Protection of Cap-dependent Protein Synthesisin Vivo and in Vitro with an eIF4G-1 Variant Highly Resistant to Cleavage by Coxsackievirus 2A Protease

Zhao, Xiaohong; Lamphear, Barry J.; Xiong, Dingding; Knowlton, Kirk U.; Rhoads, Robert E.

doi:10.1074/jbc.m212393200

Cited by 14 publications

(13 citation statements)

References 45 publications

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“…Expression and Purification of Recombinant Proteins-The expression and purification of two fragments of eIF4G-1 (NCBI accession number NP_886553) have been described previously; eIF4G(589 -1600) is aa residues 589 -1600 with an N-terminal tag consisting of thioredoxin, His 6 , and S-peptide (52), and eIF4G(1357-1600) is similar except with aa residues 1357-1600 (53). Recombinant human MNK1 and MNK2 were expressed from plasmids pET14-His 6 -Mnk1 and pET14-His 6 -Mnk2, respectively (54).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Korneeva

Song

Gram

et al. 2016

Journal of Biological Chemistry

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The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m 7 GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F.The rate of translational initiation in eukaryotic cells depends on both cis-acting features of individual mRNAs, such as the cap, poly(A) tract, and untranslated regions (UTRs), and trans-acting components, such as initiation factors, protein kinases, and microRNAs (1-3). The recruitment of capped mRNAs to 48S initiation complexes involves several discrete steps that include cap recognition by eukaryotic translation initiation factor 4E (eIF4E), 3 mRNA binding by eIF4G, ATP-dependent unwinding of 5Ј-terminal secondary structure by the helicase eIF4A in concert with eIF4B and eIF4H, recognition of the 3Ј-terminal poly(A) tract by poly(A)-binding protein (PABP), and binding of eIF4G to the 40S ribosomal subunit through eIF3. Both the primary and secondary structure of the 5Ј-UTR are capable of modulating translational efficiency (4, 5). mRNAs containing short 5Ј-UTRs with little secondary structure or terminal oligopyrimidine tracts are more efficiently translated under normal conditions with a limited level of eIF4E. Translational efficiency is diminished in mRNAs containing long, GϩC-rich, and highly structured 5Ј-UTRs because of the necessity for energy-dependent unwinding prior to start codon recognition (6). Translation of these mRNAs requires high levels of eIF4F, the complex of eIF4E, eIF4A, and eIF4G (3, 7). The availability of eIF4E to enter the eIF4F complex is regulated by the PI3K/Akt/mTOR signaling cascade; eIF4E is sequestered by binding to the 4E-BPs, but activation of the mTOR kinase causes phosphorylation of the 4E-BPs and release of eIF4E (8, 9). The activity of eIF4E can be also regulated by phosphorylation via the mitogen-activat...

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Section: Methodsmentioning

confidence: 99%

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Korneeva

Song

Gram

et al. 2016

Journal of Biological Chemistry

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“…Moreover, this assay confirmed intrinsically higher IRES stimulation and accelerated eIF4GI cleavage with CBV3 versus PV 2A pro (Fig. 6A and B -mediated eIF4GI degradation in IRES stimulation, we followed a strategy first reported by Zhao et al (56). First, we constructed expression vectors encoding eIF4GI isoforms via initiation at two of the five authentic initiating AUGs: b (41), and e (197) (numbers in paretheses correspond to the amino acid numbers of eIF4GI-a) (Fig.…”

Section: Ires Stimulation By Ev Infection Coincides With Proteolytic mentioning

confidence: 99%

“…Furthermore, we constructed eIF4G-b and eIF4GI-e variants resistant to 2A pro cleavage (eIF4G-bR and eIF4GI-eR) by altering the primary (aa 682 to 683) and secondary (aa 674 to 675) eIF4GI cleavage sites (Fig. 7A) (56). HeLa cells were transfected with plasmids expressing eIF4GI-b-eIF4GI-bR or eIF4GI-e-eIF4GI-eR or with pcDNA3.1 vector DNA.…”

Section: Ires Stimulation By Ev Infection Coincides With Proteolytic mentioning

confidence: 99%

“…Fragments encoding eIF4GI-b and -e isoforms (9,11) or N-and C-terminal cleavage products (31) were PCR amplified from full-length eIF4GI cDNA (pSPORT-eIF4GI, kindly provided by R. Lloyd) using primers 47 and 49 plus 48 and 49, or 47 and 50 plus 49 and 51, respectively, digested with HindIII and NotI, and inserted into pcDNA-myc. eIF4GI variants resistant to CBV3 2A pro cleavage (Gly6753Ala and Gly6833Glu) (56) were produced by PCR amplification of an ORF fragment with primers 52 and 53. The fragment was digested with SbfI and PflMI and used to replace a corresponding segment in eIF4GI-b or eIF4GI-e expression vectors, yielding eIF4GI-bR and eIF4GI-eR, respectively.…”

Section: Vol 80 2006 Viral Determinants Of Ires-dependent Translatimentioning

confidence: 99%

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Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by ViralcisandtransFactors

Dobrikova

Grisham

Kaiser

et al. 2006

J Virol

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Enteroviruses (EVs) overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap-independent manner at the viral internal ribosomal entry site (IRES). We have investigated the role of cis-and trans-acting viral factors in EV IRES translation in living cells. We observed that considerable portions of the viral genome, including the 5-proximal open reading frame and the 3 untranslated region, contribute to stimulation of IRESmediated translation. With the IRES in proper context, translation via internal initiation in uninfected cells is as efficient as at capped messages with short, unstructured 5 untranslated regions. IRES function is enhanced in cells infected with the EV coxsackievirus B3, but the related poliovirus has no significant stimulatory activity. This differential is due to the inherent properties of their 2A protease and is not coupled to 2A-mediated proteolytic degradation of the eukaryotic initiation factor 4G. Our results suggest that the efficiency of alternative translation initiation at EV IRESs depends on a properly configured template rather than on targeted alterations of the host cell translation machinery.Plus-strand RNA viruses are among the most common human pathogens, responsible for devastating pandemics in the past (poliomyelitis) and present (hepatitis C virus infection and dengue hemorrhagic fever). Upon entering susceptible host cells, positive-strand RNA viruses utilize their genome as a template for viral gene expression in competition with host cell mRNAs for the translation machinery. To accomplish this, many positive-strand RNA viruses alter the host cell protein synthesis apparatus and employ unorthodox translation strategies involving 5Ј untranslated region (5ЈUTR) and 3ЈUTR with uncommon structural features. This is exemplified by the Enterovirus genus of Picornaviridae.Conventional translation of eukaryotic mRNAs occurs upon formation of a ribonucleoprotein network that engages the 43S preinitiation complex (53). This network bridges the 5Ј terminus of eukaryotic mRNAs, universally modified by an m 7 Gppp cap, and a poly(A) tail. Components of the eukaryotic initiation factor 4F (eIF4F) complex mediate template closure: the cap-binding eIF4E binds to eIF4G, which interacts with the poly(A)-binding protein (PABP) (16,43,50). Since it also interacts with the RNA helicase eIF4A and via eIF3 with the 40S ribosomal subunit, eIF4G assumes a central scaffolding function for preinitiation complex assembly (20).Despite their extreme genetic austerity, enteroviruses (EVs) devote a considerable portion of their genomes to elaborate, highly structured 5Ј-and 3ЈUTRs and encode a poly(A) tail of ϳ50 nucleotides (nt) in length. The viral 5ЈUTR contains an internal ribosomal entry site (IRES) mediating 5Ј-end, capindependent translation initiation of the uncapped genome (26, 40). The onset of viral gene expression coincides with severe disruptions of the intracellular milieu, incl...

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“…The enzymes were expressed (Wang et al 2002) and purified (Zhao et al 2003) as previously described. Capped 32 P-labled oligonucleotides were subjected to digestion with either wild-type or catalytically inactive GST-hDcp2 at 37°C…”

Section: Decapping Assaysmentioning

confidence: 99%

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH₃, Se, and NH

Su¹,

Slepenkov²,

Grudzien-Nogalska³

et al. 2011

RNA

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Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an a-b CH 2 group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the b-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the b-phosphate with BH 3 or Se, or substituted at either the a-b or b-g O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m 2 7,29-O Gpp S pG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m 7 Gpp BH3 pm 7 G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t 1/2 ffi 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.

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Protection of Cap-dependent Protein Synthesisin Vivo and in Vitro with an eIF4G-1 Variant Highly Resistant to Cleavage by Coxsackievirus 2A Protease

Cited by 14 publications

References 45 publications

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by ViralcisandtransFactors

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH₃, Se, and NH

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Protection of Cap-dependent Protein Synthesisin Vivo and in Vitro with an eIF4G-1 Variant Highly Resistant to Cleavage by Coxsackievirus 2A Protease

Cited by 14 publications

References 45 publications

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5′-Terminal Cap and Hairpin

Competitive Translation Efficiency at the Picornavirus Type 1 Internal Ribosome Entry Site Facilitated by ViralcisandtransFactors

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH3, Se, and NH

Contact Info

Product

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About

Translation, stability, and resistance to decapping of mRNAs containing caps substituted in the triphosphate chain with BH₃, Se, and NH