Distinctive properties of the 5'-untranslated region of human hsp70 mRNA.
A relaxed cap-dependence of translation of the mRNA-encoding mammalian heat shock protein Hsp70 may suggest that its 5'-untranslated region (UTR) possesses an internal ribosome entry site (IRES). In this study, this possibility has been tested in transfected cells using plasmids that express dicistronic mRNAs. Using a reporter gene construct, Renilla luciferase/Photinus pyralis luciferase, we show that the 216-nt long 5'-UTR of Hsp70 mRNA acts as an IRES that directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES (100-fold over the empty vector) is similar to that of the classical picornaviral IRESs. Additional controls indicate that this high expression of the downstream reporter is not due to readthrough from the upstream cistron, nor is it due to translation of cryptic monocistronic transcripts. The effect of small deletions within the 5'-UTR of Hsp70 mRNA on the IRES activity varies in dependence on their position within the 5'-UTR sequence. With the exception of deletion of nt 33-50, it is small for the 5'-terminal half of the 5'-UTR and rather strong for the 3'-terminal section. However, neither of these small deletions abolishes the IRES activity completely. Excision of larger sections (>50 nt) by truncation of the 5'-UTR from the 5'-end or by internal deleting results in a dramatic impairment of the IRES function. Taken together, these data suggest that the IRES activity of the 5'-UTR of Hsp70 mRNA requires integrity of almost the entire sequence of the 5'-UTR. The data are discussed in terms of a model that allows a three-dimensional rather than linear mode of selection of the initiation region surrounding the start codon of Hsp70 mRNA.
Rubtsova
Maria P
MP
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia.
Sizova
Daria V
DV
Dmitriev
Sergei E
SE
Ivanov
Dmitri S
DS
Prassolov
Vladimir S
VS
Shatsky
Ivan N
IN
D020121:5' Untranslated Regions / Q000235:genetics*; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D017931:DNA Primers; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D003062:Codon; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger