Transcription-induced chimerism, a mechanism involving the transcription and intergenic splicing of two consecutive genes, has recently been estimated to account for approximately 5% of the human ...transcriptome. Despite this prevalence, the regulation and function of these fused transcripts remains largely uncharacterised.
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, RBM6 and RBM5, 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 in vitro transcription/translation.
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 RBM6 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.
In adrenergic cells norepinephrine is N‐methylated by the enzyme phenylethanolamine N‐methyltransferase (PNMT) to produce epinephrine. Prior studies have demonstrated that the PNMT gene in adrenal ...chromaffin cells may be regulated hormonally via the hypothalamic‐pituitary‐adrenal axis and neurally via stimulation of the splanchnic nerve. Further, 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. 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 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. Our findings demonstrate that CoCl2‐induced ROS have synergistic effects on the hormonal and neural activation of the PNMT promoter and suggest post‐transcriptional regulation of PNMT following hormonal or neural activation during hypoxia. This study was funded by the Natural Sciences and Engineering Research Council of Canada.
Reactive oxygen species (ROS) are an unavoidable by‐product of cellular respiration; however cells are able to respond to high levels through regulation of a number of genes. ROS are known to ...regulate catecholamine gene expression, however, currently little is known regarding the role of ROS in the regulation of the phenylethanolamine N‐methyltransferase (PNMT) gene. PNMT is the terminal enzyme in the catecholamine biosynthetic pathway involved in the synthesis of the neurotransmitter, epinephrine. To investigate the effects of ROS, an in vitro cell culture model employing the oxygen‐sensitive rat pheochromocytoma (PC12) cell line is treated with hypoxic agents cobalt chloride (CoCl2), iron chelator deferoxamine mesylate (DM) and proteasome inhibitor (MG132). CoCl2 treatment increased expression of PNMT mRNA levels at 6 and 24 h, whereas DM and MG132 treatments increased expression at 24 h. Western blot analysis showed an increase in nuclear protein levels of both the immediate early gene transcription factor (Egr1), and the hypoxia inducible factor 1α (HIF1α) following 3 h CoCl2 treatment. In contrast, only increased levels of HIF1α were observed following 3 h treatments with DM or MG132. Analysis of the wild‐type PNMT promoter‐driven luciferase activity revealed maximum activity at 24 h CoCl2, DM, and MG132 treatments. These findings provide evidence that oxidative stress can regulate PNMT gene expression.
The catecholamine epinephrine is physiologically important in cardiac function and blood pressure regulation. Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in the catecholamine ...biosynthetic pathway, responsible for epinephrine biosynthesis, and is primarily localized in the adrenal gland. In hypertensive rats, adrenal PNMT mRNA, protein and enzyme activity are elevated along with elevated levels of epinephrine, suggesting that increased expression of PNMT in the adrenal gland results in the increased adrenergic function associated with hypertension. Genetic mapping studies performed in hypertensive rats and humans have investigated the possibility that the PNMT gene may be a candidate gene for hypertension; their findings suggest that differences in expression in PNMT in hypertension are not attributed to polymorphisms within the PNMT gene. It is proposed that increased PNMT in hypertension is likely due to altered transcriptional regulation of the gene. The PNMT gene is highly regulated by key transcription factors including: Egr-1, Sp1, AP-2 and the glucocorticoid receptor. The aim of this study was to investigate the molecular mechanisms involved in the dysregulation of adrenal PNMT in a genetic model of hypertension, by examining expression of transcriptional regulators in the spontaneous hypertensive rat (SHR) in comparison to Wistar–Kyoto (WKY) normotensive controls. Results demonstrate changes in key transcription factors regulating PNMT expression within the SHR adrenal gland, coincident with elevated adrenal PNMT expression. This study suggests altered transcriptional regulation of PNMT is a contributing factor to altered adrenergic function in hypertension.
Muscle twitch threshold has been used to determine optimal stimulus intensity for somatosensory evoked potentials but neuromuscular blockade precludes the use of muscle twitch during surgery. ...Accordingly, nerve action potential (NAP) amplitude was investigated as a surrogate to muscle twitch.
The ulnar and tibial nerves were stimulated at the wrist and ankle, respectively, in 27 patients undergoing spine and brain surgery. After neuromuscular blockade was gone, the stimulus intensity for just maximal NAP amplitude recorded from Erb's point and the popliteal fossa was compared with the stimulus intensity for hypothenar and plantar foot muscle twitch threshold (times two), respectively (Wilcoxon matched pairs test).
There was no significant difference between stimulus intensity for just maximal Erb's point and popliteal fossa NAP amplitude when compared with stimulus intensity for hypothenar and plantar foot twitch threshold (times two), respectively. Eight patients required more than twitch intensity (times two) to obtain maximum NAP.
The NAP amplitude may be used to determine optimal somatosensory evoked potential stimulus intensity when muscle twitch is not visible. This method should improve the success of intraoperative somatosensory evoked potential monitoring and decrease erroneous interpretation.
Prenatal exposure to glucocorticoids (GCs) programs for hypertension later in life. The current study examined the impact of prenatal GC exposure on the post‐natal regulation of the gene encoding for ...phenylethanolamine N‐methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Results from this study show that systolic, diastolic and mean arterial blood pressure are elevated in male offspring (5 to 17 weeks of age) from dams treated with dexamethasone (DEX, +15%) or corticosterone (CORT, +12%) compared to male offspring from saline‐treated (SAL) dams. RT‐PCR analysis shows that adrenal PNMT mRNA is 1.5‐fold higher in DEX and CORT males compared to SAL males. RT‐PCR analysis of transcriptional regulators of the PNMT gene show that prenatal GC exposure increased mRNA levels of Egr‐1 (2.5‐fold), AP‐2 (1.9‐fold) and GR (2.0‐fold). In contrast, Western blot analyses show increased expression of PNMT protein (1.44‐fold, DEX), along with increased Egr‐1 (1.5 fold, DEX and CORT), AP‐2 (1.4 fold, CORT), and GR (1.7 fold, DEX and CORT) compared to SAL males. These results suggest that increased PNMT gene expression via altered transcriptional activity is a mechanism by which prenatal GC insult may program for hypertension later in life.
Phenylethanolamine N‐methyltransferase (PNMT) is the enzyme involved in the biosynthesis of the catecholamine, epinephrine. Genetic mapping studies suggest that the PNMT gene is a candidate gene for ...hypertension. Furthermore, elevated expression of PNMT is correlated with elevated blood pressure in hypertensive rats. However, the mechanism associated with changes in PNMT gene expression is not known. Recent studies have identified key transcriptional regulators of the PNMT gene, which include: Egr‐1, Sp1 and GR. The current study examined the mechanism by which dysregulation of the PNMT gene occurs in a genetic rodent model of hypertension. Results from the current study show that spontaneous hypertensive rats (SHR) have elevated systolic, diastolic and mean arterial blood pressure compared to Wistar‐Kyoto (WKY) rats at 14, 15 and 16 weeks of age. RT‐PCR analysis show that adrenal PNMT mRNA is 1.5‐fold higher in SHR compared to WKY, and have increased levels of transcription factors Egr‐1 (3.2‐fold), Sp1 (3.3‐fold) and GR (1.3‐fold). These results suggest that altered transcriptional activity may be involved in increased PNMT expression associated in the pathophysiology of hypertension.
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.
Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in the catecholamine biosynthetic pathway involved in the synthesis of epinephrine. A stressor, such as hypoxia has been shown to ...regulate expression of the PNMT gene, however, currently little is known regarding the role of reactive oxygen species (ROS) in the regulation of the PNMT gene. In the current study, an in vitro cell culture model employing the oxygen-sensitive rat pheochromocytoma (PCl2) cell line was used to investigate how hypoxia and ROS regulate PNMT gene expression. Administration of the hypoxic mimetic agent cobalt chloride (PCl2) as well as exposure to hypoxia produced a time-dependent rise in PNMT promoter-driven luciferase activity. RT-PCR revealed that pretreatment with the antioxidant N-acetyl cysteine (NAC), attenuated the hypoxia and CoCl 2 induction of intron-retaining PNMT mRNA levels. In addition, both hypoxia and PCl2 activated hypoxia-inducible factor-1α (HIF-1α), as analysis of the PNMT promoter also revealed potential hypoxia-response elements (HRE) as putative binding sites for HIF-1α. Furthermore, it was found that hypoxia and PCl2 altered both dexamethasone-induced and forskolin-induced PNMT intron-retaining mRNA levels. Taken all together, these findings provide evidence that oxidative stress can regulate PNMT gene expression.
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