The Translational Efficiencies of the Two Leishmania infantum HSP70 mRNAs, Differing in Their 3′-Untranslated Regions, Are Affected by Shifts in the Temperature of Growth through Different Mechanisms

Cristina Folgueira,Luis Quijada,Manuel Soto,Daniel R Abanades,Carlos Alonso,Jose M Requena

doi:10.1074/jbc.m505559200

Abstract

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 degrees C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 degrees C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 degrees C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 degrees C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 degrees C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 degrees C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the deltaHSP70 mutant synthesizes HSP70 at a lower rate than the wild-type parasites. Overall, our data suggest that the biological function of the HSP70-II gene is to top up HSP70 levels under conditions of stress.

Highlights

Expression in Leishmania, and other kinetoplastid protozoa, occurs by mechanisms operating almost exclusively at post-transcriptional level [3, 4]
Expression of the L. infantum HSP70 Genes—The L. infantum HSP70 locus contains six tandemly arranged genes [14] that are highly similar in sequence, and the main difference among them is found at the 3Ј-untranslated regions (3Ј-UTRs)
The levels of HSP70 mRNAs with 3Ј-UTRII remained approximately constant at the different temperatures. These results agree with our previously reported data, which demonstrated that only HSP70 –3Ј-UTRI transcripts accumulate in a temperature-dependent manner [14, 17, 18]

Summary

Introduction

Expression in Leishmania, and other kinetoplastid protozoa, occurs by mechanisms operating almost exclusively at post-transcriptional level [3, 4]. Northern blots of L. infantum RNA samples, probed with the different 3Ј-UTRs, revealed that the abundance of HSP70 transcripts with 3Ј-UTRI increases in promastigotes incubated at 37 °C (Fig. 1B).

Results

Conclusion