Schlatter Stefan, Fussenegger Martin
Institute of Biotechnology, Swiss Federal Institute of Technolog, ETH Zurich, Switzerland.
Biotechnol Bioeng. 2003 Jan 5;81(1):1-12. doi: 10.1002/bit.10549.
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 (TET(OFF)) 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 oligodeoxynucleotides. 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-controlling capacity, CNBP and La were also shown to increase cap-independent translation from polioviral internal ribosomal entry 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.
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控。翻译控制由存在于rp编码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，可阻止核糖体结合以及rp编码mRNA的翻译起始。然而，细胞核酸结合蛋白（CNBP）或La蛋白与TOP发夹结构的结合消除了TOP介导的转录阻滞并诱导rp产生。基于特定的CNBP-TOP/La-TOP相互作用，我们设计了一种翻译控制系统（TCS），用于在哺乳动物细胞中有条件地以及可调节地翻译所需的转基因mRNA。通用的TCS配置由一个在四环素响应表达系统（TET（OFF））控制下编码CNBP或La的质粒，以及一个靶标表达载体组成，该靶标表达载体在组成型P（SV40）启动子和人模型产物基因SEAP（人分泌碱性磷酸酶）（P（SV40）-TOP-SEAP-pA）之间包含一个TOP模块。TCS技术在转基因中国仓鼠卵巢（CHO）细胞中显示出出色的SEAP调控谱。除了CNBP和La之外，TOP介导的翻译控制也可以通过人工硫代磷酸酯抗TOP寡脱氧核苷酸进行调节。共聚焦激光扫描显微镜显示，FITC标记的寡脱氧核苷酸在24小时内被细胞摄取并定位在核周细胞器中。除了基于TOP的翻译控制能力外，CNBP和La还被证明可增加来自脊髓灰质炎病毒内部核糖体进入位点（IRES）的不依赖帽的翻译，并且单独的La可促进依赖帽的翻译起始。CNBP和La首次例证了RNA结合蛋白在对所需转基因进行翻译控制以及增加哺乳动物细胞中异源蛋白产生方面的潜力。我们期望这两项优势能够推动当前的基因治疗和生物制药制造策略。
