A complex IRES at the 5'-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate

Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.

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A complex IRES at the 5’-UTR of a viral mRNA assembles a functional 48S complex via an uAUG intermediate

10.1101/863761 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ritam Neupane ◽

Vera P. Pisareva ◽

Carlos F. Rodríguez ◽

Andrey V. Pisarev ◽

Israel S. Fernández

Keyword(s):

Translation Initiation ◽

Protein Production ◽

Rna Viruses ◽

Domain Architecture ◽

Translation Regulation ◽

Rna Sequences ◽

Initiation Factors ◽

Cellular Machinery ◽

Initiation Stage ◽

Viral Sequences

AbstractRNA viruses are pervasive entities in the biosphere with significant impact in human health and economically important livestock. As strict cellular parasites, RNA viruses abuse host resources, redirecting them towards viral replication needs. Taking control of the cellular apparatus for protein production is a requirement for virus progression and diverse strategies of cellular mimicry and/or ribosome hijacking evolved to ensure this control. Especially in complex eukaryotes, translation is a sophisticated process, with multiple mechanisms acting on ribosomes and mRNAs. The initiation stage of translation is specially regulated, involving multiple steps and the engagement of numerous initiation factors some of them of high complexity. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5’-UTR of a viral RNA assembles a functional translation initiation complex via an uAUG intermediate, redirecting the cellular machinery for protein production towards viral messengers. The IRES features a novel extended, multi-domain architecture, circling the 40S head, leveraging ribosomal sites not previously described to be exploited by any IRES. The structures and accompanying functional data, illustrate the importance of 5’-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs. Given the large number of new viruses metagenomic studies have uncovered, the quantity and diversity of mechanisms for translation hijacking encrypted in viral sequences may be seriously underestimated. Exploring this diversity could reveal novel avenues in the fight against these molecular pathogens.

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Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation

Journal of Biological Chemistry ◽

10.1074/jbc.m703356200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1340-1349 ◽

Cited By ~ 67

Author(s):

Mateen A. Khan ◽

Hiroshi Miyoshi ◽

Daniel R. Gallie ◽

Dixie J. Goss

Keyword(s):

Translation Initiation ◽

Wheat Germ ◽

Mrna Translation ◽

Kinetic Studies ◽

Dissociation Rate ◽

In Vitro Translation ◽

Translation Efficiency ◽

Viral Mrna ◽

Initiation Factors

Potyvirus genome linked protein, VPg, interacts with translation initiation factors eIF4E and eIFiso4E, but its role in protein synthesis has not been elucidated. We show that addition of VPg to wheat germ extract leads to enhancement of uncapped viral mRNA translation and inhibition of capped viral mRNA translation. This provides a significant competitive advantage to the uncapped viral mRNA. To understand the molecular basis of these effects, we have characterized the interaction of VPg with eIF4F, eIFiso4F, and a structured RNA derived from tobacco etch virus (TEV RNA). When VPg formed a complex with eIF4F, the affinity for TEV RNA increased more than 4-fold compared with eIF4F alone (19.4 and 79.0 nm, respectively). The binding affinity of eIF4F to TEV RNA correlates with translation efficiency. VPg enhanced eIFiso4F binding to TEV RNA 1.6-fold (178 nm compared with 108 nm). Kinetic studies of eIF4F and eIFiso4F with VPg show ∼2.6-fold faster association for eIFiso4F·VPg as compared with eIF4F·VPg. The dissociation rate was ∼2.9-fold slower for eIFiso4F than eIF4F with VPg. These data demonstrate that eIFiso4F can kinetically compete with eIF4F for VPg binding. The quantitative data presented here suggest a model where eIF4F·VPg interaction enhances cap-independent translation by increasing the affinity of eIF4F for TEV RNA. This is the first evidence of direct participation of VPg in translation initiation.

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Eukaryotic aspects of translation initiation brought into focus

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0186 ◽

2017 ◽

Vol 372 (1716) ◽

pp. 20160186 ◽

Cited By ~ 19

Author(s):

Christopher H. S. Aylett ◽

Nenad Ban

Keyword(s):

Translation Initiation ◽

Structural Information ◽

Start Codon ◽

Translation Regulation ◽

Initiation Factors ◽

Codon Recognition ◽

Initiation Of Translation ◽

Control Translation ◽

Additional Level ◽

Subunit Joining

In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits. This article is part of the themed issue ‘Perspectives on the ribosome’.

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Selective 40S footprinting reveals that scanning ribosomes remain cap-tethered in human cells

10.1101/806364 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jonathan Bohlen ◽

Kai Fenzl ◽

Günter Kramer ◽

Bernd Bukau ◽

Aurelio A. Teleman

Keyword(s):

Translation Initiation ◽

Human Cells ◽

Open Reading Frames ◽

Translation Regulation ◽

List Type ◽

Translation Efficiency ◽

Initiation Factors ◽

Upstream Open Reading Frames

SUMMARYTranslation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ∼12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.HIGHLIGHTSSelective 40S FPing visualizes regulation of translation initiation on mRNAs in vivoScanning ribosomes are cap-tethered in human cellsOnly one ribosome scans a 5’UTR at a time in human cellsRibosomes retain eIFs during early translation, allowing reinitiation after uORFs

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Near-Cognate Codons Contribute Complexity to Translation Regulation

mBio ◽

10.1128/mbio.01820-17 ◽

2017 ◽

Vol 8 (6) ◽

Author(s):

N. Louise Glass

Keyword(s):

Protein Synthesis ◽

Cell Fate ◽

Translation Initiation ◽

Cellular Response ◽

Open Reading Frames ◽

Translation Regulation ◽

Initiation Factors ◽

Translation Initiation Factors ◽

Upstream Open Reading Frames ◽

Response To Stress

ABSTRACT The interplay between translation initiation, modification of translation initiation factors, and selection of start sites on mRNA for protein synthesis can play a regulatory role in the cellular response to stress, development, and cell fate in eukaryotic species by shaping the proteome. As shown by Ivanov et al. (mBio 8:e00844-17, 2017, https://doi.org/10.1128/mBio.00844-17 !), in the filamentous fungus Neurospora crassa, both upstream open reading frames (uORFs) and near-cognate start codons negatively or positively regulate the translation of the transcription factor CPC1 and production of CPC1 isoforms, which mediate the cellular response to amino acid starvation. Dissecting the physiological roles that differentiate cellular choice of translation initiation is an important parameter to understanding mechanisms that determine cell fate via gene regulation and protein synthesis.

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IRES-mediated cap-independent translation, a path leading to hidden proteome

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz091 ◽

2019 ◽

Vol 11 (10) ◽

pp. 911-919 ◽

Cited By ~ 14

Author(s):

Yun Yang ◽

Zefeng Wang

Keyword(s):

Translation Initiation ◽

Protein Production ◽

Circular Rnas ◽

Alternative Mechanism ◽

Internal Ribosomal Entry Sites ◽

Cellular Mrnas ◽

Eukaryotic Mrnas ◽

Reporter Systems ◽

New Type ◽

Independent Translation

Abstract Most eukaryotic mRNAs are translated in a cap-dependent fashion; however, under stress conditions, the cap-independent translation driven by internal ribosomal entry sites (IRESs) can serve as an alternative mechanism for protein production. Many IRESs have been discovered from viral or cellular mRNAs to promote ribosome assembly and initiate translation by recruiting different trans-acting factors. Although the mechanisms of translation initiation driven by viral IRESs are relatively well understood, the existence of cellular IRESs is still under debate due to the limitations of translation reporter systems used to assay IRES activities. A recent screen identified > 1000 putative IRESs from viral and human mRNAs, expanding the scope and mechanism for cap-independent translation. Additionally, a large number of circular RNAs lacking free ends were identified in eukaryotic cells, many of which are found to be translated through IRESs. These findings suggest that IRESs may play a previously unappreciated role in driving translation of the new type of mRNA, implying a hidden proteome produced from cap-independent translation.

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Hepatitis C Virus Translation Regulation

International Journal of Molecular Sciences ◽

10.3390/ijms21072328 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2328 ◽

Cited By ~ 6

Author(s):

Michael Niepmann ◽

Gesche K. Gerresheim

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Translation Initiation ◽

Core Protein ◽

Mrna Translation ◽

Start Codon ◽

Translation Regulation ◽

Hcv Rna ◽

Entry Site ◽

Initiation Factors

Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5’-untranslated region (5′UTR) and part of the core protein coding sequence, and by the 3′UTR. The 5′UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional cis sequences in the viral genome, including the 3′UTR and the cis-acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNAiMet availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein–protein interactions, also connecting 5′- and 3′-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.

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Composition of Herpesvirus Ribonucleoprotein Complexes

Proceedings ◽

10.3390/proceedings2020050119 ◽

2020 ◽

Vol 50 (1) ◽

pp. 119

Author(s):

Eric S. Pringle ◽

Craig McCormick

Keyword(s):

Protein Synthesis ◽

Translation Initiation ◽

Viral Protein ◽

Lytic Replication ◽

Viral Mrna ◽

Viral Protein Synthesis ◽

Initiation Factors ◽

Ribonucleoprotein Complexes ◽

Virion Production ◽

Translation Initiation Factors

Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating messenger ribonucleotides (mRNA) that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and viral factors. These viral mRNA bear 5′-m7GTP caps and poly(A) tails that should permit the assembly of canonical host eIF4F cap-binding complexes to initiate protein synthesis. However, the precise mechanisms of translation initiation remain to be investigated for Kaposi’s sarcoma-associated herpesvirus (KSHV) and other herpesviruses. During KSHV lytic replication in lymphoid cells, the activation of caspases leads to the cleavage of eIF4G and depletion of eIF4F. Translating mRNPs depleted of eIF4F retain viral mRNA, suggesting that non-eIF4F translation initiation is sufficient to support viral protein synthesis. To identify proteins required to support viral protein synthesis, we isolated and characterized actively translating messenger ribonucleoprotein (mRNP) complexes by ultracentrifugation and sucrose-gradient fractionation followed by quantitative mass spectrometry. The abundance of host translation initiation factors available to initiate viral protein synthesis were comparable between cells undergoing KSHV lytic or latent replication. The translation initiation factors eIF4E2, NCBP1, eIF4G2, and eIF3d were detected in association with actively translating mRNP complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production. By contrast, the N6-methyladenosine methyltransferase METTL3 was required for optimal late gene expression and virion production, but was dispensable for genome replication. Furthermore, we detected several KSHV proteins in actively translating mRNP complexes that had not previously been shown to play roles in viral protein synthesis. We conclude that KSHV usurps distinct host translation initiation systems during latent and lytic phases of infection.

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