RNA/protein interactions in the 5'-untranslated leader of HSP70 mRNA in Drosophila lysates. Lack of evidence for specific protein binding.
We have investigated the hypothesis that preferential translation of the heat stress mRNAs requires the binding of a trans-acting protein factor. 32P-Labeled RNA probes covering the 5'-untranslated region (5'-UTR) of HSP70 mRNA were synthesized, and gel retardation and UV cross-linking assays performed to identify trans-acting sequence-specific RNA-binding factors. The results indicate that no HSP70 5'-UTR RNA-specific binding proteins exist. Reducing the "stringency" in the gel retardation binding analyses revealed several non-sequence-specific RNA-binding complexes. The proteins bind RNA more strongly than DNA, show minor preferences for specific sequences, but none binds more strongly to the HSP70 5'-UTR than to other non-heat stress RNAs. Ultraviolet cross-linking analysis identifies two principal HSP70 5'-UTR binding proteins, of approximately 50 and 70 kDa. The p50 binding activity is increased severalfold for all mRNAs in heat-stressed lysates, and its binding produces the primary gel-retarded complex. Further detailed analyses were performed to probe for any possible heat-influenced changes. RNA/protein interactions are not affected by capping. Neither the kinetics nor the salt sensitivity of protein binding is affected by heat. Binding analyses using partial or complete HSP70 5'-UTR give qualitatively similar conclusions. Binding analyses were also carried out with several "normal" 5'-UTRs to investigate whether a heat-induced repressor might be activated by heating. No normal mRNA-specific heat-induced binding changes are detected. We conclude that heat-induced alterations in RNA-binding proteins do not mediate preferential translation of heat stress mRNAs or repression of normal mRNAs.
Hess
M A
MA
Department of Molecular Pharmacology/Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033.
Duncan
R F
RF
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004330:Drosophila; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism
D017931:DNA Primers; D006360:Heat-Shock Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012313:RNA