Characterization of a beta-actin mRNA zipcode-binding protein
Characterization of a beta-actin mRNA zipcode-binding protein
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DOI：
10.1128/mcb.17.4.2158
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发表时间：
1997-04-01
影响因子：
5.3
通讯作者：
Singer, RH
中科院分区：
文献类型：
--
作者：
Ross, AF;Oleynikov, Y;Singer, RH
将β-肌动蛋白mRNA定位于成纤维细胞的前缘需要在mRNA的3'非翻译区中存在保守元件，包括被称为“邮政编码”的54个核苷酸元件（E.基斯劳斯基斯角Zhu和R. H，Singer，J. Cell Biol，127：441-451，1994）。为了鉴定与zipcode结合并可能在定位中起作用的蛋白质，我们进行了带移迁移率测定、UV交联和亲和纯化实验。（到编码区）具有高特异性的邮政编码的一半（ZBP-1），微测序提供了每个大约15个残基的独特肽序列，合成与来自肽的密码子相对应的简并引物，并用于PCR扩增。鸡cDNA文库的筛选导致分离出几个克隆，这些克隆提供编码67.7-kDa蛋白质的DNA序列，该蛋白质具有与几种RNA结合蛋白如hnRNPE 1和E2同源的区域，ZBP-1与120和25 kDa的蛋白质共免疫沉淀，并与RNA识别基序（RNP 1和RNP 2基序）和一个假定的REV样核输出信号（REV-like nuclear export signal）结合。120-kDa蛋白质也通过用RNA邮政编码序列的亲和纯化获得，沿着获得53-kDa蛋白质，但25-kDa蛋白质仅在免疫沉淀中出现。邮政编码中的保守序列之一的突变，在其近端一半的ACACCC元件，大大降低了其蛋白质结合和定位特性。这些数据表明，我们分离和克隆的68 kDa ZBP-1是一种RNA结合蛋白，在复合物中起定位β-肌动蛋白mRNA的作用。
Localization of beta-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the ''zipcode'' (E. Kislauskis, X. Zhu, and R. H, Singer, J. Cell Biol, 127:441-451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments, A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1), Microsequencing provided unique peptide sequences of approximately 15 residues each, Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification, Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal, Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunoprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA-binding protein that functions within a complex to localize beta-actin mRNA.