title: RNA/protein interactions in the 5'-untranslated leader of HSP70 mRNA in Drosophila lysates. Lack of evidence for specific protein binding.
abstract: 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.
authors.affiliation: Department of Molecular Pharmacology/Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033.
authors.forename: M A
authors.initials: MA
authors.lastname: Hess
authors.forename: R F
authors.initials: RF
authors.lastname: Duncan
mesh_terms: 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
chemical_list: D017931:DNA Primers; D006360:Heat-Shock Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012313:RNA