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

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

Content Provider	Europe PMC
Author	Guo, Bin Shi, Xiaorui Ma, Zhe Ji, Moxuan Tang, Chu Wang, Fu
Copyright Year	2021
Abstract	Precursor messenger RNA (pre-mRNA) splicing is critical for cell growth and development, and errors in RNA splicing frequently cause cellular dysfunction, abnormal gene expression, and a variety of human diseases. However, there is currently a lack of reliable systems to noninvasively monitor the mRNA splicing efficiency in cells and animals. Here, we described the design of a genetically engineered ratiometric dual luciferase reporter to continuously quantify the changes in mRNA splice variants in vivo. This reporter system is encoded within a single polypeptide but on separate exons, thus generating two distinct luciferase signals derived from spliced and unspliced mRNAs. With this reporter, the two kinds of luciferase in the same individual can minimize the influence of indirect factors on splicing, and the ratio of these two luciferase intensities represents the dynamic splicing efficiency of pre-mRNA. Our study offers a convenient and robust tool for the screening and identification of small molecules or trans-acting factors that affect the efficiency of specific splicing reactions.
ISSN	00219258
Volume Number	297
PubMed Central reference number	PMC8322121
Issue Number	2
PubMed reference number	34216622
Journal	The Journal of Biological Chemistry [J. Biol. Chem]
e-ISSN	1083351X
DOI	10.1016/j.jbc.2021.100933
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2021-07-01
Access Restriction	Open
Rights License	This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). © 2021 The Authors
Subject Keyword	pre-mRNA splicing luciferase reporter in vivo imaging CLB, cell lysis buffer FBS, fetal bovine serum Fluc, firefly luciferase pre-mRNA, precursor messenger RNA Rluc, renilla luciferase ROI, region of interest RT-PCR, reverse transcription–polymerase chain reaction snRNAs, small molecules of nuclear RNA
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry

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