Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines
Alternative-splicing forms of the major phase II conjugating UGT1A gene negatively regulate glucuronidation in human carcinoma cell lines
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DOI：
10.1038/tpj.2009.64
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发表时间：
2010-10-01
影响因子：
2.8
通讯作者：
Guillemette, C.
中科院分区：
文献类型：
--
作者：
Bellemare, J.;Rouleau, M.;Guillemette, C.
UDP-葡萄糖醛酸基转移酶UGT 1A基因是参与大量分子代谢的主要生物转化基因。最近，我们发现了一系列新的选择性剪接亚型，称为亚型2或UGT1As_i2，其使用选择性外显子5（5 b）。这些mRNA和相应的45 kDa蛋白的功能仍然不清楚。虽然缺乏葡萄糖醛酸基转移酶活性，但UGT1As_i2与具有酶活性的经典UGT 1A同种型（UGT1As_i1）广泛共表达。在这项研究中，我们观察到丰富的信号在人类结肠组织样本，主要是沿着肠隐窝。在人细胞中，UGT1A_i2蛋白在与UGT1As_i1相似的亚细胞区室中表达。然后在两种人结肠癌细胞系中使用合成的小干扰RNA（siRNA）研究了i2剪接形式的细胞特性，这两种细胞系显示出显著量的含外显子5a和外显子5 b的mRNA，并显示出对UGT 1As底物的酶活性。我们观察到siRNA介导的内源性i2的敲低使细胞葡萄糖醛酸化活性上调120-170%（P
The UDP-glucuronosyltransferase UGT1A gene is a major biotransformation gene involved in the metabolism of a vast array of molecules. Recently, we uncovered a new series of alternative spliced isoforms referred to as isoforms 2 or UGT1As_i2 that use an alternative exon 5 (5b). The function of such mRNAs and the corresponding 45 kDa proteins still remains unclear. Although devoid of glucuronosyltransferase activity, UGT1As_i2 are widely co-expressed with the enzymatically active and classical UGT1A isoforms (UGT1As_i1). In this study, we observed abundant signal in human colon tissue samples, predominantly along intestinal crypts. In human cells, UGT1A_i2 proteins are expressed in similar subcellular compartments as UGT1As_i1. Cellular properties of i2-spliced forms were then studied using synthetic small-interfering RNA (siRNA) in two human colon cancer cell lines that show a significant amount of exon 5a- and exon 5b-containing mRNAs and that display enzymatic activities for UGT1As substrates. We observed that siRNA-mediated knockdown of endogenous i2 upregulates cellular glucuronidation activities by 120-170% (P