Novel CNBP- and La-based translation control systems for mammalian cells
Novel CNBP- and La-based translation control systems for mammalian cells
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DOI：
10.1002/bit.10549
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发表时间：
2003-01-05
影响因子：
3.8
通讯作者：
Fussenegger, M
中科院分区：
文献类型：
--
作者：
Schlatter, S;Fussenegger, M
在非洲爪蟾的整个发育过程中，核糖体蛋白（rp）的产生在翻译水平上受到调节。翻译控制由存在于编码rp的mRNA的5'非翻译区（UTR）中的末端寡嘧啶元件（TOP）介导。TOP元件采用特定的二级结构，防止核糖体结合和rp编码mRNA的启动。然而，结合CNBP（细胞核酸结合蛋白）或La蛋白的TOP发夹结构废除TOP介导的转录阻滞，并诱导rp的生产。基于特定的CNBP-TOP/La-TOP相互作用，我们设计了一个翻译控制系统（TCS），用于哺乳动物细胞中所需转基因mRNA的条件性以及可调节的翻译。通用TCS配置由四环素应答表达系统（TETOFF）控制下的编码CNBP或La的质粒和包含组成型P-SV 40启动子和人模型产物基因SEAP（人分泌型碱性磷酸酶）之间的TOP模块的靶表达载体（P-SV 40-TOP-SEAP-pA）组成。TCS技术在转基因中国仓鼠卵巢（CHO）细胞中显示出良好的SEAP调控特性。替代CNBP和La，TOP介导的翻译控制也可以通过人工硫代磷酸酯抗TOP寡脱氧核苷酸来调节。共聚焦激光扫描显微镜显示细胞摄取的FITC标记的寡脱氧核苷酸和它们的定位在核周细胞器内24小时。除了它们基于TOP的抑制控制能力之外，CNBP和La还显示出增加从脊髓灰质炎病毒内部核糖体进入位点（IRES）的帽非依赖性翻译，并且La单独促进帽依赖性翻译起始。CNBP和La首次证实了RNA结合蛋白对所需转基因进行翻译控制并增加哺乳动物细胞中异源蛋白质产量的潜力。我们预计这两项资产将推进当前的基因治疗和生物制药制造战略。(C)2002 Wiley Periodicals，Inc.
Throughout the development of Xenopus, production of ribosomal proteins (rp) is regulated at the translational level. Translation control is mediated by a terminal oligopyrimidine element (TOP) present in the 5' untranslated region (UTR) of rp-encoding mRNAs. TOP elements adopt a specific secondary structure that prevents ribosome-binding and translation-initiation of rp-encoding mRNAs. However, binding of CNBP (cellular nucleic acid binding protein) or La proteins to the TOP hairpin structure abolishes the TOP-mediated transcription block and induces rp production. Based on the specific CNBP-TOP/La-TOP interactions we have designed a translation control system (TCS) for conditional as well as adjustable translation of desired transgene mRNAs in mammalian cells. The generic TCS configuration consists of a plasmid encoding CNBP or La under control of the tetracycline-responsive expression system (TETOFF) and a target expression vector containing a TOP module between a constitutive P-SV40 promoter and the human model product gene SEAP (human secreted alkaline phosphatase) (P-SV40-TOP-SEAP-pA). The TCS technology showed excellent SEAP regulation profiles in transgenic Chinese hamster ovary (CHO) cells. Alternatively to CNBP and La, TOP-mediated translation control can also be adjusted by artificial phosphorothioate anti-TOP oligodeoxynucleoticles. Confocal laser-scanning microscopy demonstrated cellular uptake of FITC-labeled oligodeoxynucleotides and their localization in perinuclear organelles within 24 hours. Besides their TOP-based translation-control ling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal en try sites (IRES) and La alone to boost cap-dependent translation initiation. CNBP and La exemplify for the first time the potential of RNA-binding proteins to exert translation control of desired transgenes and to increase heterologous protein production in mammalian cells. We expect both of these assets to advance current gene therapy and biopharmaceutical manufacturing strategies. (C) 2002 Wiley Periodicals, Inc.