Rational Design of an Activatable Reporter for Quantitative Imaging of RNA Aberrant Splicing In Vivo

Abstract

Pre-mRNA splicing, the process of removing introns from pre-mRNA and the arrangement of exons to produce mature transcripts, is a crucial step in the expression of most eukaryote genes. However, the splicing kinetics remain poorly characterized in living cells, mainly because current methods cannot provide the dynamic information of splicing events. Here, we developed a genetically encoded bioluminescence reporter for real-time imaging of the pre-mRNA splicing process in living subjects. We showed that the bioluminescence reporter is able to visualize the pre-mRNA aberrant splicing process in living cells in a dose-and time-dependent manner. Moreover, this reporter could provide quantitative and longitudinal information of splicing activity in response to exogenous splicing inhibitors in living animals. Our data suggest that this activatable reporter could serve as a promising tool for the highthroughput screening of splicing modulators, which would facilitate the drug development for human diseases caused by the abnormal splicing of mRNA.

Unleashing the potential of catalytic RNAs to combat mis-spliced transcripts

“…Frontiers in Bioengineering and Biotechnology frontiersin.org bioluminescence reporters are used to screen the IR splicing events while offering real time quantification (Shi et al, 2018;Zheng et al, 2019;Xie et al, 2020). Combining in silico computational methods with in vivo imaging techniques could provide accurate and reliable outcomes to ensure greater impacts in terms of detection and visualization.…”

A ratiometric dual luciferase reporter for quantitative monitoring of pre-mRNA splicing efficiency in vivo

“…Moreover, these methods are relatively time-consuming and do not lend themselves to high throughput screening of factors that affect mammalian splicing. Several reporter systems that encode luciferase (13)(14)(15) J o u r n a l P r e -p r o o f or fluorescence protein (16,17) have also been developed by our group and other researchers to visualize pre-mRNA splicing in living cells and animals. The signals derived from these luciferase enzymatic activities or protein expression are supposed to be proportional to the quantity of mRNAs, which in turn depends on the fraction of the spliced pre-mRNA.…”