Mutations in the 5' untranslated region fine-tune translational control of heterologously expressed genes

• Published in: Genes & Genetic Systems Vol. 100; p. 24-00188
• Main Authors: Kuse, Riku, Ishii, Kojiro
• Format: Journal Article
• Language: English
• Published: Japan The Genetics Society of Japan 01.01.2025; Japan Science and Technology Agency
• Subjects: 5' Untranslated regions; 5' Untranslated Regions - genetics; 5' UTR mutation; Centromere - genetics; Codon, Initiator - genetics; control of translation efficiency; fission yeast; Gene expression; Gene Expression Regulation, Fungal; Mutation; Promoter Regions, Genetic; Promoters; Protein Biosynthesis; Proteins; Ribosomal subunits; RNA, Messenger - genetics; RNA, Messenger - metabolism; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe Proteins - genetics; Translation initiation; Yeast; fission yeast; control of translation efficiency; 5' UTR mutation; gene expression
• ISSN: 1341-7568; 1880-5779; 1880-5779
• DOI: 10.1266/ggs.24-00188

Strict control of the expression levels of heterologously introduced protein-coding genes is important for the functional analysis of the protein of interest and its effective use in new situations. For this purpose, various promoters with different expression strengths, codon optimization, and expression stimulation by low-molecular-weight compounds are commonly used. However, methods to control protein expression levels by combining regulation of translation efficiency have not been studied in detail. We previously observed relatively high basal expression of Cre when it was heterologously expressed in fission yeast. Here, we used a fission yeast strain that is susceptible to centromere disruption, and thus highly sensitive to Cre levels, and report successful fine-tuning of heterologous Cre expression by modulating the Cre translation efficiency. To inhibit Cre translation initiation, we generated two mutations in the 5' untranslated region of the Cre mRNAs, both of which interfered with the scanning process of start codon recognition, mediated by specialized ribosomal subunits. These mutations successfully reduced the levels of exogenously expressed Cre to different degrees in fission yeast. Combining them with promoters of different strengths allowed us to conduct centromere disruption experiments in fission yeast. Our data indicate that modification of translational control is an additional tool in heterologous gene expression.