Artificial Protein-Responsive Riboswitches Upregulate Non-AUG Translation Initiation in Yeast

Abstract

Artificial control of gene expression is one of the core technologies for engineering biological systems. Riboswitches are cis-acting elements on mRNA that regulate gene expression in a ligand-dependent manner often seen in prokaryotes, but rarely in eukaryotes. Because of the poor variety of such elements available in eukaryotic systems, the number of artificially engineered eukaryotic riboswitches, especially of the upregulation type, is still limited. Here, we developed a design principle for upregulation-type riboswitches that utilize non-AUG initiation induced by ribosomal stalling in a ligand-dependent manner in Saccharomyces cerevisiae. Our design principle simply required the proper positioning of a near-cognate start codon relative to the RNA aptamer. Intriguingly, the CUG codon was the most preferable for non-AUG ON switches in terms of output level and switch performance. This work establishes novel choices for artificial genetic control in eukaryotes with versatile potential for industrial and biomedical applications as well as basic research.

Programmable mammalian translational modulators by CRISPR-associated proteins

“…CARTRIDGE, in contrast, does not have such restrictions and therefore can simultaneously employ a variety of Cas proteins, enabling the construction of gene circuits with far greater complexity. Another study also reported a translational activation system with SpCas9 62 , but limited its application to yeast and required extensive optimization of the sgRNA insertion position. In contrast, we validated the properties of many more Cas proteins (25 Cas proteins investigated as translational regulators) in mammalian cells, thus illustrating the potential power of CARTRIDGE and its compatibility with various CRISPR-related technologies.…”

High throughput mutagenesis and screening for yeast engineering

“…RNA-based biosensors employ noncoding mRNA sequences to sense molecules like metabolites or antibiotics [ 93 , 94 ]. Riboswitch sensors are RNA aptamers, with hairpin-like structures that when bound by a ligand, induce a conformational change in the mRNA tertiary structure, thus acting as a means of controlling translation [ 94 ].…”