Abstract

Serpins constitute a large family of protease inhibitors with regulatory functions found in all living organisms. Most plant serpins have not been functionally characterized, with the exception of Arabidopsis thaliana AtSerpin1, an inhibitor of pro-apoptotic proteases, which is involved in the regulation of the programmed cell death induction, and Cucurbita maxima CmPS1, a phloem protein, which presumably inhibits insect digestive proteases and binds RNA. CmPS1 interacts most efficiently with highly structured RNA; in particular, it forms a specific complex with tRNA. Here, we demonstrated that AtSerpin1 also forms a complex with tRNA. Analysis of tRNA species bound by AtSerpin1 and CmPS1 in the presence of tRNA excess revealed that both proteins have no strict selectivity for individual tRNAs, suggesting specific interaction of AtSerpin1 and CmPS1 proteins with elements of the secondary/tertiary structure universal for all tRNAs. Analysis of CmPS1 binding of the microRNA precursor pre-miR390 and its mutants demonstrated that the pre-miR390 mutant with a perfect duplex in the hairpin stem lost the ability to form a discrete complex with CmPS1, whereas another variant of pre-miR390 with the native unpaired nucleotide residues in the stem retained this ability. These data indicate that specific interactions of plant serpins with structured RNA are based on the recognition of structurally unique spatial motifs formed with the participation of unpaired nucleotide residues in the RNA duplexes.

中文翻译：

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体外植物丝氨酸蛋白酶抑制剂的 RNA 结合

摘要

丝氨酸蛋白酶抑制剂构成了一大类蛋白酶抑制剂，在所有生物体中都具有调节功能。大多数植物丝氨酸蛋白酶抑制剂尚未在功能上进行表征，除了拟南芥AtSerpin1（一种促凋亡蛋白酶抑制剂，它参与程序性细胞死亡诱导的调节）和最大南瓜CmPS1，一种韧皮部蛋白，可能抑制昆虫消化蛋白酶并结合 RNA。CmPS1 与高度结构化的 RNA 相互作用最有效；特别是，它与 tRNA 形成特定的复合物。在这里，我们证明 AtSerpin1 也与 tRNA 形成复合物。在 tRNA 过量存在的情况下，对 AtSerpin1 和 CmPS1 结合的 tRNA 种类的分析表明，这两种蛋白质对单个 tRNA 没有严格的选择性，这表明 AtSerpin1 和 CmPS1 蛋白与所有 tRNA 通用的二级/三级结构元素的特异性相互作用。对 microRNA 前体 pre-miR390 及其突变体的 CmPS1 结合分析表明，在发夹茎中具有完美双链体的 pre-miR390 突变体失去了与 CmPS1 形成离散复合物的能力，而在茎中具有天然未配对核苷酸残基的pre-miR390的另一种变体保留了这种能力。这些数据表明植物丝氨酸蛋白酶抑制剂与结构化 RNA 的特定相互作用是基于对在 RNA 双链体中未配对核苷酸残基的参与下形成的结构上独特的空间基序的识别。