m7GpppG cap dependence for efficient translation of Drosophila 70-kDa heat-shock-protein (Hsp70) mRNA.
To investigate whether preferential translation of the heat-shock mRNAs occurs via cap-independent translation, the requirement for the m7GpppG cap structure for efficient translation of 70-kDa heat-shock-protein (Hsp70) mRNA was quantified by in vitro translation and by in vivo translation following electroporation. Hsp70 mRNA was transcribed in vitro with and without a cap structure. Translation in the rabbit reticulocyte or wheat germ lysate was reduced about 70% when the cap was absent. For comparison, translation of uncapped encephalomyocarditis virus 5'-untranslated-region-containing mRNA was equal to or greater than capped mRNA, whereas translation of several non-heat-shock mRNAs was reduced by 85-95% when capping was omitted. Cap-dependent translational stimulation of Hsp70 is not due to increased stability, is not a kinetic effect, and requires the methylated GpppG. To confirm the in vitro analyses, capped and uncapped mRNA were introduced into Drosophila tissue culture cells by electroporation, followed by heat shock. Paralleling the in vitro results, uncapped Hsp70 mRNA translation was 70-80% reduced relative to the capped form. Complementary experiments in which eIF-4 was inactivated in vitro using either m7GTP cap analogue or foot-and-mouth-disease virus L protease expression likewise indicated that the cap-dependent translation pathway is required for optimal Hsp mRNA translation. Since cellular Hsp70 mRNA translation during heat shock is very efficient, it is unlikely that translation via a cap-independent pathway is the principal basis for preferential translation.
Song
H J
HJ
University of Southern California School of Pharmacy, Department of Molecular Pharmacology and Toxicology, Los Angeles 90033, USA.
Gallie
D R
DR
Duncan
R F
RF
D000818:Animals; D002474:Cell-Free System; D015226:Dinucleoside Phosphates / Q000502:physiology*; D004330:Drosophila / Q000166:cytology / Q000235:genetics*; D018274:Electroporation; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D010448:Peptide Initiation Factors / Q000037:antagonists & inhibitors; D014176:Protein Biosynthesis; D012315:RNA Caps; D012333:RNA, Messenger / Q000737:chemistry; D012156:Reticulocytes / Q000502:physiology; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014908:Triticum / Q000235:genetics
D015226:Dinucleoside Phosphates; D018840:HSP70 Heat-Shock Proteins; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; C075833:eIF-4; C062229:7-methyl-diguanosine triphosphate