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.
Svitkin
Y V
YV
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada.
Pause
A
A
Haghighat
A
A
Pyronnet
S
S
Witherell
G
G
Belsham
G J
GJ
Sonenberg
N
N
D020121:5' Untranslated Regions / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D039601:Eukaryotic Initiation Factor-4A; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D039502:Nuclear Cap-Binding Protein Complex; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010849:Picornaviridae / Q000235:genetics; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps; D020365:RNA Helicases / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D020121:5' Untranslated Regions; C100383:CBC2 protein, S cerevisiae; D039502:Nuclear Cap-Binding Protein Complex; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012315:RNA Caps; D012333:RNA, Messenger; D012367:RNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases