesis ◽  
Luciferase Reporter ◽  
Cis Acting ◽  
The Core ◽  
Rna Transfection ◽  
Alternative Translation ◽  
Internal Translation

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5′-untranslated region (5′UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597–599 and 603–605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344–596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome–HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5′UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF.

scholarly journals Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells

2012 ◽  
Vol 442 (1) ◽  
pp. 199-207 ◽  
Author(s):  
Sofia Origanti ◽  
Shannon L. Nowotarski ◽  
Theresa D. Carr ◽  
Suzanne Sass-Kuhn ◽  
Lan Xiao ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Translation Initiation ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Regulatory Sequences ◽  
Minor Effect ◽  
Dependent Manner ◽  
Cis Acting ◽  
Treatment And Prevention ◽  
Effector Pathways

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

scholarly journals The yeast eIF2 kinase Gcn2 facilitates H2O2-mediated feedback inhibition of both protein synthesis and ER oxidative folding during recombinant protein production

2021 ◽  
Author(s):  
Veronica Gast ◽  
Kate Campbell ◽  
Cecilia Picazo Campos ◽  
Martin Engqvist ◽  
Verena Siewers ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Recombinant Protein ◽  
Translation Initiation ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Feedback Inhibition ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Oxidative Folding ◽  
Amylase Production

AbstractRecombinant protein production is a known source of oxidative stress. Knowledge of which ROS are involved or the specific growth phase in which stress occurs however remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, micro-cultivation and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find Gcn2-dependent phosphorylation of eIF2α to increase alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains, but not in the wild-type. Gcn2-deficiency furthermore reduced intracellular H2O2 levels and the unfolded protein response whilst expression of antioxidants and the ER disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding to be coupled and subject to feedback inhibition by H2O2.ImportanceReactive oxygen species (ROS) accumulate during recombinant protein production both in yeast and Chinese hamster ovary cells, two of the most popular organisms used in the multi-million dollar protein production industry. Here we document increased H2O2 in the cytosol of yeast cells producing α-amylase. Since H2O2 predominantly targets the protein synthesis machinery and activates the translation initiation factor kinase Gcn2, we removed Gcn2, resulting in increased recombinant α-amylase production in two different previously engineered high-producing protein production strains. Removal of this negative feed-back loop thus represents a complementary strategy for improving recombinant protein production efforts currently used in yeast. Gcn2-deficiency also increased the expression of antioxidant genes and the ER-foldase PDI1, suggesting that protein synthesis and ER oxidative folding are linked and feed-back regulated via H2O2. Identification of additional components in this complex regulation may further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

The role of IRES trans-acting factors in regulating translation initiation

2010 ◽  
Vol 38 (6) ◽  
pp. 1581-1586 ◽  
Author(s):  
Helen A. King ◽  
Laura C. Cobbold ◽  
Anne E. Willis
Keyword(s):  
Translation Initiation ◽  
Alternative Form ◽  
Structural Element ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Cis Acting ◽  
Initiation Factors ◽  
Eukaryotic Initiation Factors ◽  
Cellular Pathways

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.

scholarly journals uORFs: Important Cis-Regulatory Elements in Plants

2020 ◽  
Vol 21 (17) ◽  
pp. 6238
Author(s):  
Ting Zhang ◽  
Anqi Wu ◽  
Yaping Yue ◽  
Yu Zhao
Keyword(s):  
Gene Expression ◽  
Translation Initiation ◽  
Translational Control ◽  
Translational Regulation ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Post Translational Modification ◽  
Cis Acting ◽  
Eukaryotic Mrnas

Gene expression is regulated at many levels, including mRNA transcription, translation, and post-translational modification. Compared with transcriptional regulation, mRNA translational control is a more critical step in gene expression and allows for more rapid changes of encoded protein concentrations in cells. Translation is highly regulated by complex interactions between cis-acting elements and trans-acting factors. Initiation is not only the first phase of translation, but also the core of translational regulation, because it limits the rate of protein synthesis. As potent cis-regulatory elements in eukaryotic mRNAs, upstream open reading frames (uORFs) generally inhibit the translation initiation of downstream major ORFs (mORFs) through ribosome stalling. During the past few years, with the development of RNA-seq and ribosome profiling, functional uORFs have been identified and characterized in many organisms. Here, we review uORF identification, uORF classification, and uORF-mediated translation initiation. More importantly, we summarize the translational regulation of uORFs in plant metabolic pathways, morphogenesis, disease resistance, and nutrient absorption, which open up an avenue for precisely modulating the plant growth and development, as well as environmental adaption. Additionally, we also discuss prospective applications of uORFs in plant breeding.

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