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RNA. 2005 Mar;11(3):261-74. Epub 2005 Jan 20.

Mutational analysis of the DEAD-box RNA helicase eIF4AII characterizes its interaction with transformation suppressor Pdcd4 and eIF4GI.

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1
Laboratory of Cancer Prevention, Bldg. 576, Rm. 101, National Cancer Institute, Frederick, MD 21702, USA.

Abstract

Eukaryotic initiation factor (eIF) 4A unwinds secondary and tertiary structures in the 5'-untranslated region of mRNA, permitting translation initiation. Programmed cell death 4 (Pdcd4) is a novel transformation suppressor and eIF4A-binding partner that inhibits eIF4A helicase activity and translation. To elucidate the regions of eIF4A that are functionally significant in binding to Pdcd4, we generated point mutations of eIF4A. Two-hybrid analysis revealed that five eIF4A mutants completely lost binding to Pdcd4 while four eIF4A mutants retained wild-type levels of binding. The residues that, when mutated, inactivated Pdcd4 binding specified ATP binding, ATP hydrolysis, or RNA binding. With the exception of the Q-motif mutant eIF4AP56L, the eIF4A mutants inactivated for Pdcd4 binding were inactivated for binding to eIF4G (GM, GC, or both) and for enhancing translation. Several eIF4A mutants showing wild-type level binding to Pdcd4 were also inactivated for binding to eIF4G and for enhancing translation. Thus, significant dissociation of eIF4A's Pdcd4- and eIF4G-binding regions appears to occur. Because three of the four eIF4A mutants that retained Pdcd4 binding also suppressed translation activity in a dominant-negative manner, the structure that defines the Pdcd4-binding domain of eIF4A may be necessary but is insufficient for translation. A structural homology model of eIF4A shows regions important for binding to Pdcd4 and/or eIF4G lying on the perimeters of the hinge area of eIF4A. A competition experiment revealed that Pdcd4 competes with C-terminal eIF4G for binding to eIF4A. In summary, the Pdcd4-binding domains on eIF4A impact both binding to eIF4G and translation initiation in cells.

PMID:
15661843
PMCID:
PMC1370716
DOI:
10.1261/rna.7191905
[Indexed for MEDLINE]
Free PMC Article
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