Heterogeneity within animal thioredoxin reductases - Evidence for alternative first exon splicing
Heterogeneity within animal thioredoxin reductases - Evidence for alternative first exon splicing
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DOI：
10.1074/jbc.m004750200
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发表时间：
2001-02-02
影响因子：
4.8
通讯作者：
Gladyshev, VN
中科院分区：
文献类型：
--
作者：
Sun, QA;Zappacosta, F;Gladyshev, VN
动物硫氧还蛋白还原酶（TRs）是一种含硒半胱氨酸的黄素酶，利用NADPH还原硫氧还蛋白和其他蛋白质和非蛋白质底物。已知三种类型的哺乳动物TR，其中TR 1是细胞溶质酶，TR 3是线粒体酶。以前的特点TR 1和TR 3发生作为同源二聚体的55-57-kDa的亚基，我们在这里报告，TR 1分离小鼠肝脏，小鼠肝肿瘤，和人类T细胞系表现出广泛的异质性检测电泳，免疫印迹和质谱分析。特别地，检测到TR 1的67-kDa条带。此外，一种新形式的小鼠TR 1 cDNA编码一个67 kDa的硒蛋白亚基与一个额外的N-末端序列被确定。随后的同源性分析揭示了小鼠和大鼠TR 1 mRNA的三种不同的同种型。这些形式在5'序列上不同，这是由于前三个外显子的交替使用，但具有共同的下游序列。类似地，对于人TR 3和果蝇TR观察到多种mRNA形式的表达。在这些基因中，选择性第一外显子剪接导致预测的线粒体和胞质蛋白的形成。此外，人TR 3基因与儿茶酚-O-甲基转移酶（COMT）基因在互补DNA链上重叠，使得线粒体TR 3和膜结合COMT mRNA具有共同的第一外显子序列;然而，预测的胞质TR 3和可溶性COMT形式的转录起始位点被相似的30个内切酶分开，因此，本研究证明了TRs内的显著异质性，至少部分是由采用第一外显子可变剪接的进化保守遗传机制引起的。TR基因内的多个转录起始位点可能与动物硫氧还蛋白还原酶的表达和/或细胞器和细胞类型特异性定位的复杂调控有关。
Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits, We report here that TR1 isolated from mouse liver, mouse liver tumor, and a human T-cell line exhibited extensive heterogeneity as detected by electrophoretic, immunoblot, and mass spectrometry analyses. In particular, a 67-kDa band of TR1 was detected. Furthermore, a novel form of mouse TR1 cDNA encoding a 67-kDa selenoprotein subunit with an additional N-terminal sequence was identified. Subsequent homology analyses revealed three distinct isoforms of mouse and rat TR1 mRNA These forms differed in 5' sequences that resulted from the alternative use of the first three exons but had common downstream sequences. Similarly, expression of multiple mRNA forms was observed for human TR3 and Drosophila TR, In these genes, alternative first exon splicing resulted in the formation of predicted mitochondrial and cytosolic proteins. In addition, a human TR3 gene overlapped with the gene for catechol-O-methyltransferase (COMT) on a complementary DNA strand, such that mitochondrial TR3 and membrane-bound COMT mRNAs had common first exon sequences; however, transcription start sites for predicted cytosolic TR3 and soluble COMT forms were separated by similar to 30 kilobases, Thus, this study demonstrates a remarkable heterogeneity within TRs, which, at least in part, results from evolutionary conserved genetic mechanisms employing alternative first exon splicing. Multiple transcription start sites within TR genes may be relevant to complex regulation of expression and/or organelle- and cell type-specific location of animal thioredoxin reductases.