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The Wilms' tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron - PubMed

  • ️Sun Jan 01 2006

. 2006 Jun 15;20(12):1597-608.

doi: 10.1101/gad.1402306. Epub 2006 May 31.

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The Wilms' tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron

Yeou-cherng Bor et al. Genes Dev. 2006.

Abstract

The Wilms' tumor 1 (WT1) gene plays an important role in mammalian urogenital development, and dysregulation of this gene is observed in many human cancers. Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of unspliced RNA containing a CTE and specifically promotes the association of this RNA with polyribosomes. These studies provide further support for links between different steps in RNA metabolism and for the existence of post-transcriptional operons.

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Figures

Figure 1.
Figure 1.

(A) Nucleotide sequence alignment of a cellular element, 5A1, with the non-protein-coding T12 mRNA sequence. Identical nucleotides are indicated by vertical lines. (B) Schematic drawing of reporter constructs used in this study. The reporter plasmids contain the HIV-1 gagpol coding sequence with expression driven by the CMV promoter. The resulting unspliced transcript, containing a complete intron, is retained in the nucleus without the presence of a CTE. In the presence of a CTE, the unspliced mRNA is exported to the cytoplasm, allowing the expression of HIV p24 from the gag gene. (SD) Splice donor; (SA) splice acceptor; (pA) polyadenylation signal. The dotted line below the spliced message depicts the removed intron. (C) Expression of p24 from pCMVGagPol-5A1 or pCMVGagPol-CTE is enhanced by cotransfection of plasmids expressing Tap/NXT1 or WT1(+KTS). 293T cells (3 × 106 cells in a 10-cm culture dish) were transfected with 15 μg of pCMVGagPol-5A1 or 5 μg of pCMVGagPol-CTE plasmids and 0.25 μg of pCMVSEAP alone or together with 2 μg of pCMVTap and 1 μg of pCDNA3FLAGNXT1 or 5 μg of WT1(+KTS) expression vectors. At 72 h post-transfection, supernatants were collected and analyzed for p24 levels and SEAP activity. The p24 values were normalized to SEAP values that serve as a control for transfection efficiency. The values shown are averages of duplicate transfections. (D) Expression of p24 from pCMVGagPol-SIRT7 or pCMVGagPol-ACTN4 is enhanced by cotransfection of plasmids expressing Tap/NXT1 or WT1(+KTS). Transfections were performed and analyzed as described in C.

Figure 2.
Figure 2.

Effects of WT1(+KTS), WT1(−KTS), or EGR1 on p24 expression from pCMVGagPol-CTE reporter constructs in 293T cells. Cells (3 × 106) were cotransfected with 5 μg of pCMVGagPol-CTE, 0.25 μg of pCMVSEAP, and increasing amounts of plasmids expressing either WT1(+KTS) or WT1(−KTS) (A), or EGR1 proteins (D). (B,C) The same set of data shown in A was plotted as “fold increase” on different scales. At 72 h post-transfection, supernatants were harvested and analyzed for p24 levels and SEAP activity. The p24 values shown have been normalized for SEAP activity. The data represent the average of two independent transfections.

Figure 3.
Figure 3.

(A,B) Overexpression of WT1(−KTS) inhibits the enhancement effects of WT1(+KTS) in transfected 293T cells. Cells were cotransfected with 5 μg of pCMVGagPol-CTE, 0.25 μg of pCMVSEAP, and increasing amounts of WT1(−KTS) (A) or WT1(+KTS) (B), while the amount of the plasmid expressing the other isoform was kept constant at 1 μg. (C,D) Neither exon 5 nor the specific KTS amino acid sequence is important for the post-transcriptional function of WT1. Cells were cotransfected with 5 μg of pCMVGagPol-CTE, 0.25 μg of pCMVSEAP, and increasing amounts of WT1 constructs, as indicated in the figure. In all cases, the overall amount of plasmid transfected was kept constant using an empty vector. Transfections and analyses were performed as described in the legend for Figure 2.

Figure 4.
Figure 4.

Northern blot analyses of total (A) and cytoplasmic (B) mRNA, and protein synthesis levels (C) of Pr55Gag in transfected cells. (A,B) 293T Cells (1 × 107 in 15-cm culture dish) were transfected with 15 μg of pCMVGagPol-CTE and 5 μg of pCMVSEAP in the absence or presence of 15 μg of WT1(+KTS) or WT1(−KTS) plasmids. As a control, cells were also transfected with 15 μg of pCMVGagPol-RRE and 5 μg of pCMVSEAP in the presence or absence of 5 μg of pCMVRev. Sixty-five hours post-transfection, total and cytoplasmic poly(A)+ mRNA was isolated from the transfected cells as described in Materials and Methods. The blot contains 5 μg of poly(A)+ mRNA per lane and was hybridized with 32P-labeled GagPol and SEAP probes. The values shown in the panels marked RRE, on the left side of each figure, represent the fold difference in the levels of the GagPol RNA bands between transfections without and with Rev. The values under the CTE panels (the right side of the figure) represent the fold difference in the levels of the GagPol-CTE RNA in the presence of the indicated proteins compared with a transfection with pCMVGagPol-CTE alone. All values were normalized using the SEAP band. (C) Pulse-chase analysis of Pr55Gag protein expressed from pCMVGagPol-CTE(myr− pro−) in transfected 293T cells. Cells (3 × 106) were transfected with 5 μg of pCMVGagPolCTE(myr− pro−) with or without 5 μg of WT1(+KTS), or 5 μg of WT1(−KTS). Thirty-six hours post-transfection, cells were pulsed with 35S-translabel (methionine and cysteine) for 20 min and chased for 10 h. Lysates were made, 35S-labeled GST-p24 was added as a recovery control, and immunoprecipitation was performed using an anti-p24 monoclonal antibody (183-H12-5C). The precipitates were analyzed on a 15% SDS-PAGE using a PhosphorImager. The lanes marked P contained the samples from the pulse and the lanes marked C contained the samples that were pulse-chased. The locations of the immunoprecipitated Pr55Gag band (and derivative) and the control GST-p24 protein are indicated. The relative normalized pixel intensity of the total Pr55Gag protein bands is shown under each lane. The data have been normalized using the intensity of the recovery control protein GST-p24 in each sample.

Figure 5.
Figure 5.

Polyribosome profile analysis of GagPol-CTE mRNA in transfected 293T cells by sucrose gradient centrifugation. (A) 293T Cells (8 × 106) were transfected with 15 μg of pCMVGagPol-CTE in the absence or presence of 15 μg of WT1(+KTS) plasmid. Forty-eight hours post-transfection, cells were harvested and cytoplasmic extracts were subjected to sucrose gradient centrifugation as described in Materials and Methods. The gradients were fractionated and the OD 254 was measured using a continuous flow cell. An in vitro transcribed gag RNA (IVTgag) was then added into each fraction as a control for recovery of RNA before RNA was isolated from each fraction. The isolated RNA was then analyzed for GagPol-CTE, SEAP mRNA, and IVTgag RNA using Northern blots. PhosphorImager analysis of the blot was used to quantitate the intensity of the bands. The measured intensity of each GagPol-CTE and SEAP band was then corrected for recovery, using the IVTgag RNA band in each fraction. The values shown for each fraction are the percentage of total GagPol-CTE or SEAP mRNA that was detected in that fraction. (B) The graphs show the distribution of GagPol-CTE (left) and SEAP mRNA (right) in the CTE (white bars) and CTE + WT1(+KTS) gradients (black bars).

Figure 6.
Figure 6.

WT1(+KTS) is associated with polyribosomes in transfected 293T cells. (A) Cells (8 × 106) were cotransfected with 15 μg of pCMVGagPol-CTE, 2.5 μg of pCMVSEAP, and 15 μg of WT1(+KTS) plasmids and were subjected to polyribosome analysis as described in Materials and Methods. The gradient was fractionated and the OD 254 of each fraction was measured. Proteins from each fraction were resolved by SDS-PAGE and analyzed by Western blotting using the anti-T7 antibody and 125I-Protein A. Optical density profiles of the gradient fractions are shown at the top. (B) The experiment was performed as in A, except that 15 mM EDTA was added to the lysate before loading onto sucrose gradients containing 15 mM EDTA.

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