6 research outputs found
Role of the 5ⲠUTR region in ERG and Tmprss2:ERG variants expression.
<p>(<b>A</b>) The native 5ⲠUTR of ERG-1b, ERG-1c and T1:E4 were replaced with a common one from the expression vector (in orange), and an optimized Kozak sequence. The replaced ATGs are in exon 3 (M3), 1c (M1c) and 4 (M4a) (<b>B</b>) Context of multiple in-frame ATG used as start codons in various ERG and Tmprss2:ERG variants. The sequence around the ATG is aligned to a consensus Kozak sequence, with the important conserved positions at -3 (R) and +4 (G) highlighted in red (counting the A as +1). Context is evaluated as âstrongâ (S) if both positions are conserved, and âweakâ (W) if they are not. An expanded analysis of ATGs in the 5â˛UTR region of ERG and Tmprss2:ERG is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>C</b>) WB analysis of the variants and mutants represented in (A). Improving the M3 context favors its use at the expenses of the M4a ATG. (<b>D</b>) uORFs that might influence translation efficiency of ERG variants in the 5ⲠUTRs of ERG-1b and several common Tmprss2:ERG variants. Red ticks indicate ATGs, boxes below indicate the putative uORF generated and their approximate length (drawing not to scale). Red boxes indicate strong ATG contexts and green weak ones (as defined in (B)). More details about the uORFs are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s004" target="_blank">Table S1</a>. (<b>E</b>) Effect of independently mutating uORFsâ ATG in ERG-1b: mutation of the first ATG in exon 2 releases suppression of translation and increases levels of ERG from the ATG in exon 4. (<b>F</b>) Expression of ERG and Tmprss2:ERG variants<b>.</b> Full-length cDNAs for the variants, including their entire 5ⲠUTRs, were expressed and lysates from transiently transfected HeLa cells were analyzed by western blot.</p
5ⲠUTR Control of Native ERG and of Tmprss2:ERG Variants Activity in Prostate Cancer
<div><p>ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5ⲠUTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF) in their 5ⲠUTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4) with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4) fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumorâassociated and normal ERG.</p> </div
Mapping of translation initiation of ERG isoforms.
<p>(<b>A</b>) N-terminal heterogeneity in human native ERG variants. The 5Ⲡregions for the indicated variants are reproduced. Boxes represent exons with the ORF in blue. Red ticks below the exons indicate in-frame ATGs in exons 1c and exons 3 to 5. The out-of-frame products caused by exon skipping, from the otherwise in frame ATGs are indicated in red. (<b>B</b>) Western Blot (WB) of transient expression of the variants in HeLa cells. Antibody C20 recognizes exogenous protein and an endogenous band at âź44 KDa corresponding in size to Î4 variants. Antibody C17 preferentially recognizes exogenous ERG bands. (<b>C</b>) Mutations that independently eliminate the 3 in-frame ATGs in exon 4 were introduced into the cDNA of T1:E4 and analyzed as above. (<b>D</b>), Similarly, mutations were introduced to eliminate the ATGs in exon 3 (ERG-1b) or in exon 1c (ERG-1c) by themselves or in combination with mutations in the first in-frame ATG (M4a) in exon 4 (ERG-1b and ERG-1c).</p
Human ERG gene structure and main isoforms.
<p>(<b>A</b>) Top: The âź300 Kb human ERG locus, drawn roughly to scale. Approximate intron sizes are indicated, along with exons position (bars). Red indicates first exons, blue common alternative ones and gray uncommon ones. Middle: Exon structure, with exon sizes at the bottom. Blue boxes indicate the main predicted ORFs, white boxes the untranslated regions and gray the uncommon exons. Red circles indicate polyA sites. Bottom: alignment of the exons forming the main ORF (ERG-1b) with the proteinâs domains. Numbers indicate size in amino acids. PNTâ=âpointed domain, ADâ=âalternative domain, Etsâ=âEts domain, TADâ=âtransactivation domain. Asterisk and circle indicate position of the first and second ATG. (<b>B</b>) Human ERG main variants. Alignment of exons forming the 30 main RNA variants of human ERG. Blue indicates the ORF, light blue the additional region from the ATG in exon 3. For each variant, the proposed name is indicated next to previous nomenclature (if available). The proposed protein name is reported along the predicted size in aa and KDa. Variants derived from the alternative usage of promoter 1a and 1b are paired as they lead to related mRNAs and identical proteins.</p
Differential ERG variant expression in normal tissues and PCa cells.
<p>Quantification by qPCR of alternative ERG variants in normal tissue (<b>A, D</b>), primary PCa samples (<b>B, E</b>) and PCa cell lines. (<b>C, F</b>). (<b>AâC</b>) Quantification of promoter usage. Primer sets specific for the 3 different ERG promoters and for the Tmprss2:ERG fusion where used, along with a primer set spanning exons 5â7 to quantify total ERG. Normal tissues do not express the fusion product. Expression of variants 1a and 1c is virtually undetectable in the PCa cell lines analyzed. For panel C, open symbols indicate LnCap, DU145 and C4-2 (not expressing Tmprss2:ERG fusion), full symbols indicate VCap and NCI-H660 (expressing Tmprss2:ERG fusion). See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> AâC. (<b>DâF</b>) Quantification by qPCR of alternative polyadenylation usage. Primer sets specific for the 3 different polyA sites where used, along with a primer set to quantify total ERG and one set to quantify total exon 11 levels in order to infer 11SpA usage. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone.0049721.s001" target="_blank">Fig. S1</a> DâF. In all cases, each indicated value represents averages of âĽ3 independent experiments and is presented as ÎC(t) normalized to the housekeeping gene GAPDH, therefore a âhighâ ÎC(t) value means low levels of expression and a âlowâ value means high level of expression. Horizontal bars indicate the mean. Asterisks indicate that the product was not detected in the sample and would therefore be equivalent to an experimental point at the top of the table, but it is not reflected by the mean.</p
Activity of ERG and Tmprss2:ERG variants.
<p>(<b>A</b>) Stable expression of ERG variants in NIH-3T3 cells. Expression was analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049721#pone-0049721-g003" target="_blank">Fig 3</a>. (<b>BâC</b>) Cells from (A) were assayed for their invasion potential using a matrigel invasion assay (B) and for their motility using a wound healing assay (C). Representative images are shown. (<b>D</b>) Quantification of (C), using ImageJ software (âĽ4 independent experiments). Bars indicate standard deviations. (<b>E</b>) Transient transcriptional activation of ERG-dependent promoters. Firefly luciferase cDNA was cloned downstream of the VE-Cadherin or MMP-1 promoters as depicted. The VE-Cadherin (red) and the MMP-1 (blue) promoters contain respectively 4 and 11 Ets binding sites (blue boxes, not to scale). Luciferase reporters were transiently co-expressed in HeLa cells with Tmprss2:ERG variants. A renilla luciferase construct was used to normalize for variation in transfection efficiency. Dual-luciferase assay was performed and activity is represented as fold-activity over that of co-transfected empty vector. Data represent averages of at least 3 independent experiments, with bars indicating standard deviations.</p