Distinctive Properties of the 5′-Untranslated Region of Human Hsp70 mRNA

Abstract

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.

Internal ribosome entry site drives cap-independent translation of reaper and heat shock protein 70 mRNAs in Drosophila embryos

“…The resemblance and similar behavior of rpr and Dm-hsp70 mRNA 5ЈUTRs correlates with the ability of both rpr and Dm-hsp70 mRNAs to be translated under heat shock and X-ray irradiation conditions, as our polysome analysis showed. A very recent finding suggests that human-hsp70 mRNA might also support IRES activity (Rubtsova et al 2003), in contradiction to previously published evidence (Vivinus et al 2001). Although the IRES capability of human-hsp70 5ЈUTR remains a matter of controversy, more definitive evidence has been provided to support that human-hsp70 mRNA uses a shunting mechanism (Yueh and Schneider 2000).…”

Differential Cleavage of eIF4GI and eIF4GII in Mammalian Cells

“…Another mRNA classified as IRES-containing is Hsp70 mRNA (30). The dependence of translation of this mRNA on some initiation factors is low, and translation was significant when eIF4GI was cleaved by 2A pro (17).…”

“…This is the case for Hsp70 mRNA (17), which may contain an IRES element at its 5Ј-untranslated region (30). Alternatively, it may be translated by a "shunting mechanism" under heat shock conditions (31).…”

Mutation of Eif4g3, encoding a eukaryotic translation initiation factor, causes male infertility and meiotic arrest of mouse spermatocytes

“…A number of IRES-containing mRNAs are translated by a cap-independent mechanism. This might be the case for human HSPA mRNA, which is reported to contain an IRES element at its 5Јuntranslated region (Rubtosova et al, 2003) and is dependent on the integrity of EIF4G3 for in vitro translation (Castello et al, 2006). The mouse Hspa2 also has a long 5Ј untranslated leader sequence, although no common DNA sequence in the 5Ј untranslated region is shared between mouse Hspa2 and other Hspa genes (Dix et al, 1996b).…”