Modulation of translation-initiation in CHO-K1 cells by rapamycin-induced heterodimerization of engineered eIF4G fusion proteins.
Translation-initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine-tunes the flow of information from gene to protein. In eukaryotes, translation-initiation is typically initiated at a 7-methyl-guanylic acid cap posttranscriptionally linked to the 5' end of mRNAs. Alternative cap-independent translation-initiation involves 5' untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation-initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G(Delta) and (Delta)eIF4G to redirect the cellular translation machinery exclusively to its own IRES-containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap- and IRES-dependent transgene mRNAs in mammalian cells. eIF4G(Delta) and (Delta)eIF4G were fused C- and N-terminally to the FK506-binding protein (FKBP) and the FKBP-rapamycin-binding domain (FRB) of the human FKBP-rapamycin-associated protein (FRAP), respectively. Rapamycin-induced heterodimerization of eIF4G(Delta)-FKBP and FRB-(Delta)eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose-dependent manner. Rigorous quantitative expression analysis of cap- and IRES-dependent SEAP- (human placental secreted alkaline phosphatase) and luc- (Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization-induced heterologous eIF4G in Chinese hamster ovary (CHO-K1) cells.
Schlatter
Stefan
S
Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, CH-8093 Zurich, Switzerland.
Senn
Claudia
C
Fussenegger
Martin
M
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D019281:Dimerization; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology*; D014162:Transfection
D017931:DNA Primers; D039603:Eukaryotic Initiation Factor-4G; D011993:Recombinant Fusion Proteins; D020123:Sirolimus