Preferential translation of heat shock mRNAs in HeLa cells deficient in protein synthesis initiation factors eIF-4E and eIF-4 gamma.
Expression of antisense RNA against eukaryotic translation initiation factor 4E (eIF-4E) in HeLa cells causes a reduction in the levels of both eIF-4E and eIF-4 gamma (p220) and a concomitant decrease in the rates of both cell growth and protein synthesis (De Benedetti, A., Joshi-Barve, S., Rinker-Schaffer, C., and Rhoads, R. E. (1991) Mol. Cell Biol. 11, 5435-5445). The synthesis of most proteins in the antisense RNA-expressing cells (AS cells) is decreased, but certain proteins continue to be synthesized. In the present study, we identified many of these as stress-inducible or heat shock proteins (HSPs). By mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by reactivity with monoclonal antibodies generated against human HSPs, four of these were shown to be HSP 90, HSP 70, HSP 65, and HSP 27. The steady-state levels of HSP 90, 70, and 27 were elevated in relation to total protein in AS cells. Pulse labeling and immunoprecipitation indicated that HSP 90 and HSP 70 were synthesized more rapidly in AS cells than in control cells. The accelerated synthesis of HSPs in the AS cells was not due, however, to increased mRNA levels; the levels of HSP 90 and 70 mRNAs either remained the same or decreased after induction of antisense RNA expression. Actin mRNA, a typical cellular mRNA, was found on high polysomes in control cells but shifted to smaller polysomes in AS cells, as expected from the general decrease in translational initiation caused by eIF-4E and eIF-4 gamma depletion. HSP 90 and 70 mRNAs showed the opposite behavior; they were associated with small polysomes in control cells but shifted to higher polysomes in AS cells. These results demonstrate that HSP mRNAs have little or no requirement in vivo for the cap-recognition machinery and suggest that these mRNAs may utilize an alternative, cap-independent mechanism of translational initiation.
Joshi-Barve
S
S
Department of Biochemistry, University of Kentucky, Lexington 40536.
De Benedetti
A
A
Rhoads
R E
RE
D015152:Blotting, Northern; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D039561:Eukaryotic Initiation Factor-4E; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism*
D039561:Eukaryotic Initiation Factor-4E; D006360:Heat-Shock Proteins; D010448:Peptide Initiation Factors; D000072317:Polychlorinated Dibenzodioxins; D016372:RNA, Antisense; D012333:RNA, Messenger; C077382:eIF-4gamma