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Evaluation of the dual luciferase assay under screening conditions. ( A ) Increasing volumes of yeast (strain BY4741) culture expressing FlucUUG ( d ) and RlucAUG ( j ) from plasmid pFuugRaug were added to lysis buffer, and the resulting luciferase activities measured. Points are fit to a straight line with R > 0.99 for both Renilla and firefly luciferase activities. ( B ) BY4741 transformed with pFuugRaug was grown with various concentrations of cycloheximide, and the luciferase activities were measured after 4 h ( d , FlucUUG; j , RlucAUG). ( C )
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Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compo...
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Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis.
In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate
AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide impo...
Citations
... Initiation from the correct start codon is critical for translation fidelity, and EIF1 and 1A are important regulators of this process [30][31][32][33]. Natural and induced fluctuations in the levels or activity of these factors affect initiation site stringency [30,[34][35][36], particularly in the context of non-AUG translation of upstream open reading frames (uORFs) [37,38] (reviewed in [39]). Interestingly, CoV2 and other coronaviruses encode for multiple conserved uORFs that are actively translated during infection [21,40]. ...
A better mechanistic understanding of virus-host dependencies can help reveal vulnerabilities and identify opportunities for therapeutic intervention. Of particular interest are essential interactions that enable production of viral proteins, as those could target an early step in the virus lifecycle. Here, we use subcellular proteomics, ribosome profiling analyses and reporter assays to detect changes in protein synthesis dynamics during SARS-CoV-2 (CoV2) infection. We identify specific translation factors and molecular chaperones that are used by CoV2 to promote the synthesis and maturation of its own proteins. These can be targeted to inhibit infection, without major toxicity to the host. We also find that CoV2 non-structural protein 1 (Nsp1) cooperates with initiation factors EIF1 and 1A to selectively enhance translation of viral RNA. When EIF1/1A are depleted, more ribosomes initiate translation from a conserved upstream CUG start codon found in all genomic and subgenomic viral RNAs. This results in higher translation of an upstream open reading frame (uORF1) and lower translation of the main ORF, altering the stoichiometry of viral proteins and attenuating infection. Replacing the upstream CUG with AUG strongly inhibits translation of the main ORF independently of Nsp1, EIF1, or EIF1A. Taken together, our work describes multiple dependencies of CoV2 on host biosynthetic networks and proposes a model for dosage control of viral proteins through Nsp1-mediated control of translation start site selection.
... Furthermore, a considerable number of non-AUG uORFs whose mutation significantly changed reporter expression may not be translated, as insertion of the stalling CGA dicodon sequence did not reduce expression. Thus, at least during log-phase growth, many non-AUG uORFs appear to have little influence on gene expression, consistent with the relatively low rate of translation initiation at near-cognate start codons (Kolitz et al., 2009;Takacs et al., 2011). As non-AUG uORFs have been predicted in many other species, our results suggest that most of them may also play limited roles in regulating expression. ...
Upstream open-reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.
... Furthermore, a considerable number of non-AUG uORFs whose mutation significantly changed reporter expression may not be translated, as insertion of the stalling CGA dicodon sequence did not reduce expression. Thus, at least during log-phase growth, many non-AUG uORFs appear to have little influence on gene expression, consistent with the relatively low rate of translation initiation at near-cognate start codons (Kolitz et al., 2009;Takacs et al., 2011). As non-AUG uORFs have been predicted in many other species, our results suggest that most of them may also play limited roles in regulating expression. ...
Upstream open-reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.
... Furthermore, a considerable number of non-AUG uORFs whose mutation significantly changed reporter expression may not be translated, as insertion of the stalling CGA dicodon sequence did not reduce expression. Thus, at least during log-phase growth, many non-AUG uORFs appear to have little influence on gene expression, consistent with the relatively low rate of translation initiation at near-cognate start codons (Kolitz et al., 2009;Takacs et al., 2011). As non-AUG uORFs have been predicted in many other species, our results suggest that most of them may also play limited roles in regulating expression. ...
Upstream open-reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that both uORF sequences and locations within transcript leaders predict their effect on gene expression. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as predictive as uORF sequences.
... A daunting task as any intervention with this mechanism will affect the translation of a multitude of mRNAs. A large screen in yeast yielded two related quinoline-containing compounds that improved translation from non-canonical start codons (Takacs et al., 2011). However, this was achieved at the expense of fidelity of translation from the canonical AUG start codon. ...
Disruption of axon-glia interactions in the peripheral nervous system has emerged as a major cause of arthrogryposis multiplex congenita (AMC), a condition characterized by multiple congenital postural abnormalities involving the major joints. Several genes crucially important to the biology of Schwann cells have now been implicated with AMC. One such gene is LGI4 which encodes a secreted glycoprotein. LGI4 is expressed and secreted by Schwann cells and binds its receptor ADAM22 on the axonal membrane to drive myelination. Homozygous mutations in LGI4 or ADAM22 results in severe congenital hypomyelination and joint contractures in mice. Recently bi-allelic LGI4 loss of function mutations has been described in three unrelated families with severe AMC. Two individuals in a fourth, non-consanguineous family were found to be compound heterozygous for two LGI4 missense mutations. It is not known how these missense mutations affect the biology of LGI4. Here we investigated whether these missense mutations affected the secretion of the protein, its ADAM22 binding capacity, or its myelination-promoting function. We demonstrate that the mutations largely affect the progression of the mutant protein through the endomembrane system resulting in severely reduced expression. Importantly, binding to ADAM22 and myelination-promoting activity appear largely unaffected, suggesting that treatment with chemical chaperones to improve secretion of the mutant proteins might prove beneficial.
... Thus, at least during log-phase growth, many non-AUG uORFs appear to have little influence on gene expression, consistent with the relatively low rate of translation initiation at near-cognate start codons (Kolitz et al., 2009;Takacs et al., 2011). As non-AUG uORFs have been predicted in many other species, our results suggest that most of them may also play limited roles in regulating expression. ...
Upstream open reading frames (uORFs) are potent cis-acting regulators of mRNA translation and nonsense-mediated decay (NMD). While both AUG- and non-AUG initiated uORFs are ubiquitous in ribosome profiling studies, few uORFs have been experimentally tested. Consequently, the relative influences of sequence, structural, and positional features on uORF activity have not been determined. We quantified thousands of yeast uORFs using massively parallel reporter assays in wildtype and ∆upf1 yeast. While nearly all AUG uORFs were robust repressors, most non-AUG uORFs had relatively weak impacts on expression. Machine learning regression modeling revealed that uORF functions are strongly impacted by both their sequences and locations within transcript leaders. Indeed, alternative transcription start sites highly influenced uORF activity. These results define the scope of natural uORF activity, identify features associated with translational repression and NMD, and suggest that the locations of uORFs in transcript leaders are nearly as important as uORF sequences.
... For example, TISs with high translation efficiency tend to have Kozak sequences (i.e., a purine [A or G] and a guanine at the −3 and +4 positions, respectively; +1 denotes the first base of the AUG start site) (Kozak 1984), and mammalian transcripts also have M (i.e., A or C), M and C at the −2, −4, and +5 positions, respectively (Noderer et al. 2014). Trans-acting factors such as eIF1 and eIF1A play critical roles in distinguishing AUG from non-AUG start codons (Takacs et al. 2011;Lind and Åqvist 2016). ...
Translation initiation is a key step determining protein synthesis. Studies have uncovered a number of alternative translation initiation sites (TISs) in mammalian mRNAs and showed their roles in reshaping the proteome. However, the extent to which alternative TISs affect gene expression across plants remains largely unclear. Here, by profiling initiating ribosome positions, we globally identified in vivo TISs in tomato and Arabidopsis and found thousands of genes with more than one TIS. Of the identified TISs, >19% and >20% were located at unannotated AUG and non-AUG sites, respectively. CUG and ACG were the most frequently observed codons at non-AUG TISs, a phenomenon also found in mammals. In addition, although alternative TISs were usually found in both orthologous genes, the TIS sequences were not conserved, suggesting the conservation of alternative initiation mechanisms but flexibility in using TISs. Unlike upstream AUG TISs, the presence of upstream non-AUG TISs was not correlated with the translational repression of main open reading frames, a pattern observed across plants. Also, the generation of proteins with diverse N-terminal regions through the use of alternative TISs contributes to differential subcellular localization, as mutating alternative TISs resulted in the loss of organelle localization. Our findings uncovered the hidden coding potential of plant genomes and, importantly, the constraint and flexibility of translational initiation mechanisms in the regulation of gene expression across plant species.
... A mutation in eIF1A with the opposite effect on fidelity was shown to confer heightened discrimination genome-wide against AUGs in poor sequence context [19]. The use of NCC start sites in a reporter mRNA in yeast was also shown to be enhanced by two small molecules identified in a highthroughput screen [20], indicating that external agents can modulate the fidelity of start codon recognition. ...
... Changes in the fidelity of the start codon recognition in response to external or internal stimuli could rapidly modify the proteome by changing the balance of translation of uORFs, N-terminal extensions, and main ORFs. In an attempt to test this hypothesis, we used the same dual-luciferase reporter assay used for the highthroughput chemical screen for compounds that alter the start codon recognition in S. cerevisiae [20] to search for other external stimuli that produce similar effects. We found that growth temperature modulates the use of near-cognate start codons in both the luciferase reporter system and an orthogonal lacZ-based system. ...
... We previously developed and validated a dual-luciferase assay to calculate the efficiency of utilization of nearcognate codons (NCCs) as translational start sites in yeast [20]. In this assay, Renilla luciferase (R-Luc) and Firefly luciferase (F-Luc) are expressed using separate promoters and transcription terminators from a single low-copy plasmid (Fig. 1a). ...
Background:
Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature.
Results:
Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation.
Conclusions:
Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.
... Such classes of drugs are an active field of research. For example, drug screens have identified compounds that decrease the fidelity of start codon initiation [93]. Moreover, ataluren (Translarna), a drug promoting premature stop-codon read-through [94], is in clinical trials for treatment of diseases caused by nonsense mutations, such as Duchenne muscular dystrophy and cystic fibrosis [95,96]. ...
Ribosomopathies are congenital diseases with defects in ribosome assembly and are characterized by elevated cancer risks. Additionally, somatic mutations in ribosomal proteins have recently been linked to a variety of cancers. Despite a clear correlation between ribosome defects and cancer, the molecular mechanisms by which these defects promote tumorigenesis are unclear. In this review, we focus on the emerging mechanisms that link ribosomal defects in ribosomopathies to cancer progression. This includes functional “onco-specialization” of mutant ribosomes, extra-ribosomal consequences of mutations in ribosomal proteins and ribosome assembly factors, and effects of ribosomal mutations on cellular stress and metabolism. We integrate some of these recent findings in a single model that can partially explain the paradoxical transition from hypo- to hyperproliferation phenotypes, as observed in ribosomopathies. Finally, we discuss the current and potential strategies, and the associated challenges for therapeutic intervention in ribosome-mutant diseases.
... If fidelity is impaired, combining L-leucine with a drug that corrects fidelity defects may be required. Indeed, drugs targeting ribosomal fidelity are an active field of research: Drug screens have identified compounds that decrease the fidelity of start codon initiation (170). Moreover, ataluren (Translarna), a drug promoting premature stop-codon readthrough (171), has received market authorization from the European Commission and is in clinical trials for treatment of diseases caused by nonsense mutations, such as Duchenne muscular dystrophy and cystic fibrosis (172,173). ...
A wealth of novel findings, including congenital ribosomal mutations in ribosomopathies and somatic ribosomal mutations in various cancers, have significantly increased our understanding of the relevance of ribosomes in oncogenesis. Here, we explore the growing list of mechanisms by which the ribosome is involved in carcinogenesis—from the hijacking of ribosomes by oncogenic factors and dysregulated translational control, to the effects of mutations in ribosomal components on cellular metabolism. Of clinical importance, the recent success of RNA polymerase inhibitors highlights the dependence on “onco-ribosomes” as an Achilles' heel of cancer cells and a promising target for further therapeutic intervention.
Significance: The recent discovery of somatic mutations in ribosomal proteins in several cancers has strengthened the link between ribosome defects and cancer progression, while also raising the question of which cellular mechanisms such defects exploit. Here, we discuss the emerging molecular mechanisms by which ribosomes support oncogenesis, and how this understanding is driving the design of novel therapeutic strategies. Cancer Discov; 7(10); 1069–87. ©2017 AACR.
