Global PUS1 mRNA targets detection by STAMP

Recently, several studies revealed that pseudourine exists on mRNA. However, identification of ? sites for specific PUS remains a certain ambiguity. PUS1, an important pseudouridine synthase, plays a critical role in cell growth and tumorigenesis of HCC. Here, we utilized an IP-free approach, Surveying Targets by APOBEC-Mediated Profiling (STAMP) and analyzed C-to-U editing sites to realize PUS1-bound mRNAs. And then, we identified the potential mRNA pseudouridylation targets of PUS1 by intersecting editing sites' host genes with these that known to pseudouridylated. Overall design: To delineate the landscape of mRNA targets of PUS1, we utilized an IP-free approach, STAMP, wherein APOBEC1 was fused to PUS1 and facilitated C-to-U editing of PUS1-bound mRNAs. Subsequently, we performed RNA-seq to analyze the whole transcriptome, followed by detection of C-to-U editing. The editing sites only emerged in PUS1-APOBEC1 overexpressed Hep3B cells were considered for further analysis. Since pseudouridylated mRNAs have been extensively identified by high-throughput sequencing methods, to further reduce false-positive, we intersected editing sites' host genes with these that known to pseudouridylated using StarBase V2.0 (https://starbase.sysu.edu.cn/starbase2/).