The development of a sensitive fluorescent protein-based transcript reporter for high throughput screening of negative modulators of lncRNAs

Genes & Diseases
Zongyue ZengXingye Wu

Abstract

While the human genome is pervasively transcribed, <2% of the human genome is transcribed into protein-coding mRNAs, leaving most of the transcripts as noncoding RNAs, such as microRNAs and long-noncoding RNAs (lncRNAs), which are critical components of epigenetic regulation. lncRNAs are emerging as critical regulators of gene expression and genomic stability. However, it remains largely unknown about how lncRNAs are regulated. Here, we develop a highly sensitive and dynamic reporter that allows us to identify and/or monitor negative modulators of lncRNA transcript levels in a high throughput fashion. Specifically, we engineer a fluorescent fusion protein by fusing three copies of the PEST destruction domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of the codon-optimized bilirubin-inducible fluorescent protein, designated as dBiFP, and show that the dBiFP protein is highly destabilized, compared with the commonly-used eGFP protein. We further demonstrate that the dBiFP signal is effectively down-regulated when the dBiFP and mouse lncRNA H19 chimeric transcript is silenced by mouse H19-specific siRNAs. Therefore, our results strongly suggest that the dBiFP fusion protein may serve as a sensitive and dynamic ...Continue Reading

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Citations

Feb 13, 2021·NAR Genomics and Bioinformatics·Saba NafeesCatherine A Wakeman
Aug 12, 2021·Laboratory Investigation; a Journal of Technical Methods and Pathology·Xi WangZongyue Zeng

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Methods Mentioned

BETA
RNA-seq
PCR
Assay
transfection
bioluminescence imaging
fluorescence imaging

Software Mentioned

Genescript
Image J

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