Characterization of multimeric complexes formed by the human PTB1 protein on RNA
Characterization of multimeric complexes formed by the human PTB1 protein on RNA
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作者：
Clerte, C;Hall, KB
多聚嘧啶束结合蛋白（PTO 或 hnRNP 1）在真核细胞中具有多种已知功能，包括选择性剪接事件期间的外显子排除、mRNA 稳定以及病毒翻译和复制的调节。 PTB包含四个RNA结合域（RBD或RRM），所有这些都可以潜在地结合RNA，但它们在PTO的各种生物学功能中的作用尚不清楚。我们研究了人 PTB1 在两种靶 RNA 上形成的复合物的特性：大鼠 GABA(A) 受体、γ 2 亚基前 mRNA 和丙型肝炎病毒 3' 非翻译 RNA。 GABA RNA 在内含子和外显子中包含四个多聚嘧啶束，而 HCV NTR 包含一个 75 nt 富含 U 的束和一个高度结构化的 3' 末端。电泳迁移率变动分析表明，PTB1 蛋白首先以纳摩尔亲和力与两种 RNA 结合，但随后的蛋白质添加导致形成更高阶复合物。化学计量实验表明，最终的复合物每条 RNA 链含有多达 8 个 PTB1 蛋白。含有两个串联 RBD 的蛋白质构建体也结合这两个 RNA，但具有不同的亲和力和化学计量。核酸酶保护测定表明，PTB1 可以保护 GABA RNA 中的多聚嘧啶束，由 RBD3 和 RBD4 组成的构建体也能起到保护作用；然而，含有 RBD1 和 RBD2 的构建体增强了结合 RNA 的切割。 PTB1的结合机制是全长蛋白所独有的；这些模式似乎包括与 RNA 的直接关联以及较弱的分子间蛋白质关联。
Polypyrimidine tract binding protein (PTO or hnRNP 1) has several known functions in eukaryotic cells, including exon exclusion during alternative splicing events, mRNA stabilization, and regulation of viral translation and replication. PTB contains four RNA Binding Domains (RBDs, or RRMs), all of which can potentially bind RNA, but their roles in the various biological functions of PTO are not clear. We investigate the properties of the complexes formed by human PTB1 on two target RNAs: the rat GABA(A) receptor,gamma 2 subunit pre-mRNA and the Hepatitis C Virus 3' NonTranslated RNA. The GABA RNA contains four polypyrimidine tracts in the intron and exon, while the HCV NTR contains a 75-nt U-rich tract and a highly structured 3'-terminus. Electrophoretic mobility shift assays show that PTB1 protein first binds to both RNAs with nanomolar affinities, but subsequent protein addition leads to formation of higher-order complexes. Stoichiometry experiments show that the ultimate complexes contain up to eight PTB1 proteins per RNA strand. Protein constructs containing two tandem RBDs also bind the two RNAs, but with different affinities and stoichiometries. Nuclease protection assays show that PTB1 protects the polypyrimidine tracts in the GABA RNA, as does a construct consisting of RBD3 and RBD4; however, a construct containing RBD1 and RBD2 enhances cleavage of bound RNA. The binding mechanisms of PTB1 are unique to the full-length protein; these modes appear to include direct association with the RNA as well as weaker intermolecular protein associations.