Point mutations modify the response of poliovirus RNA to a translation initiation factor: A comparison of neurovirulent and attenuated strains

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.

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mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor

Journal of Virology ◽

10.1128/jvi.75.21.10272-10280.2001 ◽

2001 ◽

Vol 75 (21) ◽

pp. 10272-10280 ◽

Cited By ~ 79

Author(s):

Pinghui Feng ◽

David N. Everly ◽

G. Sullivan Read

Keyword(s):

Translation Initiation ◽

Mammalian Cells ◽

Mrna Decay ◽

Point Mutations ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Viral Mrnas ◽

Cellular Translation ◽

Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

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The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.4990 ◽

1995 ◽

Vol 15 (9) ◽

pp. 4990-4997 ◽

Cited By ~ 443

Author(s):

S Mader ◽

H Lee ◽

A Pause ◽

N Sonenberg

Keyword(s):

Growth Factors ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Interactions ◽

Translation Initiation ◽

Binding Proteins ◽

Point Mutations ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Regulatory Pathways

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of cap-dependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4 gamma, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.

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Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation

Molecular and Cellular Biology ◽

10.1128/mcb.25.13.5480-5491.2005 ◽

2005 ◽

Vol 25 (13) ◽

pp. 5480-5491 ◽

Cited By ~ 36

Author(s):

Chingakham Ranjit Singh ◽

Cynthia Curtis ◽

Yasufumi Yamamoto ◽

Nathan S. Hall ◽

Dustin S. Kruse ◽

...

Keyword(s):

Amino Acid ◽

Translation Initiation ◽

Translational Control ◽

Point Mutations ◽

Mrna Translation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Preinitiation Complex ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation

ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAi Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.

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The translation initiation factor eIF-4B contains an RNA-binding region that is distinct and independent from its ribonucleoprotein consensus sequence

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2307-2316.1994 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2307-2316

Author(s):

N Méthot ◽

A Pause ◽

J W Hershey ◽

N Sonenberg

Keyword(s):

Translation Initiation ◽

Rna Binding ◽

Consensus Sequence ◽

Point Mutations ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Rna Recognition Motif ◽

Helicase Activity ◽

Binding Region ◽

Stimulation Of

eIF-4B is a eukaryotic translation initiation factor that is required for the binding of ribosomes to mRNAs and the stimulation of the helicase activity of eIF-4A. It is an RNA-binding protein that contains a ribonucleoprotein consensus sequence (RNP-CS)/RNA recognition motif (RRM). We examined the effects of deletions and point mutations on the ability of eIF-4B to bind a random RNA, to cooperate with eIF-4A in RNA binding, and to enhance the helicase activity of eIF-4A. We report here that the RNP-CS/RRM alone is not sufficient for eIF-4B binding to RNA and that an RNA-binding region, located between amino acids 367 and 423, is the major contributor to RNA binding. Deletions which remove this region abolish the ability of eIF-4B to cooperate with eIF-4A in RNA binding and the ability to stimulate the helicase activity of eIF-4A. Point mutations in the RNP-CS/RRM had no effect on the ability of eIF-4B to cooperate with eIF-4A in RNA binding but significantly reduced the stimulation of eIF-4A helicase activity. Our results indicate that the carboxy-terminal RNA-binding region of eIF-4B is essential for eIF-4B function and is distinct from the RNP-CS/RRM.

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