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

2019 ◽  
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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Ritam Neupane ◽  
Vera P Pisareva ◽  
Carlos F Rodriguez ◽  
Andrey V Pisarev ◽  
Israel S Fernández
Keyword(s):  
Translation Initiation ◽  
Functional Data ◽  
Protein Production ◽  
Domain Architecture ◽  
Translation Regulation ◽  
Viral Mrna ◽  
Rna Sequences ◽  
Internal Ribosomal Entry Sites ◽  
Initiation Factors ◽  
Initiation Stage

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.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

scholarly journals Hijacking the translation apparatus by RNA viruses

2002 ◽  
Vol 158 (3) ◽  
pp. 395-399 ◽  
Author(s):  
Martin Bushell ◽  
Peter Sarnow
Keyword(s):  
Host Cell ◽  
Translation Initiation ◽  
Rna Viruses ◽  
Viral Mrnas ◽  
Viral Genomes ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Translation Apparatus ◽  
Cellular Mrnas ◽  
Successful Amplification

As invading viruses do not harbor functional ribosomes in their virions, successful amplification of the viral genomes requires that viral mRNAs compete with cellular mRNAs for the host cell translation apparatus. Several RNA viruses have evolved remarkable strategies to recruit the host translation initiation factors required for the first steps in translation initiation by host cell mRNAs. This review describes the ways that three families of RNA viruses effectively usurp limiting translation initiation factors from the host.

scholarly journals Eukaryotic aspects of translation initiation brought into focus

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160186 ◽  
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’.

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

2019 ◽  
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

scholarly journals Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

2020 ◽  
Author(s):  
Christopher P. Lapointe ◽  
Rosslyn Grosely ◽  
Alex G. Johnson ◽  
Jinfan Wang ◽  
Israel S. Fernández ◽  
...  
Keyword(s):  
Single Molecule ◽  
Causative Agent ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Start Codon ◽  
Eukaryotic Translation ◽  
Initiation Factors ◽  
Cellular Machinery ◽  
Eukaryotic Translation Initiation Factors ◽  
Molecular Framework

ABSTRACTSARS-CoV-2 recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. Using extract-based and reconstitution experiments, we demonstrate that NSP1 inhibits translation initiation on model human and SARS-CoV-2 mRNAs. NSP1 also specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we demonstrate that eukaryotic translation initiation factors (eIFs) modulate the interaction: NSP1 rapidly and stably associates with most ribosomal pre-initiation complexes in the absence of mRNA, with particular enhancement and inhibition by eIF1 and eIF3j, respectively. Using model mRNAs and an inter-ribosomal-subunit FRET signal, we elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 associates with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.SIGNIFICANCE STATEMENTSARS-CoV-2 is the causative agent of the COVID-19 pandemic. A molecular framework for how SARS-CoV-2 manipulates host cellular machinery to facilitate infection is needed. Here, we integrate biochemical and single-molecule strategies to reveal molecular insight into how NSP1 from SARS-CoV-2 inhibits translation initiation. NSP1 directly binds to the small (40S) subunit of the human ribosome, which is modulated by human initiation factors. Further, NSP1 and mRNA compete with each other to bind the ribosome. Our findings suggest that the presence of NSP1 on the small ribosomal subunit prevents proper accommodation of the mRNA. How this competition disrupts the many steps of translation initiation is an important target for future studies.

scholarly journals Near-Cognate Codons Contribute Complexity to Translation Regulation

mBio ◽  
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.

scholarly journals Hepatitis C Virus Translation Regulation

2020 ◽  
Vol 21 (7) ◽  
pp. 2328 ◽  
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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