Identification of the NAD+-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain
Identification of the NAD+-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain
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DOI：
10.1006/bbrc.2000.3246
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发表时间：
2000-08-28
影响因子：
3.1
通讯作者：
Kellermayer, M
中科院分区：
文献类型：
--
作者：
Nagy, E;Henics, T;Kellermayer, M
越来越多的证据表明，代谢酶可能充当多功能蛋白质，在细胞代谢中发挥多种作用。这些功能包括 NAD(+) 依赖性脱氢酶的 RNA 结合活性。此前，我们已将糖酵解酶甘油醛-3-磷酸脱氢酶 (GAPDH) 表征为优先于富含腺嘌呤-尿嘧啶序列的 RNA 结合蛋白。在本研究中，我们使用通过缺失诱变产生的 GST-GAPDH 融合蛋白来寻找 RNA 结合域。我们确定，GAPDH N 端 43 个氨基酸残基（对应于 NAD(+) 结合折叠的第一个单核苷酸结合结构域）足以赋予 RNA 结合作用。我们还提供证据表明，这个单一结构域虽然保留了大部分 RNA 结合活性，但失去了序列特异性。我们的结果表明NAD（+）依赖性脱氢酶和（二）核苷酸结合代谢酶识别RNA的分子基础，据报道这些酶具有不同特异性的RNA结合活性。为了支持这一预测，我们还鉴定了 NAD(+) 依赖性脱氢酶家族的其他成员，这些成员以前没有在体外作为 RNA 结合蛋白进行核酸结合的历史。根据我们的发现，我们建议将 NAD(+) 结合结构域添加到 RNA 结合结构域/基序列表中。 (C) 2000 年学术出版社。
There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs. (C) 2000 Academic Press.