Fig 2. Validation of the reporter genes used in the screen.

(A) The Fluc-int-WT reporter RNA is spliced. PCR amplification was performed on the Fluc-WT or Fluc-int-WT construct (lanes 1 and 3, respectively), or on the reverse-transcription reaction (RT) performed with extracted RNAs from HeLa cells transfected with the Fluc-int-WT construct (lane 2) in the presence of radioactive dCTP(α³³P). The amplified fragments were electrophoresed through an acrylamide gel to demonstrate that the intron introduced in the firefly luciferase cDNA is efficiently spliced out. The position of each species is indicated on the right side of the gel and a quantification of the relative proportion of pre-mRNA (black box) and mRNA (spotted box) is shown on the right panel. (B) Luciferase expression associated with the constructs used in this study. Firefly luciferase activity normalized by renilla luciferase measured in wells of a 96-well plate containing untransfected HeLa cells, or HeLa cells transfected with pRluc and pFluc-WT, pFluc-int-WT, pFluc-int-UGA, pFluc-int-UAG, or pFluc-int-UAA constructs. (C) Measure of the firefly luciferase (Fluc) and renilla luciferase (Rluc) mRNAs by RT-PCR from HeLa cells transfected with pRluc and pFluc-WT, Fluc-int-WT, Fluc-int-UGA, Fluc-int-UAG, or Fluc-int-UAA constructs. (D) The extent of NMD is shown on a bar plot depicting levels of Fluc-int-PTC RNAs measured by quantitative RT-PCR in the absence and in the presence of cycloheximide (+CHX). The values shown are from two independent experiments. Error bar = S.D., Student t-test: **P<0.01; ***P<0.001.