Novel CNBP- and La-based translation control systems for mammalian cells.

Abstract

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.