Identification of bona-fide targets of PUM by using RIP-seq and BRIC-seq. Homo sapiens

We report transcripts of which stabilities are regulated by human Pumilio proteins (PUM). PUM is an RNA-binding protein that binds specifically to 5'-UGUAHAUA-3' sequence and is a degradation factor of cytoplasmic mRNA by recruiting CCR4-NOT deadenylase. Although many RIP-seq, RIP-chip, CLIP-seq identified mRNAs bound to PUM, we still do not know which mRNAs are degraded by PUM. Here we identify bona-fide target mRNAs of PUM in a human cell line. Bona-fide targets of the RBP are defined by two criteria. One criterion is mRNAs bound to the PUM, which are determined by RNA immunoprecipitation sequencing (RIP-seq). The other criterion is mRNAs degraded by the PUM, which are determined by 5'-bromo-uridine (BrU) immunoprecipitation chase-deep sequencing analysis (BRIC-seq). BRIC-seq enables the determination of genome-wide RNA stability by chasing chronological decreases of BrU-labeled RNAs under physiologically non-disturbed condition. An RNA half-life of each transcript is calculated from the decreasing BrU-labeled RNA. By comparing the RNA half-lives between in normal cells and in the PUM KD cells, we can identify mRNA of which stability is regulated by the PUM. Merging the results of RIP-seq and BRIC-seq will yield the bona-fide targets of the PUM.