Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Thomas, Adri A. M.; Rijnbrand, René; Voorma, Harry O.

doi:10.1099/0022-1317-77-2-265

Cited by 7 publications

(4 citation statements)

References 36 publications

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“…Addition of intact eIF2 resulted in a slight stimulation in overall translation as expected (Fig. 10B) and a significant increase in translation (34%, p Ͻ 0.006) from the upstream AUG which was evidenced by the increase in the ratio of preCAT:CAT (lane f); also in good agreement with previous studies (23). In contrast, addition of caspase 3-treated eIF2 did not stimulate translation in general or specifically increase translation from the upstream AUG (lane e).…”

Section: Figsupporting

confidence: 92%

“…The selection of the initiation codon in mRNAs containing the FMDV 5Ј-UTR has been shown to be influenced by the activity of eIF2 and the amount of ternary complex eIF2⅐Met-tRNA⅐GTP (23,28) in a mechanism that may involve direct binding of eIF2 to the viral IRES RNA element (29). In previous studies, it was shown that the addition of eIF2 to in vitro translation lysates programmed with reporter mRNAs containing the FMDV 5Ј-UTR resulted in an increase in initiation from the upstream AUG (23).…”

Section: Figmentioning

confidence: 99%

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Identification of Caspase 3-mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2α in Apoptosis

Marissen¹,

Guo²,

Thomas³

et al. 2000

Journal of Biological Chemistry

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Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2␣, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2␣ by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2␣ and this could be inhibited by addition of Ac-DEVD-CHO in vitro.Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2␣ revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2⅐2B complex was used as substrate, only caspase 3 was capable of eIF2␣ cleavage, which was not affected by phosphorylation of the ␣ subunit. The eIF2⅐GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the Cterminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2␣ results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2␣ that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.Initiation of protein synthesis in mammalian cells is a highly regulated process that requires multiple translation initiation factors. The eukaryotic translation initiation factor 2 (eIF2) 1 plays a key role in the initiation of translation. eIF2 is a heterotrimeric protein consisting of three subunits ␣, ␤, and ␥, which forms a so-called ternary complex with GTP and the initiator Met-tRNA. The ternary complex interacts with the 40 S ribosomal subunit thereby forming a 43 S preinitiation complex that is capable of recognizing the proper start codon of the mRNA. It has been shown in yeast that eIF2 itself is involved in this process of start codon selection (1) indicating its importance in translation initiation. Upon joining of the 60 S ribosomal subunit to the 43 S preinitiation complex, the GTP moiety is hydrolyzed and an eIF2⅐GDP complex is released from the ribosome (2). Participation of the eIF2⅐GDP complex in a new round of translation requires the exchange of the GDP moiety for a new GTP molecule, a process carried out by the guanine nucleotide exchange factor, eIF2B. The global rate of protein synthesis in eukaryotes is mainly regulated by the specific phosphorylation of Ser-51 ...

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Section: Figsupporting

confidence: 92%

Section: Figmentioning

confidence: 99%

Identification of Caspase 3-mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2α in Apoptosis

Marissen¹,

Guo²,

Thomas³

et al. 2000

Journal of Biological Chemistry

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“…Shortly afterwards, the requirements for different eIFs were investigated, revealing that eIF2 was necessary for EMCV mRNA translation [7]. Since then, all experiments with picornavirus mRNAs have provided overwhelming evidence for requirement of eIF2 for the initiation of picornavirus protein synthesis in cell free systems and in culture cells transfected with these mRNAs [8], [9], [10]. The elegant experiments by Pestova et al .…”

Section: Introductionmentioning

confidence: 99%

Translation of Viral mRNA without Active eIF2: The Case of Picornaviruses

et al. 2011

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Previous work by several laboratories has established that translation of picornavirus RNA requires active eIF2α for translation in cell free systems or after transfection in culture cells. Strikingly, we have found that encephalomyocarditis virus protein synthesis at late infection times is resistant to inhibitors that induce the phosphorylation of eIF2α whereas translation of encephalomyocarditis virus early during infection is blocked upon inactivation of eIF2α by phosphorylation induced by arsenite. The presence of this compound during the first hour of infection leads to a delay in the appearance of late protein synthesis in encephalomyocarditis virus-infected cells. Depletion of eIF2α also provokes a delay in the kinetics of encephalomyocarditis virus protein synthesis, whereas at late times the levels of viral translation are similar in control or eIF2α-depleted HeLa cells. Immunofluorescence analysis reveals that eIF2α, contrary to eIF4GI, does not colocalize with ribosomes or with encephalomyocarditis virus 3D polymerase. Taken together, these findings support the novel idea that eIF2 is not involved in the translation of encephalomyocarditis virus RNA during late infection. Moreover, other picornaviruses such as foot-and-mouth disease virus, mengovirus and poliovirus do not require active eIF2α when maximal viral translation is taking place. Therefore, translation of picornavirus RNA may exhibit a dual mechanism as regards the participation of eIF2. This factor would be necessary to translate the input genomic RNA, but after viral RNA replication, the mechanism of viral RNA translation switches to one independent of eIF2.

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“…The T 7 TS-HRV/ EMCV/FMDV-CAT plasmids were constructed by inserting the 5 ′ UTR-CAT sequences as Eco RV-Bam HI fragments into a blunt-ended Hin dIII/ Bgl II-digested T 7 TS plasmid. The HRV fragment contained the 3 ′ 592 bp of the 5 ′ UTR (32), the EMCV 5 ′ UTR was a 3 ′ 607-bp fragment (22), and the FMDV 5 ′ UTR was a 530-bp fragment described earlier (39). The PV-CAT sequence (26) was inserted as a Hin dIII-Bam HI fragment into the Hin dIII/ Bgl II-digested T 7 TS, resulting in T 7 TS-PV-CAT.…”

Section: Construction Of the Plasmidsmentioning

confidence: 99%

Vector Design for Optimal Protein Expression

2001

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Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is essential for stability, as well as for translation efficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of the sequences [5'UTR, 3'UTR, and poly(A) signal] are suggested.

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Recognition of the initiation codon for protein synthesis in foot-and-mouth disease virus RNA

Cited by 7 publications

References 36 publications

Identification of Caspase 3-mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2α in Apoptosis

Identification of Caspase 3-mediated Cleavage and Functional Alteration of Eukaryotic Initiation Factor 2α in Apoptosis

Translation of Viral mRNA without Active eIF2: The Case of Picornaviruses

Vector Design for Optimal Protein Expression

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