Identification of mRNA targets of PUM-mediated mRNA decay

human Pumilio (PUM) is an RNA-binding protein and plays a pivotal role in gene expression by modulating the stability of transcripts, however, the identification of degradation targets of PUMs remains difficult. We defined degradation targets of PUMs by two criteria. One criterion is the mRNA binds to PUMs, which we determined by RNA immunoprecipitation sequencing (RIP-seq). The other criterion is the mRNA stability is decreased by PUMs, which we determined by bromo-uridine (BrU) immunoprecipitation chase sequencing (BRIC-seq). BRIC-seq enables the determination of genome-wide RNA stability by monitoring the decrease in BrU-labeled RNA abundance over time from which the RNA half-life of each transcript is calculated. By comparing the half-lives of transcripts in control cells with the half-lives of those in PUM-depleted cells, we can identify mRNAs of which stability is regulated by PUMs. Combining the results of the RIP-seq and BRIC-seq analyses yields the stability-regulated mRNAs by PUMs.