The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure.

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.

[1]  C. Hellen,et al.  Physical Association of Eukaryotic Initiation Factor 4G (eIF4G) with eIF4A Strongly Enhances Binding of eIF4G to the Internal Ribosomal Entry Site of Encephalomyocarditis Virus and Is Required for Internal Initiation of Translation , 2000, Molecular and Cellular Biology.

[2]  J. Hershey,et al.  2 The Pathway and Mechanism of Initiation of Protein Synthesis , 2000 .

[3]  A. Gingras,et al.  6 Regulation of Ribosomal Recruitment in Eukaryotes , 2000 .

[4]  D. Mckay,et al.  Crystallographic structure of the amino terminal domain of yeast initiation factor 4A, a representative DEAD-box RNA helicase. , 1999, RNA.

[5]  D. Herschlag,et al.  Effects of oligonucleotide length and atomic composition on stimulation of the ATPase activity of translation initiation factor elF4A. , 1999, RNA.

[6]  U. Baumann,et al.  Crystal structure of the ATPase domain of translation initiation factor 4A from Saccharomyces cerevisiae--the prototype of the DEAD box protein family. , 1999, Structure.

[7]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[8]  J. de la Cruz,et al.  Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families. , 1999, Trends in biochemical sciences.

[9]  W. Merrick,et al.  Biochemical and Kinetic Characterization of the RNA Helicase Activity of Eukaryotic Initiation Factor 4A* , 1999, The Journal of Biological Chemistry.

[10]  R. Jackson,et al.  unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA. , 1999, Genes & development.

[11]  A. Gingras,et al.  Human eukaryotic translation initiation factor 4G (eIF4G) recruits Mnk1 to phosphorylate eIF4E , 1999, The EMBO journal.

[12]  A. Gingras,et al.  eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. , 1999, Annual review of biochemistry.

[13]  N. Sonenberg,et al.  A newly identified N‐terminal amino acid sequence of human eIF4G binds poly(A)‐binding protein and functions in poly(A)‐dependent translation , 1998, The EMBO journal.

[14]  C. Hellen,et al.  Translation Eukaryotic Initiation Factor 4G Recognizes a Specific Structural Element within the Internal Ribosome Entry Site of Encephalomyocarditis Virus RNA* , 1998, The Journal of Biological Chemistry.

[15]  W. Merrick,et al.  Purification and Characterization of a New Eukaryotic Protein Translation Factor , 1998, The Journal of Biological Chemistry.

[16]  D. Herschlag,et al.  The DEAD box protein eIF4A. 2. A cycle of nucleotide and RNA-dependent conformational changes. , 1998, Biochemistry.

[17]  N. Sonenberg,et al.  A Novel Functional Human Eukaryotic Translation Initiation Factor 4G , 1998, Molecular and Cellular Biology.

[18]  U. Stahl,et al.  The protein family of RNA helicases. , 1998, Critical reviews in biochemistry and molecular biology.

[19]  R. Jackson,et al.  A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. , 1998, Genes & development.

[20]  A. Prats,et al.  Alternative Translation of the Proto-oncogene c-mycby an Internal Ribosome Entry Site* , 1997, The Journal of Biological Chemistry.

[21]  N. Sonenberg,et al.  Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A , 1997, Molecular and cellular biology.

[22]  N. Sonenberg,et al.  eIF4G Dramatically Enhances the Binding of eIF4E to the mRNA 5′-Cap Structure* , 1997, The Journal of Biological Chemistry.

[23]  M. Hentze,et al.  Starting at the Beginning, Middle, and End: Translation Initiation in Eukaryotes , 1997, Cell.

[24]  N. Sonenberg,et al.  Assays for eukaryotic translation factors that bind mRNA. , 1997, Methods.

[25]  A. Sachs,et al.  Association of the yeast poly(A) tail binding protein with translation initiation factor eIF‐4G. , 1996, The EMBO journal.

[26]  C. Hellen,et al.  Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes , 1996, Molecular and cellular biology.

[27]  G. Dreyfuss,et al.  General RNA binding proteins render translation cap dependent. , 1996, The EMBO journal.

[28]  N. Sonenberg,et al.  RNA-protein interactions in regulation of picornavirus RNA translation. , 1996, Microbiological reviews.

[29]  A. Prats,et al.  Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region. , 1996, Cancer research.

[30]  R. Jackson,et al.  Internal initiation of translation in eukaryotes: the picornavirus paradigm and beyond. , 1995, RNA.

[31]  N. Sonenberg,et al.  La autoantigen alleviates translational repression by the 5' leader sequence of the human immunodeficiency virus type 1 mRNA , 1994, Journal of virology.

[32]  A. Gingras,et al.  Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function , 1994, Nature.

[33]  E. Wimmer,et al.  Effect of mutations downstream of the internal ribosome entry site on initiation of poliovirus protein synthesis , 1994, Journal of virology.

[34]  N. Sonenberg,et al.  Dominant negative mutants of mammalian translation initiation factor eIF‐4A define a critical role for eIF‐4F in cap‐dependent and cap‐independent initiation of translation. , 1994, The EMBO journal.

[35]  N. Sonenberg,et al.  The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis , 1993, Molecular and cellular biology.

[36]  V. Agol,et al.  La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate , 1993, Journal of virology.

[37]  Eugene V. Koonin,et al.  Helicases: amino acid sequence comparisons and structure-function relationships , 1993 .

[38]  N. Sonenberg,et al.  The p46 subunit of eukaryotic initiation factor (eIF)-4F exchanges with eIF-4A. , 1993, The Journal of biological chemistry.

[39]  N. Sonenberg,et al.  Remarks on the mechanism of ribosome binding to eukaryotic mRNAs. , 1993, Gene expression.

[40]  N. Sonenberg,et al.  Helicases and RNA unwinding in translation , 1993 .

[41]  N. Sonenberg,et al.  mRNAs containing extensive secondary structure in their 5′ non‐coding region translate efficiently in cells overexpressing initiation factor eIF‐4E. , 1992, The EMBO journal.

[42]  H. van Steeg,et al.  Interaction of initiation factors with the cap structure of chimaeric mRNA containing the 5'-untranslated regions of Semliki Forest virus RNA is related to translational efficiency. , 1992, European journal of biochemistry.

[43]  P Linder,et al.  ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[44]  N. Sonenberg,et al.  Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF‐4A. , 1992, The EMBO journal.

[45]  A. Nomoto,et al.  Internal ribosome entry site within hepatitis C virus RNA , 1992, Journal of virology.

[46]  P Linder,et al.  D‐E‐A‐D protein family of putative RNA helicases , 1992, Molecular microbiology.

[47]  N. Sonenberg,et al.  RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B , 1991, Molecular and cellular biology.

[48]  K. Hilse,et al.  A 60-kDa protein from rabbit reticulocytes specifically recognizes the capped 5' end of beta-globin mRNA. , 1991, European journal of biochemistry.

[49]  R. Jackson.  The ATP requirement for initiation of eukaryotic translation varies according to the mRNA species. , 1991, European journal of biochemistry.

[50]  The absence of a m7G cap on beta-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation. , 1990, The Journal of biological chemistry.

[51]  N. Sonenberg,et al.  Translation initiation factor-dependent extracts from Saccharomyces cerevisiae. , 1990, Biochimica et biophysica acta.

[52]  N. Sonenberg,et al.  Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F , 1990, Molecular and cellular biology.

[53]  H. Trachsel,et al.  The 5'-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts. , 1990, Gene.

[54]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[55]  V. Blinov,et al.  Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. , 1989, Nucleic acids research.

[56]  P. Blackshear,et al.  Rat ornithine decarboxylase gene. Nucleotide sequence, potential regulatory elements, and comparison to the mouse gene. , 1989, The Journal of biological chemistry.

[57]  N. Sonenberg,et al.  Activation of double-stranded RNA-dependent kinase (dsl) by the TAR region of HIV-1 mRNA: A novel translational control mechanism , 1989, Cell.

[58]  N. Sonenberg,et al.  Mutational analysis of the 5′ non‐coding region of human immunodeficiency virus type 1: effects of secondary structure on translation. , 1988, The EMBO journal.

[59]  S. Jobling,et al.  Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G. , 1988, The Journal of biological chemistry.

[60]  N. Sonenberg,et al.  Identification of the 80-kDa protein that crosslinks to the cap structure of eukaryotic mRNAs as initiation factor eIF-4B. , 1988, Archives of biochemistry and biophysics.

[61]  F Sherman,et al.  mRNA structures influencing translation in the yeast Saccharomyces cerevisiae , 1988, Molecular and cellular biology.

[62]  N. Sonenberg,et al.  Photochemical cross-linking of cap binding proteins to eucaryotic mRNAs: effect of mRNA 5' secondary structure , 1985, Molecular and cellular biology.

[63]  R. Abramson,et al.  ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors. , 1985, The Journal of biological chemistry.

[64]  N. Sonenberg,et al.  Insertion mutagenesis to increase secondary structure within the 5′ noncoding region of a eukaryotic mRNA reduces translational efficiency , 1985, Cell.

[65]  V. Agol,et al.  Differences between translation products of tick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cells and rabbit reticulocytes: involvement of membranes in the processing of nascent precursors of flavivirus structural proteins. , 1984, Virology.

[66]  A. Rich,et al.  5'-Conformation of capped alfalfa mosaic virus ribonucleic acid 4 may reflect its independence of the cap structure or of cap-binding protein for efficient translation. , 1983, Biochemistry.

[67]  K. A. Lee,et al.  Involvement of eukaryotic initiation factor 4A in the cap recognition process. , 1983, The Journal of biological chemistry.

[68]  M. Morgan,et al.  New initiation factor activity required for globin mRNA translation. , 1983, The Journal of biological chemistry.

[69]  N Sonenberg,et al.  mRNA secondary structure as a determinant in cap recognition and initiation complex formation. ATP-Mg2+ independent cross-linking of cap binding proteins to m7I-capped inosine-substituted reovirus mRNA. , 1983, The Journal of biological chemistry.

[70]  K. A. Lee,et al.  Inactivation of cap-binding proteins accompanies the shut-off of host protein synthesis by poliovirus. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[71]  N. Sonenberg,et al.  ATP/Mg++-dependent cross-linking of cap binding proteins to the 5' end of eukaryotic mRNA. , 1981, Nucleic acids research.

[72]  N. Sonenberg,et al.  A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5'-terminal cap in mRNA. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[73]  V. Agol,et al.  Complete translation of encephalomyocarditis virus RNA and faithful cleavage of virus‐specific proteins in a cell‐free system from Krebs‐2 cells , 1978, FEBS letters.

[74]  B. Erni,et al.  Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors. , 1977, Journal of molecular biology.

[75]  J. Lelong,et al.  Functional identity of a mouse ascites and a rabbit reticulocyte initiation factor required for natural mRNA translation , 1977, Nature.

[76]  R. Benne,et al.  Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. , 1976, The Journal of biological chemistry.

[77]  A. Smith,et al.  Specificity in initiation of protein synthesis in a fractionated mammalian cell-free system. , 1973, Nature: New biology.