Bert Andrew G, Grépin Renaud, Vadas Mathew A, Goodall Gregory J
Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science (IMVS), Adelaide, SA, Australia.
RNA. 2006 Jun;12(6):1074-83. doi: 10.1261/rna.2320506. Epub 2006 Apr 6.
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.
双顺反子报告质粒，如含有双荧光素酶的pR-F质粒，已被广泛用于检测细胞和病毒的5'非翻译区（UTR）的内部核糖体进入位点（IRES）活性。我们发现，含有来自缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）、原癌基因c-myc、X连锁凋亡抑制蛋白（XIAP）、血管内皮生长因子受体-1（VEGFR-1）或早期生长反应蛋白-1（Egr-1）UTR的5'UTR的pR-F双顺反子报告基因，主要由于质粒中SV40增强子元件激活的隐蔽启动子活性而产生下游荧光素酶。使用双顺反子或无帽RNA进行的RNA转染实验避免了隐蔽启动子活性的复杂性，结果表明HIF-1α、VEGF、c-myc和XIAP的UTR确实具有一定的IRES活性，尽管其活性远低于病毒脑心肌炎病毒（EMCV）的IRES。含有这些细胞UTR的转染单顺反子RNA的翻译在存在5'帽的情况下大大增强，这引发了关于这些检测中IRES介导的翻译强度或机制的问题。
