Assessing IRES activity in the HIF-1alpha and other cellular 5' UTRs

Abstract

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.

FIGURE 1.

Effect of the VEGF-R1 and Egr-1 5′ UTRs on activity of the downstream reporter in a dicistronic gene. (A) Schematic illustration of the dicistronic reporter genes R-F, R-vegfr1-F, and R-egr-F. (B) Relative enhancement of expression of the downstream reporter enzyme by the VEGF-R1 and Egr-1 5′ UTRs. HeLa cells were transiently transfected with the reporter genes as indicated. Renilla and firefly luciferase activities were determined 24 h post-transfection and expressed as ratios of firefly to Renilla luciferase, relative to the ratio for R-F, which was given a value of 1. Data are presented as the mean (± SEM) of triplicate samples from three independent experiments. (C) Effect of removal of the promoter and enhancer (−Prom−Enh) or removal of just the proximal promoter (−Prom). HeLa cells were transiently transfected with the reporter genes as indicated, and Renilla and firefly luciferase activities were determined 24 h post-transfection. The amount of each luciferase activity is shown as a percentage relative to the amount produced from the intact form of the plasmid (Prom + Enh).

FIGURE 2.

Cryptic promoter activity in the 5′ UTRs of HIF-1α, VEGF, c-myc, and XIAP is activated by the viral enhancer region in a dicistronic reporter plasmid. (A) Schematic illustration of the dicistronic reporter gene showing variants with or without SV40 proximal promoter or enhancer regions. (B) Effect of removal of the enhancer (−Enh) or the proximal promoter (−Prom). HeLa cells were transiently transfected with the reporter genes as indicated, and Renilla and firefly luciferase activities were determined 24 h post-transfection. The individual Renilla and firefly luciferase activities are shown, expressed as a percentage of Renilla activity from the corresponding intact dicistronic form of the plasmid. The ratios of firefly activity to Renilla activity are tabulated below the histograms. Data are presented as the mean (± SEM) of triplicate samples from at least three independent experiments.

FIGURE 3.

Knockdown of dicistronic transcripts by RNAi targeting the Renilla luciferase coding region does not reduce firefly luciferase expression from reporters containing cellular UTRs. The indicated plasmids were cotransfected with pCMV-SPORT-βgal, with or without 1 pmol of Renilla Luciferase siRNA mix as indicated. R + F indicates cotransfection of monocistronic Renilla and firefly reporters. Renilla and firefly luciferase activities were determined 24 h post-transfection, and data were normalized to β-galactosidase activity to control for any variations in transfection efficiency. The amount of each luciferase activity is shown as a percentage relative to the amount produced in the absence of Renilla siRNA. Data are shown as the mean (± SEM) of a total of five samples from two independent experiments.

FIGURE 4.

Relative IRES activities determined by transfection of in vitro transcribed dicistronic mRNAs. (A) Denaturing gel electrophoresis of in vitro transcribed mRNAs. Two micrograms (2 μg) of each mRNA as indicated was electrophoresed on a denaturing formaldehyde agarose gel and visualized by staining with ethidium bromide. Because a proportion (∼50%) of R-xiap-F in vitro transcripts terminate prematurely within the XIAP UTR, this mRNA was additionally purified by isolation of poly(A+) RNA on oligo d(T) magnetic beads, and 1 μg of the purified mRNA was electrophoresed on a denaturing formaldehyde agarose gel and visualized by staining with ethidium bromide in the panel on the right. (B) Capped, polyadenylated dicistronic mRNAs were transfected into HeLa cells, and Renilla and firefly luciferase activities were determined 15 h post-transfection. The ratios of firefly luciferase to Renilla luciferase are shown relative to the ratio for R-F, which was given a value of 1. Data are presented as the mean (± SEM) of triplicate samples from a minimum of three transfections.

FIGURE 5.

IRES-dependent translation from the cellular IRESs is weak compared to cap-initiated translation. (A) Schematic illustration showing the monocistronic and dicistronic mRNAs. (B) Comparison of the firefly activities produced from monocistronic vs. dicistronic mRNAs. Capped, polyadenylated monocistronic and dicistronic firefly luciferase mRNAs were prepared in vitro. For each transfection a constant molar amount of firefly and Renilla cistron was used. (For transfection with monocistronic mRNAs, this was an equimolar mixture of Renilla and firefly mRNAs, and each equal to the molar amount transcript used for transfection of dicistronic mRNA.) β-Galactosidase reporter was included in each transfection to normalize for any variations in transfection efficiency. The luciferase and β-galactosidase activities were measured at 15 h after transfection. Firefly luciferase activity is shown relative to F, the firefly luciferase mRNA with a short vector-derived 5′ UTR.

FIGURE 6.

Time course of luciferase activity in cells transfected with various uncapped firefly luciferase mRNAs. Uncapped monocistronic firefly luciferase mRNAs with the indicated 5′ UTRs were transcribed in vitro and cotransfected with capped monocistronic Renilla luciferase mRNA into HeLa cells. Luciferase activities were determined at the various times indicated and are expressed relative to the activity at 4 h. Data are shown as mean (± SEM) from a minimum of three transfections.

FIGURE 7.

The effect of various 5′ UTRs on translation of capped and uncapped monocistronic mRNAs. Capped and uncapped monocistronic firefly luciferase mRNAs with the indicated 5′ UTRs were transcribed in vitro and transfected into HeLa cells, along with an equimolar amount of control monocistronic Renilla luciferase mRNA for normalization of transfection efficiencies. Luciferase activities were determined 4 h post-transfection and are expressed relative to the activity from the capped F mRNA, which is the firefly luciferase mRNA with a short vector-derived 5′ UTR. Data are presented as the mean (± SEM) of triplicate samples from at least three independent experiments.