Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs.
Dicistronic reporter plasmids, such as the dual luciferase-containing pR-F plasmid, have been widely used to assay cellular and viral 5' untranslated regions (UTRs) for IRES activity. We found that the pR-F dicistronic reporter containing the 5' UTRs from HIF-1alpha, VEGF, c-myc, XIAP, VEGFR-1, or Egr-1 UTRs all produce the downstream luciferase predominantly as a result of cryptic promoter activity that is activated by the SV40 enhancer elements in the plasmid. RNA transfection experiments using dicistronic or uncapped RNAs, which avoid the complication of cryptic promoter activity, indicate that the HIF-1alpha, VEGF, c-myc, and XIAP UTRs do have some IRES activity, although the activity was much less than that of the viral EMCV IRES. The translation of transfected monocistronic RNAs containing these cellular UTRs was greatly enhanced by the presence of a 5' cap, raising questions as to the strength or mechanism of IRES-mediated translation in these assays.
Bert
Andrew G
AG
Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science (IMVS), Adelaide, SA, Australia.
Grépin
Renaud
R
Vadas
Mathew A
MA
Goodall
Gregory J
GJ
D020121:5' Untranslated Regions; D051766:Early Growth Response Protein 1 / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D017930:Genes, Reporter; D016259:Genes, myc; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
D020121:5' Untranslated Regions; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D012313:RNA; D040281:Vascular Endothelial Growth Factor Receptor-1