A flexible, high-throughput system for studying live mRNA translation with HiBiT technology

TL;DR

This paper introduces a new method using HiBiT technology to study how mRNA is translated into proteins in real time, both in cells and in lab settings.

Contribution

The study adapts HiBiT technology to monitor live mRNA translation, enabling tailored screening of mRNA features for specific proteins.

Findings

HiBiT technology can detect differences in mRNA features like cap, 5′UTR, and poly(A) length affecting translation.

Optimal mRNA composition varies depending on the target protein, emphasizing the need for protein-specific screening.

The system allows real-time monitoring of mRNA translation in live cells and in vitro.

Abstract

HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present, HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here, we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap, 5′UTR, modified nucleotide composition, coding sequence optimization and poly(A) length, and their effects on mRNA translation over time. Importantly, using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest, highlighting the importance of screening methods tailored to the protein of interest, and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics.

Graphical Abstract

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