Necessity of flanking repeats R1′ and R8′ of human Pumilio1 protein for RNA binding
Necessity of flanking repeats R1′ and R8′ of human Pumilio1 protein for RNA binding
复制标题
人 Pumilio1 蛋白侧翼重复序列 R1 和 R8 对 RNA 结合的必要性
DOI：
10.1021/acs.biochem.1c00445
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发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
T. Sera*
中科院分区：
文献类型：
--
作者：
K. Nakamura;T. Nakao;T. Mori;S. Ohno;Y. Fujita;K. Masaoka;K. Sakabayashi;K. Mori;T. Tobimatsu;T. Sera*
人Pumilio（hPUM）是一种结构分析良好的RNA结合蛋白，最近已用于人工RNA结合。结构分析显示，从R1到R8的重复序列中第12、13和16位的氨基酸分别与八核苷酸RNA靶中的一个特定RNA碱基接触。然而，N-和C-末端侧翼重复R1′和R8′的功能仍不清楚。在这里，我们报告如何重复有助于整体RNA结合。我们首先制备了R1′和/或R8′缺失的三个突变体，然后使用凝胶位移测定法分析RNA结合。分析表明，所有缺失突变体与其靶标的结合都低于原始hPUM，但R1′的贡献大于R8′，不像果蝇PUM。接下来我们研究了R1′或R8′的哪些氨基酸残基负责RNA结合。通过对蛋白质三级结构的详细分析，我们发现每个重复序列中都有一个疏水核心。因此，我们将每个核心中的所有疏水性氨基残基突变为丙氨酸。凝胶位移分析表明，两个疏水核都对RNA结合有贡献，特别是R1′的疏水核对RNA结合有显著影响。在本研究中，我们证明了侧翼R1′和R8′重复序列对于hPUM的RNA结合是必不可少的，并且表明疏水R1′-R1相互作用可以稳定整个hPUM结构。
Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1′ and R8′, however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1′ and/or R8′ were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1′ contributed more than R8′, unlikeDrosophilaPUM. We next investigated which amino acid residues of R1′ or R8′ were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1′ had a significant influence. In the present study, we demonstrated that the flanking R1′ and R8′ repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1′–R1 interactions may stabilize the whole hPUM structure.