Global PUS1 mRNA targets detection by STAMP

Recently, several studies revealed that pseudourine exists on mRNA. However, identification of ψ 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. 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/).