Identification of Arabidopsis CCR4-NOT Complexes with Pumilio RNA-Binding Proteins, APUM5 and APUM2
Identification of Arabidopsis CCR4-NOT Complexes with Pumilio RNA-Binding Proteins, APUM5 and APUM2
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DOI：
10.1093/pcp/pcz089
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发表时间：
2019-09-01
影响因子：
4.9
通讯作者：
Chiba, Yukako
中科院分区：
文献类型：
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作者：
Arae, Toshihiro;Morita, Kotone;Chiba, Yukako
CCR 4/CAF 1是真核生物中广泛保守的去腺苷酸酶。它们形成一个大的复合体，包括作为支架蛋白的NOT 1和各种NOT蛋白，这些蛋白是多水平基因表达控制的核心成分。CCR 4-NOT复合物还含有几种RNA结合蛋白作为辅助蛋白，其是CCR 4/CAF 1去腺苷酸酶的靶识别所需的。AtCCR 4 a/B是人CCR 4在拟南芥中的直向同源物，具有多种生理功能。AtCCR 4亚型可能具有与每种生理效应相关的特异性靶mRNA;然而，AtCCR 4不具有RNA结合能力。因此，识别与AtCCR 4a/B相互作用的因子对于理解其作为基因表达调节剂的功能以及靶mRNA识别机制是必不可少的。在这里，我们确定了推定的组分AtCCR 4-NOT复合物使用免疫共沉淀结合质谱使用FLAG标记的AtCCR 4 b和随后的验证与酵母双杂交试验。有趣的是，在酵母双杂交试验中，11种AtCAF 1亚型中有4种与AtCCR 4 b和AtNOT 1相互作用，而2种亚型仅与AtNOT 1相互作用。这些结果表明，拟南芥具有多种CCR 4-NOT复合物与各种组合的去腺苷酸酶。我们还发现，RNA结合蛋白拟南芥Pumilio 5和2与AtCCR 4 a/B在细胞质中的相互作用，有几个焦点。
CCR4/CAF1 are widely conserved deadenylases in eukaryotes. They form a large complex that includes NOT1 as a scaffold protein and various NOT proteins that are core components of multiple levels of gene expression control. The CCR4-NOT complex also contains several RNA-binding proteins as accessory proteins, which are required for target recognition by CCR4/CAF1 deadenylases. AtCCR4a/b, orthologs of human CCR4 in Arabidopsis, have various physiological effects. AtCCR4 isoforms are likely to have specific target mRNAs related to each physiological effect; however, AtCCR4 does not have RNA-binding capability. Therefore, identifying factors that interact with AtCCR4a/b is indispensable to understand its function as a regulator of gene expression, as well as the target mRNA recognition mechanism. Here, we identified putative components of the AtCCR4-NOT complex using co-immunoprecipitation in combination with mass spectrometry using FLAG-tagged AtCCR4b and subsequent verification with a yeast two-hybrid assay. Interestingly, four of 11 AtCAF1 isoforms interacted with both AtCCR4b and AtNOT1, whereas two isoforms interacted only with AtNOT1 in yeast two-hybrid assays. These results imply that Arabidopsis has multiple CCR4-NOT complexes with various combinations of deadenylases. We also revealed that the RNA-binding protein Arabidopsis Pumilio 5 and 2 interacted with AtCCR4a/b in the cytoplasm with a few foci.