Effective non-viral leader for cap-independent translation in a eukaryotic cell-free system

Abstract

The 61 nt 5'-untranslated region (5'-UTR) of mRNA encoding for a light-emitting protein of hydroid polyp Obelia longissima, obelin, is shown to provide a high level of cap-independent translation of heterologous mRNAs in cell-free translation systems based on wheat germ extracts. The inhibition of translation typically observed when excess mRNA is present or produced in a eukaryotic system (the so-called self-inhibition phenomenon) is found abated with mRNA constructs carrying the obelin mRNA leader. The role of the sequestration of a limiting initiation factor, probably eIF4F, in the self-inhibition phenomenon and the possible independence of the obelin mRNA leader from eIF4F are discussed. We propose the obelin mRNA leader be used for effective cap-independent translation in eukaryotic cell-free systems, including combined transcription-translation systems with uncontrolled phage polymerase-catalyzed accumulation of mRNA.