Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster.
We have identified the initiation factor 4A (eIF4A) in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A.
Hernández
Greco
G
Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid and C.S.I.C., Madrid, Spain.
Lalioti
Vassiliki
V
Vandekerckhove
Joel
J
Sierra
José M
JM
Santarén
Juan F
JF
D017398:Alternative Splicing / Q000235:genetics; D000818:Animals; D030562:Databases, Protein; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D039601:Eukaryotic Initiation Factor-4A / Q000235:genetics / Q000378:metabolism*; D039562:Eukaryotic Initiation Factor-4F / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007553:Isotope Labeling; D007814:Larva / Q000235:genetics / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism; D020871:RNA Stability / Q000502:physiology; D014921:Wings, Animal / Q000378:metabolism*
D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D019476:Insect Proteins; D010448:Peptide Initiation Factors; C031799:eIF-4B; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases