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

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

Zongyue Zeng
Bo Huang
Shifeng Huang
Ruyi Zhang
Shujuan Yan
Xinyi Yu
Yi Shu
Chen Zhao
Jiayan Lei
Wenwen Zhang
Chao Yang
Ke Wu
Ying Wu
Liping An
Xiaojuan Ji
Cheng Gong
Chengfu Yuan
Linghuan Zhang
Wei Liu
Yixiao Feng
Bo Zhang
Zhengyu Dai
Yi Shen
Xi Wang
Wenping Luo
Rex C. Haydon
Hue H. Luu
Lan Zhou
Russell R. Reid
Tong-Chuan He
Xingye Wu
Genes & Diseases第5卷, 第1期pp.62-74纸质出版 2018-03-01在线发表 2018-02-21
185001

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 transcript reporter to monitor the inhibition of lncRNAs by microRNAs, synthetic regulatory RNA molecules, RNA binding proteins, and/or small molecule inhibitors so that novel and efficacious inhibitors targeting the epigenetic circuit can be discovered to treat human diseases such as cancer and other chronic disorders.

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BiFPGreen fluorescent proteinHigh throughput screeninglncRNANoncoding RNATranscript reporter assay