RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours

<p>Abstract</p> <p>Background</p> <p>Transcription-induced chimerism, a mechanism involving the transcription and intergenic splicing of two consecutive genes, has recently been estimated to account for ~5% of the human transcriptome. Despite this prevalence, the regulation and function of these fused transcripts remains largely uncharacterised.</p> <p>Results</p> <p>We identified three novel transcription-induced chimeras resulting from the intergenic splicing of a single RNA transcript incorporating the two neighbouring 3p21.3 tumour suppressor locus genes, <it>RBM6 </it>and <it>RBM5</it>, which encode the RNA Binding Motif protein 6 and RNA Binding Motif protein 5, respectively. Each of the three novel chimeric transcripts lacked exons 3, 6, 20 and 21 of RBM6 and exon 1 of RBM5. Differences between the transcripts were associated with the presence or absence of exon 4, exon 5 and a 17 nucleotide (nt) sequence from intron 10 of RBM6. All three chimeric transcripts incorporated the canonical splice sites from both genes (excluding the 17 nt intron 10 insertion). Differential expression was observed in tumour tissue compared to non-tumour tissue, and amongst tumour types. In breast tumour tissue, chimeric expression was associated with elevated levels of RBM6 and RBM5 mRNA, and increased tumour size. No protein expression was detected by <it>in vitro </it>transcription/translation.</p> <p>Conclusion</p> <p>These results suggest that RBM6 mRNA experiences altered co-transcriptional gene regulation in certain cancers. The results also suggest that RBM6-RBM5 transcription-induced chimerism might be a process that is linked to the tumour-associated increased transcriptional activity of the <it>RBM6 </it>gene. It appears that none of the transcription-induced chimeras generates a protein product; however, the novel alternative splicing, which affects putative functional domains within exons 3, 6 and 11 of RBM6, does suggest that the generation of these chimeric transcripts has functional relevance. Finally, the association of chimeric expression with breast tumour size suggests that RBM6-RBM5 chimeric expression may be a potential tumour differentiation marker.</p

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours-0

<p><b>Copyright information:</b></p><p>Taken from "RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours"</p><p>http://www.biomedcentral.com/1471-2164/8/348</p><p>BMC Genomics 2007;8():348-348.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2174484.</p><p></p>ze of both genes, in relation to the distance between them, is indicated in base-pairs. Vertical numbers represent chromosomal markers. Sideways arrows represent translation start sites. . Scale diagram comparing intragenic distances to the intergenic distance. Vertical bars represent exons, solid horizontal lines represent introns and the dotted line represents the intergenic region. The sideways arrows represent the translation start sites, within exon 2 of both genes

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours-3

<p><b>Copyright information:</b></p><p>Taken from "RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours"</p><p>http://www.biomedcentral.com/1471-2164/8/348</p><p>BMC Genomics 2007;8():348-348.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2174484.</p><p></p>ption/translation system in the presence of [S] methionine. Products were electrophoresed through a denaturing polyacrylamide gel. pcDNA3.RBM10 and the antisense pcDNA3.RBM5(-) constructs were used as positive controls for the T7 and SP6 polymerases, respectively. Reaction minus template ("no template") was used as a negative reaction control

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours-6

<p><b>Copyright information:</b></p><p>Taken from "RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours"</p><p>http://www.biomedcentral.com/1471-2164/8/348</p><p>BMC Genomics 2007;8():348-348.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2174484.</p><p></p> RBM6E8F and RBM5E7R and inner primers RBM6E17F and RBM5E4R, showing the ~550 bp amplicon in lane 2. Lane 1: DNA ladder (1 kb, Invitrogen). . Schematic of the amplified 550 bp fragment, showing the splicing out of RBM6 exons 20 and 21, and RBM5 exon 1. Blue boxes indicate exons of RBM6, orange boxes indicate exons of RBM5, dotted lines delineate site of intragenic splicing. . Agarose gel of the nested RT-PCR results for full-length RBM6-RBM5 chimeric transcripts, using outer primers RBM6Fb and RBM5E7R and inner primers RBM6Fc and RBM5E5R. The three chimeric transcripts are from different experiments. Chimeric transcripts 1 and 3 were amplified from Jurkat cells, whereas chimeric transcript 2 was amplified from skeletal muscle tumour. M1: 1 kb DNA ladder (Invitrogen). M2: 1 kb DNA ladder (New England Biolabs). Arrow identifies the faint amplicon observed for chimeric transcript 3. . Schematic detailing the splicing patterns associated with the three chimeric transcripts. Numbered boxes represent exons, dotted lines represent alternative splice sites. Arrows: delineate putative translation start codons of longest ORFs. Stop signs: represent putative translation stop codons of the longest ORFs. The triangle in chimeric transcript 2 indicates the site of the 17 nucleotide insertion (represented by the red line) from RBM6 intron 10. Not drawn to scale

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours-4

<p><b>Copyright information:</b></p><p>Taken from "RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours"</p><p>http://www.biomedcentral.com/1471-2164/8/348</p><p>BMC Genomics 2007;8():348-348.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2174484.</p><p></p>inserted regions from within intron 2, putative translation start sites (arrows) and putative stop codons (stop signs) [7]. Boxes represent exons and are not drawn to scale. Exon 3 (which is present in all RBM6 variants but absent in all RBM6-RBM5 chimeric transcripts) is depicted in blue, while exon 6 (which is differentially spliced in RBM6 variants and the RBM6-RBM5 chimeras, and contains the first of two RRM domains) is depicted in red. The forward and backward hatched boxes drawn as alternatively spliced sequences from intron 2, represent two different sequences, while the solid black box represents the same sequence in both RBM6C and RBM6D

RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours-2

<p><b>Copyright information:</b></p><p>Taken from "RBM6-RBM5 transcription-induced chimeras are differentially expressed in tumours"</p><p>http://www.biomedcentral.com/1471-2164/8/348</p><p>BMC Genomics 2007;8():348-348.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2174484.</p><p></p> . Expression in purchased human tissue samples: lung – large cell carcinoma; ovary – cystadenoma; skeletal muscle – malignant fibrous histiocytoma, poorly differentiated; skin – malignant melanoma; spleen – malignant lymphoma; uterus – adenocarcinoma, well differentiated. . Expression in purchased human tissue samples: bone – osteoblastic osteosarcoma; brain – astrocytoma, moderately differentiated; lymph node – non-Hodgkin's lymphoma of tonsil; pancreas – insulinoma; prostate – adenocarcinoma, moderately differentiated; and testis – seminoma. . Expression in three tumour samples from the Ontario Cancer Research Network, and an unmatched purchased non-tumour sample. . Expression in human cancer cell lines: GLC20 – small cell lung carcinoma; MDA-MB-231 – pleural effusion of a breast adenocarcinoma; JKM1 – Jurkat T lymphoblastic leukemia subclone; MCF-7 – pleural effusion of a breast adenocarcinoma; TF-1 – erythroblastic leukemia; A431 – epidermoid carcinoma; HeLa – cervical carcinoma; BT-474 – invasive ductal breast carcinoma; K-562 – chronic myelogenous leukemia; Raji – Burkitt's lymphoma. NT: non-tumour tissue, T: tumour tissue