Identification of RNA-Binding Protein Targets with HyperTRIBE in Saccharomyces cerevisiae

Autor: Weilan Piao, Chong Li, Pengkun Sun, Miaomiao Yang, Yansong Ding, Wei Song, Yunxiao Jia, Liqun Yu, Yanming Lu, Hua Jin
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 24, Iss 10, p 9033 (2023)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
DOI: 10.3390/ijms24109033
Popis: As a master regulator in cells, RNA-binding protein (RBP) plays critical roles in organismal development, metabolism and various diseases. It regulates gene expression at various levels mostly by specific recognition of target RNA. The traditional CLIP-seq method to detect transcriptome-wide RNA targets of RBP is less efficient in yeast due to the low UV transmissivity of their cell walls. Here, we established an efficient HyperTRIBE (Targets of RNA-binding proteins Identified By Editing) in yeast, by fusing an RBP to the hyper-active catalytic domain of human RNA editing enzyme ADAR2 and expressing the fusion protein in yeast cells. The target transcripts of RBP were marked with new RNA editing events and identified by high-throughput sequencing. We successfully applied HyperTRIBE to identifying the RNA targets of two yeast RBPs, KHD1 and BFR1. The antibody-free HyperTRIBE has competitive advantages including a low background, high sensitivity and reproducibility, as well as a simple library preparation procedure, providing a reliable strategy for RBP target identification in Saccharomyces cerevisiae.
Databáze: Directory of Open Access Journals
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