scholarly journals Structural characterization of ribosome recruitment and translocation by type IV IRES

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jason Murray ◽  
Christos G Savva ◽  
Byung-Sik Shin ◽  
Thomas E Dever ◽  
V Ramakrishnan ◽  
...  
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Post Translational Modification ◽  
Entry Site ◽  
Small Ribosomal Subunit ◽  
Initiation Factors ◽  
Type Iv ◽  
Peptidyl Transfer ◽  
Initiation Of Translation

Viral mRNA sequences with a type IV IRES are able to initiate translation without any host initiation factors. Initial recruitment of the small ribosomal subunit as well as two translocation steps before the first peptidyl transfer are essential for the initiation of translation by these mRNAs. Using electron cryomicroscopy (cryo-EM) we have structurally characterized at high resolution how the Cricket Paralysis Virus Internal Ribosomal Entry Site (CrPV-IRES) binds the small ribosomal subunit (40S) and the translocation intermediate stabilized by elongation factor 2 (eEF2). The CrPV-IRES restricts the otherwise flexible 40S head to a conformation compatible with binding the large ribosomal subunit (60S). Once the 60S is recruited, the binary CrPV-IRES/80S complex oscillates between canonical and rotated states (<xref ref-type="bibr" rid="bib19">Fernández et al., 2014</xref>; <xref ref-type="bibr" rid="bib34">Koh et al., 2014</xref>), as seen for pre-translocation complexes with tRNAs. Elongation factor eEF2 with a GTP analog stabilizes the ribosome-IRES complex in a rotated state with an extra ~3 degrees of rotation. Key residues in domain IV of eEF2 interact with pseudoknot I (PKI) of the CrPV-IRES stabilizing it in a conformation reminiscent of a hybrid tRNA state. The structure explains how diphthamide, a eukaryotic and archaeal specific post-translational modification of a histidine residue of eEF2, is involved in translocation.

scholarly journals Dissemination of Internal Ribosomal Entry Sites (IRES) Between Viruses by Horizontal Gene Transfer

Viruses ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 612
Author(s):  
Yani Arhab ◽  
Alexander G. Bulakhov ◽  
Tatyana V. Pestova ◽  
Christopher U.T. Hellen
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Viral Evolution ◽  
Internal Ribosomal Entry Site ◽  
Type I ◽  
Cell Type Specificity ◽  
Entry Site ◽  
Type Iv ◽  
Initiation Of Translation ◽  
Ribosomal Entry

Members of Picornaviridae and of the Hepacivirus, Pegivirus and Pestivirus genera of Flaviviridae all contain an internal ribosomal entry site (IRES) in the 5′-untranslated region (5′UTR) of their genomes. Each class of IRES has a conserved structure and promotes 5′-end-independent initiation of translation by a different mechanism. Picornavirus 5′UTRs, including the IRES, evolve independently of other parts of the genome and can move between genomes, most commonly by intratypic recombination. We review accumulating evidence that IRESs are genetic entities that can also move between members of different genera and even between families. Type IV IRESs, first identified in the Hepacivirus genus, have subsequently been identified in over 25 genera of Picornaviridae, juxtaposed against diverse coding sequences. In several genera, members have either type IV IRES or an IRES of type I, II or III. Similarly, in the genus Pegivirus, members contain either a type IV IRES or an unrelated type; both classes of IRES also occur in members of the genus Hepacivirus. IRESs utilize different mechanisms, have different factor requirements and contain determinants of viral growth, pathogenesis and cell type specificity. Their dissemination between viruses by horizontal gene transfer has unexpectedly emerged as an important facet of viral evolution.

scholarly journals Translation Initiation Factor eIF4B Interacts with a Picornavirus Internal Ribosome Entry Site in both 48S and 80S Initiation Complexes Independently of Initiator AUG Location

1999 ◽  
Vol 73 (9) ◽  
pp. 7505-7514 ◽  
Author(s):  
Kerstin Ochs ◽  
RenéC. Rust ◽  
Michael Niepmann
Keyword(s):  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Translation Initiation Factor ◽  
Entry Site ◽  
Small Ribosomal Subunit ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Mouth Disease Virus ◽  
Energy Dependent

ABSTRACT Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation.

scholarly journals Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry.

1996 ◽  
Vol 16 (12) ◽  
pp. 6859-6869 ◽  
Author(s):  
T V Pestova ◽  
C U Hellen ◽  
I N Shatsky
Keyword(s):  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Entry Site ◽  
Independent Manner ◽  
Initiation Factors ◽  
Virus Rna ◽  
Initiation Of Translation ◽  
Initiation Factor 2 ◽  
Ribosomal Entry

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5' untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap- and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.

scholarly journals Unr Is Required In Vivo for Efficient Initiation of Translation from the Internal Ribosome Entry Sites of both Rhinovirus and Poliovirus

2003 ◽  
Vol 77 (6) ◽  
pp. 3353-3359 ◽  
Author(s):  
Oréda Boussadia ◽  
Michael Niepmann ◽  
Laurent Créancier ◽  
Anne-Catherine Prats ◽  
François Dautry ◽  
...  
Keyword(s):  
Rna Binding ◽  
Es Cells ◽  
Internal Ribosomal Entry Site ◽  
Embryonic Stem ◽  
Encephalomyocarditis Virus ◽  
Entry Site ◽  
Initiation Of Translation ◽  
Mouth Disease Virus

ABSTRACT Translation of picornavirus RNAs is mediated by internal ribosomal entry site (IRES) elements and requires both standard eukaryotic translation initiation factors (eIFs) and IRES-specific cellular trans-acting factors (ITAFs). Unr, a cytoplasmic RNA-binding protein that contains five cold-shock domains and is encoded by the gene upstream of N-ras, stimulates translation directed by the human rhinovirus (HRV) IRES in vitro. To examine the role of Unr in translation of picornavirus RNAs in vivo, we derived murine embryonic stem (ES) cells in which either one (−/+) or both (−/−) copies of the unr gene were disrupted by homologous recombination. The activity of picornaviral IRES elements was analyzed in unr +/+, unr +/−, and unr −/− cell lines. Translation directed by the HRV IRES was severely impaired in unr −/− cells, as was that directed by the poliovirus IRES, revealing a requirement for Unr not previously observed in vitro. Transient expression of Unr in unr −/− cells efficiently restored the HRV and poliovirus IRES activities. In contrast, the IRES elements of encephalomyocarditis virus and foot-and-mouth-disease virus are not Unr dependent. Thus, Unr is a specific regulator of HRV and poliovirus translation in vivo and may represent a cell-specific determinant limiting replication of these viruses.

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 262-272 ◽  
Author(s):  
Rastislav Horos ◽  
Hanna IJspeert ◽  
Dagmar Pospisilova ◽  
Regine Sendtner ◽  
Charlotte Andrieu-Soler ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Binding ◽  
Fetal Liver ◽  
Internal Ribosomal Entry Site ◽  
Ribosomal Subunit ◽  
Translation Efficiency ◽  
Entry Site ◽  
Diamond Blackfan Anemia ◽  
Ribosomal Entry

Abstract Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

scholarly journals Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

2003 ◽  
Vol 23 (2) ◽  
pp. 687-698 ◽  
Author(s):  
Victoria G. Kolupaeva ◽  
Ivan B. Lomakin ◽  
Tatyana V. Pestova ◽  
Christopher U. T. Hellen
Keyword(s):  
Conformational Changes ◽  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Initiation Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Central Domain ◽  
C Terminus ◽  
Initiation Of Translation

ABSTRACT Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3′ border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

scholarly journals Conformational Changes of the Small Ribosomal Subunit During Elongation Factor G-dependent tRNA–mRNA Translocation

2004 ◽  
Vol 343 (5) ◽  
pp. 1183-1194 ◽  
Author(s):  
Frank Peske ◽  
Andreas Savelsbergh ◽  
Vladimir I. Katunin ◽  
Marina V. Rodnina ◽  
Wolfgang Wintermeyer
Keyword(s):  
Conformational Changes ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Elongation Factor G ◽  
Small Ribosomal Subunit ◽  
Factor G

scholarly journals Functional and Structural Similarities between the Internal Ribosome Entry Sites of Hepatitis C Virus and Porcine Teschovirus, a Picornavirus

2004 ◽  
Vol 78 (9) ◽  
pp. 4487-4497 ◽  
Author(s):  
Andrey V. Pisarev ◽  
Louisa S. Chard ◽  
Yoshihiro Kaku ◽  
Helen L. Johns ◽  
Ivan N. Shatsky ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Ribosomal Subunit ◽  
Binary Complex ◽  
Entry Site ◽  
High Sequence Identity ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation ◽  
Porcine Teschovirus ◽  
Hcv Ires

ABSTRACT Initiation of protein synthesis on picornavirus RNA requires an internal ribosome entry site (IRES). Typically, picornavirus IRES elements contain about 450 nucleotides (nt) and use most of the cellular translation initiation factors. However, it is now shown that just 280 nt of the porcine teschovirus type 1 Talfan (PTV-1) 5′ untranslated region direct the efficient internal initiation of translation in vitro and within cells. In toeprinting assays, assembly of 48S preinitiation complexes from purified components on the PTV-1 IRES was achieved with just 40S ribosomal subunits plus eIF2 and Met-tRNAi Met. Indeed, a binary complex between 40S subunits and the PTV-1 IRES is formed. Thus, the PTV-1 IRES has properties that are entirely different from other picornavirus IRES elements but highly reminiscent of the hepatitis C virus (HCV) IRES. Comparison between the PTV-1 IRES and HCV IRES elements revealed islands of high sequence identity that occur in regions critical for the interactions of the HCV IRES with the 40S ribosomal subunit and eIF3. Thus, there is significant functional and structural similarity between the IRES elements from the picornavirus PTV-1 and HCV, a flavivirus.

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