Tad smith insurance boulder12/6/2023 A causal role for ERG in neoplastic transformation of prostate epithelium. Klezovitch O., Risk M., Coleman I., Lucas J.M., Null M., True L.D., Nelson P.S., Vasioukhin V. Gasi Tandefelt D., Boormans J., Hermans K., Trapman J. The impact of translocations and gene fusions on cancer causation. The induced TMPRSS2-ERG fusion RNA level by antisense chimeric RNA ‘asB-35′ was used as the relative 100%. The dashed line marks the most effective bulge size. Error bars represent standard deviations. The average band intensities from three independent experiments were plotted as a line graph against the bulge size. Quantitation was done using ImageJ software. ( D) All experiments were repeated independently thrice starting from cell transfection to RT-PCR and quantifications. ( C) RT-PCR results by chimeric RNAs designed to target location D. ( B) RT-PCR results by chimeric RNAs designed to target location C. ( A) RT-PCR results of induced TMPRSS2-ERG transcripts by chimeric RNAs designed to target location B. No transfection was used as the negative control for RT-PCR reactions. RT-PCR was then performed to detect the level of induced TMPRSS2-ERG fusion RNA. LNCaP cells were transfected with designed chimeric RNAs and treated with 900 nM of DHT for three days. A set of ten different antisense chimeric RNAs were designed to create different bulge sizes when annealed to each targeted location described in Figure 1. The bulge size regulated the efficiency of RNA-mediated gene fusion. In these cases, a bulge of 35 nt will be created when the chimeric RNAs form an RNA/DNA duplex with the genomic sequences. The targeted sequences contained a 75-nt ERG gene and a 52-nt TMPRSS2 gene. ( D) Examples of genomic sequences targeted by antisense chimeric RNA asB-35, asC-35, and as-D35. T and A♼ wobble-pair known to have Watson–Crick-like geometry in a DNA double helix.The intergenic DNA stem may include a high-energy G An intergenic DNA stem can occur when the TMPRSS2 sequence is complementary to the ERG sequence near the junction site. ( C) The putative three-way junction formed between the targeted genomic DNA locations (black) and the designed antisense chimeric RNAs (green/blue). As these locations are in the introns, the designed chimeric RNAs targeting them contain only intronic sequences and no exonic sequences. ( B) Three independent target locations used in our previous study where the designer chimeric RNAs are known to induce TMPRSS2-ERG gene fusion. Our study highlights the effects of two essential elements in the proposed three-way junction: (1) the unpaired bulges linking between the RNA/DNA duplex and the intergenic DNA stem and (2) the chimeric RNA length for forming the RNA/DNA duplex. The three-way junction model consists of the RNA/DNA duplex and the intergenic DNA stem formed by the genomic TMPRSS2 sequence complementary to the genomic ERG sequence. Lower panel: schematic illustration of a three-way junction formed between genomic DNA and chimeric RNA. Both TMPRSS2 and ERG genes are on the minus strand of chromosome 21, separated by 3 Mb, an intra-chromosomal configuration prone to rearrangements. ( A) Upper panel: chromosomal locations of TMPRSS2 and ERG genes. TMPRSS2-ERG chimeric RNA gene fusion genomic recombination prostate cancer.Ī model of three-way junction formation in RNA-mediated TMPRSS2-ERG gene fusion. The knowledge could also facilitate the development of useful genomic technology for manipulating mammalian genomes. These empirically determined parameters provide important insight for searching cellular RNAs that may initiate oncogenic fusion genes. These observations were consistent regardless of the target locations within TMPRSS2 and ERG genes. The optimal length of unpaired bulges was about 35 nt, while the optimal chimeric RNA length was about 50 nt for targeting. Our results indicate that both the chimeric RNA lengths and the sizes of unpaired bulges play important roles in inducing TMPRSS2-ERG gene fusion. In this study, we determined the important parameters for chimeric RNA-mediated gene fusion using TMPRSS2-ERG fusion gene as the model. Central to this RNA-mediated gene fusion mechanism is a proposed three-way junction formed by RNA/DNA hybrid and the intergenic DNA stem formed by target genes. Previously, we provided the direct evidence that expression of a designed chimeric RNA can drive the formation of TMPRSS2-ERG gene fusion. The mechanisms that create such oncogenic fusion genes are not well understood. One common genetic alteration in cancer is gene fusion resulting from chromosomal translocations.
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