scholarly journals The 3′ Untranslated Region of a Plant Viral RNA Directs Efficient Cap-Independent Translation in Plant and Mammalian Systems

Pathogens ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 28 ◽  
Author(s):  
Jelena Kraft ◽  
Mariko Peterson ◽  
Sung Cho ◽  
Zhaohui Wang ◽  
Alice Hui ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Plant Viruses ◽  
Translation Initiation Factor ◽  
Viral Rna ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Translational Machinery ◽  
Rich Domain ◽  
Reticulocyte Lysate ◽  
Independent Translation

Many plant viral RNA genomes lack a 5′ cap, and instead are translated via a cap-independent translation element (CITE) in the 3′ untranslated region (UTR). The panicum mosaic virus-like CITE (PTE), found in many plant viral RNAs, binds and requires the cap-binding translation initiation factor eIF4E to facilitate translation. eIF4E is structurally conserved between plants and animals, so we tested cap-independent translation efficiency of PTEs of nine plant viruses in plant and mammalian systems. The PTE from thin paspalum asymptomatic virus (TPAV) facilitated efficient cap-independent translation in wheat germ extract, rabbit reticulocyte lysate, HeLa cell lysate, and in oat and mammalian (BHK) cells. Human eIF4E bound the TPAV PTE but not a PTE that did not stimulate cap-independent translation in mammalian extracts or cells. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) footprinting revealed that both human and wheat eIF4E protected the conserved guanosine (G)-rich domain in the TPAV PTE pseudoknot. The central G plays a key role, as it was found to be required for translation and protection from SHAPE modification by eIF4E. These results provide insight on how plant viruses gain access to the host’s translational machinery, an essential step in infection, and raise the possibility that similar PTE-like mechanisms may exist in mRNAs of mammals or their viruses.

scholarly journals Impaired Binding of Standard Initiation Factors Mediates Poliovirus Translation Attenuation

2003 ◽  
Vol 77 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Kerstin Ochs ◽  
Amandus Zeller ◽  
Lanja Saleh ◽  
Gergis Bassili ◽  
Yutong Song ◽  
...  
Keyword(s):  
Single Point ◽  
Translation Initiation Factor ◽  
Viral Rna ◽  
Initiation Factor ◽  
Single Point Mutation ◽  
Translation Efficiency ◽  
Entry Site ◽  
Polypyrimidine Tract Binding Protein ◽  
Domain V ◽  
Impaired Binding

ABSTRACT In the oral poliovirus vaccine, three attenuated virus strains generated by Albert Sabin are used. However, insufficient genetic stability of these strains causes major problems in poliovirus eradication. In infected cells, translation of the plus-strand poliovirus RNA genome is directed by the internal ribosome entry site (IRES), a cis-acting RNA element that facilitates the cap-independent binding of ribosomes to an internal site of the viral RNA. In each Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence attenuation. Here we report how these decisive mutations in the IRES confer a reduction in poliovirus translation efficiency. These single-nucleotide exchanges impair the interaction of the standard translation initiation factor eIF4G with the IRES domain V. Moreover, binding of eIF4B and the polypyrimidine tract-binding protein and the association of ribosomes with the viral RNA are affected by these mutations. However, the negative effects of the IRES mutations are completely relieved by addition of purified eIF4F. This indicates that eIF4G is the crucial factor that initially binds to the poliovirus IRES and recruits the IRES to the other components of the translational apparatus, while impaired binding of eIF4G plays a key role in attenuation of poliovirus neurovirulence.

scholarly journals The RNA of Maize Chlorotic Mottle Virus, an Obligatory Component of Maize Lethal Necrosis Disease, Is Translated via a Variant Panicum Mosaic Virus-Like Cap-Independent Translation Element

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Elizabeth J. Carino ◽  
Kay Scheets ◽  
W. Allen Miller
Keyword(s):  
Translation Initiation ◽  
Untranslated Region ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Mottle Virus ◽  
Resistance Strategies ◽  
Chlorotic Mottle Virus ◽  
Maize Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Independent Translation

ABSTRACT Maize chlorotic mottle virus (MCMV) combines with a potyvirus in maize lethal necrosis disease (MLND), a serious emerging disease worldwide. To inform resistance strategies, we characterized the translation initiation mechanism of MCMV. We report that MCMV RNA contains a cap-independent translation element (CITE) in its 3′ untranslated region (UTR). The MCMV 3′ CITE (MTE) was mapped to nucleotides 4164 to 4333 in the genomic RNA. 2′-Hydroxyl acylation analyzed by primer extension (SHAPE) probing revealed that the MTE is a distinct variant of the panicum mosaic virus-like 3′ CITE (PTE). Like the PTE, electrophoretic mobility shift assays (EMSAs) indicated that eukaryotic translation initiation factor 4E (eIF4E) binds the MTE despite the absence of an m7GpppN cap structure, which is normally required for eIF4E to bind RNA. Using a luciferase reporter system, mutagenesis to disrupt and restore base pairing revealed that the MTE interacts with the 5′ UTRs of both genomic RNA and subgenomic RNA1 via long-distance kissing stem-loop interaction to facilitate translation. The MTE stimulates a relatively low level of translation and has a weak, if any, pseudoknot, which is present in the most active PTEs, mainly because the MTE lacks the pyrimidine-rich tract that base pairs to a G-rich bulge to form the pseudoknot. However, most mutations designed to form a pseudoknot decreased translation activity. Mutations in the viral genome that reduced or restored translation prevented and restored virus replication, respectively, in maize protoplasts and in plants. In summary, the MTE differs from the canonical PTE but falls into a structurally related class of 3′ CITEs. IMPORTANCE In the past decade, maize lethal necrosis disease has caused massive crop losses in East Africa. It has also emerged in China and parts of South America. Maize chlorotic mottle virus (MCMV) infection is required for this disease. While some tolerant maize lines have been identified, there are no known resistance genes that confer immunity to MCMV. In order to improve resistance strategies against MCMV, we focused on how the MCMV genome is translated, the first step of gene expression by all positive-strand RNA viruses. We identified a structure (cap-independent translation element) in the 3′ untranslated region of the viral RNA genome that allows the virus to usurp a host translation initiation factor, eIF4E, in a way that differs from host mRNA interactions with the translational machinery. This difference indicates eIF4E may be a soft target for engineering of—or breeding for—resistance to MCMV.

scholarly journals 5′-UTR recruitment of the translation initiation factor eIF4GI or DAP5 drives cap-independent translation of a subset of human mRNAs

2020 ◽  
Vol 295 (33) ◽  
pp. 11693-11706 ◽  
Author(s):  
Solomon A. Haizel ◽  
Usha Bhardwaj ◽  
Ruben L. Gonzalez ◽  
Somdeb Mitra ◽  
Dixie J. Goss
Keyword(s):  
Translation Initiation ◽  
Tumor Hypoxia ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Luciferase Reporter ◽  
Binding Studies ◽  
Specific Subset ◽  
Independent Translation

During unfavorable conditions (e.g. tumor hypoxia or viral infection), canonical, cap-dependent mRNA translation is suppressed in human cells. Nonetheless, a subset of physiologically important mRNAs (e.g. hypoxia-inducible factor 1α [HIF-1α], fibroblast growth factor 9 [FGF-9], and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eukaryotic translation initiation factor 4GI (eIF4GI) and of its homolog, death-associated protein 5 (DAP5), are elevated. By examining the 5′ UTRs of HIF-1α, FGF-9, and p53 mRNAs and using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, we demonstrate here that eIF4GI and DAP5 specifically bind to the 5′ UTRs of these cap-independently translated mRNAs. Surprisingly, we found that the eIF4E-binding domain of eIF4GI increases not only the binding affinity but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5′ UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we conclude that eIF4GI or DAP5 is critical for recruitment of a specific subset of mRNAs to the ribosome, providing mechanistic insight into their cap-independent translation.

Translational control by mTOR-independent routes: how eIF6 organizes metabolism

2016 ◽  
Vol 44 (6) ◽  
pp. 1667-1673 ◽  
Author(s):  
Annarita Miluzio ◽  
Sara Ricciardi ◽  
Nicola Manfrini ◽  
Roberta Alfieri ◽  
Stefania Oliveto ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Insulin Sensitivity ◽  
Fatty Acid Synthesis ◽  
Translational Control ◽  
Metabolic Diseases ◽  
Lipid Synthesis ◽  
Acid Synthesis ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Translational Machinery

Over the past few years, there has been a growing interest in the interconnection between translation and metabolism. Important oncogenic pathways, like those elicited by c-Myc transcription factor and mTOR kinase, couple the activation of the translational machinery with glycolysis and fatty acid synthesis. Eukaryotic initiation factor 6 (eIF6) is a factor necessary for 60S ribosome maturation. eIF6 acts also as a cytoplasmic translation initiation factor, downstream of growth factor stimulation. eIF6 is up-regulated in several tumor types. Data on mice models have demonstrated that eIF6 cytoplasmic activity is rate-limiting for Myc-induced lymphomagenesis. In spite of this, eIF6 is neither transcriptionally regulated by Myc, nor post-transcriptionally regulated by mTOR. eIF6 stimulates a glycolytic and fatty acid synthesis program necessary for tumor growth. eIF6 increases the translation of transcription factors necessary for lipogenesis, such as CEBP/β, ATF4 and CEBP/δ. Insulin stimulation leads to an increase in translation and fat synthesis blunted by eIF6 deficiency. Paradoxycally, long-term inhibition of eIF6 activity increases insulin sensitivity, suggesting that the translational activation observed upon insulin and growth factors stimulation acts as a feed-forward mechanism regulating lipid synthesis. The data on the role that eIF6 plays in cancer and in insulin sensitivity make it a tempting pharmacological target for cancers and metabolic diseases. We speculate that eIF6 inhibition will be particularly effective especially when mTOR sensitivity to rapamycin is abrogated by RAS mutations.

scholarly journals Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

1999 ◽  
Vol 19 (9) ◽  
pp. 5861-5871 ◽  
Author(s):  
Sheri Uma ◽  
Vanitha Thulasiraman ◽  
Robert L. Matts
Keyword(s):  
Heat Shock ◽  
Environmental Conditions ◽  
De Novo ◽  
Clofibric Acid ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Α Subunit ◽  
Eukaryotic Translation ◽  
Initiation Factor 2 ◽  
Reticulocyte Lysate

ABSTRACT The heme-regulated kinase of the α subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI.

scholarly journals A Six-Nucleotide Segment within the 3′ Untranslated Region of Hibiscus Chlorotic Ringspot Virus Plays an Essential Role in Translational Enhancement

2002 ◽  
Vol 76 (3) ◽  
pp. 1144-1153 ◽  
Author(s):  
Dora Chin-Yen Koh ◽  
D. X. Liu ◽  
Sek-Man Wong
Keyword(s):  
Gene Expression ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Viral Gene Expression ◽  
Plant Viruses ◽  
Ringspot Virus ◽  
Viral Gene ◽  
Entry Site ◽  
Viral Mrnas ◽  
Translational Machinery

ABSTRACT RNA plant viruses use various translational regulatory mechanisms to control their gene expression. Translational enhancement of viral mRNAs that leads to higher levels of protein synthesis from specific genes may be essential for the virus to successfully compete for cellular translational machinery. The control elements have yet to be analyzed for members of the genus Carmovirus, a small group of plant viruses with positive-sense RNA genomes. In this study, we examined the 3′ untranslated region (UTR) of hibiscus chlorotic ringspot virus (HCRSV) genomic RNA (gRNA) and subgenomic RNA (sgRNA) for its role in the translational regulation of viral gene expression. The results showed that the 3′ UTR of HCRSV significantly enhanced the translation of several open reading frames on gRNA and sgRNA and a viral gene in a bicistronic construct with an inserted internal ribosome entry site. Through deletion and mutagenesis studies of both the bicistronic construct and full-length gRNA, we demonstrated that a six-nucleotide sequence, GGGCAG, that is complementary to the 3′ region of the 18S rRNA and a minimal length of 180 nucleotides are required for the enhancement of translation induced by the 3′ UTR.

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