An element within the 5′ untranslated region of human Hsp70 mRNA which acts as a general enhancer of mRNA translation

Abstract

The untranslated regions of mRNAs encoding heat-shock proteins have been reported to contain elements important to the post-transcriptional regulation of these key components of the stress response. In this report we describe an element from the 5 H UTR of human Hsp70 mRNA that increases the efficiency of mRNA translation. Cloning of this region upstream of the coding sequence of two different reporter genes (firefly luciferase and chloramphenicol acetyltransferase) increases expression of the reporter under normal cell culture conditions by up to an order of magnitude. This effect was observed in three different promoter contexts (HSP, SV40 and CMV) and in six cell lines. The increase in protein production is not accompanied by any alteration in mRNA levels, suggesting that the element facilitates translation. 5 H or 3 H truncated sequences are ineffective in enhancing reporter expression, suggesting that the activity arises from the secondary structure of the element, rather than from some smaller defined motif. Computer analysis of this region revealed that it is able to form stable secondary structures (DG < 2292.6 kJ´mol 21 ). The Hsp70 element does not seem to act as an internal ribosome entry site. Incorporation of the sequence into plasmids used for DNA vaccination produces increased antibody responses, confirming that the sequence is functional in primary cells. These data suggest that the 5 H UTR of human Hsp70 mRNA plays an important role in determining Hsp70 expression levels, and that it contains an element of general utility in enhancing recombinant protein expression systems.

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

“…No large section of the 5Ј-UTR can be excised without a dramatic deleterious effect on the expression of the second cistron. This conclusion nicely correlates with data of Vivinus et al (9), who have recently shown the importance of the integrity of Hsp70 5Ј-UTR for its enhancing effect on translation of reporter genes in monocistronic constructions.…”

“…The attempts to experimentally identify a specific structure within the relatively short 5Ј-UTR of BiP mRNA (210 nt) have been unsuccessful (7). Even more surprising is that the 5Ј-UTR of mammalian Hsp70 mRNA has been reported to be devoid of IRES properties (8,9), despite the fact that its length, GC-content, and relaxed cap-dependence (10,11) are similar to the 5Ј-UTR of BiP mRNA, and the corresponding mRNAs encode proteins with related functions (chaperones). The high GϩC content of the 5Ј-UTR of mammalian Hsp70 mRNA seems more compatible with operation via an IRES than by means of the classical scanning mechanism.…”

empty pericarp2 Encodes a Negative Regulator of the Heat Shock Response and Is Required for Maize Embryogenesis

“…We propose that the 5Ј UTR of the emp2 transcript harbors an element(s) important for its efficient translation. It has been demonstrated in both Drosophila and human that a lengthy and intact 5Ј UTR is required for the preferential translation of hsp70 transcripts under stress conditions (Lindquist and Petersen, 1990;Hess and Duncan, 1996;Vivinus et al, 2001). If the 5Ј UTR of emp2 has a function similar to that of hsp70, this may provide a mechanism to regulate the accumulation of EMP2 in response to heat stress, assuming that emp2 is transcribed constitutively (data not shown).…”

Extrathymic TCR Gene Rearrangement in Human Small Intestine: Identification of New Splice Forms of Recombination Activating Gene-1 mRNA with Selective Tissue Expression

“…There are examples of a posttranscriptional regulatory role for the 5ЈUTR. The 5ЈUTR of human heat shock protein 70 mRNA contains an element that increases the translation efficiency at 42°C compared with at 37°C (37). Relatively little is known about the role(s) of selective expression of mRNAs differing only in the 5ЈUTR.…”