Functional analysis of mRNA scavenger decapping enzymes
Functional analysis of mRNA scavenger decapping enzymes
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DOI：
10.1261/rna.7660804
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发表时间：
2004-09-01
期刊：
影响因子：
4.5
通讯作者：
Kiledjian, M
中科院分区：
文献类型：
--
作者：
Liu, SW;Jiao, XF;Kiledjian, M
真核细胞主要利用外核糖核酸酶和脱帽酶来降解它们的mRNA。已经确定了两种主要的脱帽酶。hDcp2蛋白催化与RNA片段相连的5'帽的水解，而清道夫脱帽酶（dcp）在缺乏RNA片段的帽结构上起作用。dcp是组氨酸三联体（HIT）水解酶家族的一员，可催化m(7)GpppN的裂解。HIT蛋白是同源二聚体，含有两个保守的100个氨基酸的HIT折叠结构域，它们具有独立的活性位点，每个活性位点都足以结合和水解同源底物。我们对DcpS酶进行了功能表征，并证明与先前描述的HIT蛋白不同，DcpS是一种模块化蛋白，它既需要核心HIT在羧基端折叠，也需要蛋白质的氨基端序列来进行帽结合和水解。有趣的是，即使存在过量的eIF4E， DcpS也能有效地竞争和水解帽结构，这意味着DcpS可以减轻eIF4E和帽结构之间复合物的积累，否则这些复合物会随着mRNA的衰变而积累。利用免疫荧光显微镜，我们证明了DcpS主要是一种核蛋白，在细胞质中检测到低水平的蛋白。此外，对内源性hDcp2蛋白的分析表明，除了细胞质病灶外，它也存在于细胞核中。这些数据表明，这两种脱帽酶都包含在核室中，这表明它们可能在细胞核中发挥比以前所认识到的更大的功能。
Eukaryotic cells primarily utilize exoribonucleases and decapping enzymes to degrade their mRNA. Two major decapping enzymes have been identified. The hDcp2 protein catalyzes hydrolysis of the 5' cap linked to an RNA moiety, whereas the scavenger decapping enzyme, DcpS, functions on a cap structure lacking the RNA moiety. DcpS is a member of the histidine triad (HIT) family of hydrolases and catalyzes the cleavage of m(7)GpppN. HIT proteins are homodimeric and contain two conserved 100-amino-acid HIT fold domains with independent active sites that are each sufficient to bind and hydrolyze cognate substrates. We carried out a functional characterization of the DcpS enzyme and demonstrate that unlike previously described HIT proteins, DcpS is a modular protein that requires both the core HIT fold at the carboxyl-terminus and sequences at the amino-terminus of the protein for cap binding and hydrolysis. Interestingly, DcpS can efficiently compete for and hydrolyze the cap structure even in the presence of excess eIF4E, implying that DcpS could function to alleviate the accumulation of complexes between eIF4E and cap structure that would otherwise accumulate following mRNA decay. Using immunofluorescence microscopy, we demonstrate that DcpS is predominantly a nuclear protein, with low levels of detected protein in the cytoplasm. Furthermore, analysis of the endogenous hDcp2 protein reveals that in addition to the cytoplasmic foci, it is also present in the nucleus. These data reveal that both decapping enzymes are contained in the nuclear compartment, indicating that they may fulfill a greater function in the nucleus than previously appreciated.