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.
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.
Folgueira
Cristina
C
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.
Quijada
Luis
L
Soto
Manuel
M
Abanades
Daniel R
DR
Alonso
Carlos
C
Requena
Jose M
JM
D020413:3' Untranslated Regions; D000483:Alleles; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002874:Chromosome Mapping; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006358:Hot Temperature; D047468:Immunoprecipitation; D007313:Insecta; D018314:Leishmania infantum / Q000378:metabolism*; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011132:Polyribosomes / Q000737:chemistry / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D020413:3' Untranslated Regions; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D013395:Sucrose; D012313:RNA; D004247:DNA