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Updated: Jul 4, 2026

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
Published on: February 18, 2022
Selection of mRNA 5'-untranslated region sequence with high translation efficiency through ribosome display
Masayasu Mie1, Shun Shimizu, Fumio Takahashi
1Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-28 4259 Nagatsuta, Midori-ku, Yokohama, Kanagawa 226-8501, Japan.
Abstract:
The 5'-untranslated region (5'-UTR) of mRNAs functions as a translation enhancer, promoting translation efficiency. Many in vitro translation systems exhibit a reduced efficiency in protein translation due to decreased translation initiation. The use of a 5'-UTR sequence with high translation efficiency greatly enhances protein production in these systems. In this study, we have developed an in vitro selection system that favors 5'-UTRs with high translation efficiency using a ribosome display technique. A 5'-UTR random library, comprised of 5'-UTRs tagged with a His-tag and Renilla luciferase (R-luc) fusion, were in vitro translated in rabbit reticulocytes. By limiting the translation period, only mRNAs with high translation efficiency were translated. During translation, mRNA, ribosome and translated R-luc with His-tag formed ternary complexes. They were collected with translated His-tag using Ni-particles. Extracted mRNA from ternary complex was amplified using RT-PCR and sequenced. Finally, 5'-UTR with high translation efficiency was obtained from random 5'-UTR library.
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