Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.
Identification and characterization of alternative promoters, transcripts and protein isoforms of zebrafish R2 gene.
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DOI：
10.1371/journal.pone.0024089
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Cui Z
中科院分区：
文献类型：
--
作者：
Shang H;Li Q;Feng G;Cui Z
核糖核苷酸还原酶（RNR）是脱氧核糖核苷三磷酸从头合成的限速酶。RNR亚基的表达与DNA的复制和修复密切相关。哺乳动物RNR M2亚基（R2）由于其S期特异性表达和有丝分裂后期降解，只在正常细胞的DNA复制中起作用。在此，我们证明了通过斑马鱼的替代启动子、剪接和聚腺苷化位点来控制R2的表达。鉴定出3个功能性R2启动子，产生6个具有不同5 '末端的转录本变体。近端启动子包含一个保守的E2F结合位点和两个CCAAT盒子，它们在细胞周期中对R2基因的转录至关重要。DNA损伤可以诱导远端启动子的活性，通过选择性剪接产生四种转录物变体。此外，发现两个新的剪接变体编码不同的n -截短的R2同工异构体，其中含有酶活性残基，但没有其蛋白质水解所必需的KEN box。这两个n截短的R2亚型留在细胞质中，并能够与RNR M1亚基（R1）相互作用。因此，我们的研究结果表明，在细胞周期和遗传毒性应激下，斑马鱼R2基因的差异表达和功能受到多层机制的控制。
Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress.
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