Genome-wide CRISPR screen for comprehensive identification of human factors involved in alternative polyadenylation based on differential localization of CD47 protein

At least 70% of the human protein-coding genes possess more than one poly(A) site (PAS) and undergo a phenomenon known as alternative polyadenylation (APA), leading to distinct transcripts from a single gene. Previously, it has been demonstrated that shortening or lengthening of mRNA, resulting from the APA, could be observed in physiological and pathological conditions. Nevertheless, factors and cellular mechanisms responsible for the APA are largely unknown. CD47 protein localization was previously demonstrated to be regulated by APA. Here, we showed that core 3′ end processing factors regulate the different CD47 mRNA transcripts. This finding led us to develop an immunofluorescence staining method that simultaneously detects cell surface and intracellular CD47 protein and visualizes altered APA. Using this method, we detected the alterations of CD47 protein localization upon the depletion of the long 3′ untranslated region (UTR) mRNA isoform of CD47 or CFIm25. To systematically elucidate potential novel regulators of APA, we integrated an approach using immunofluorescence staining of cell surface and intracellular CD47 with genome-wide CRISPR screen coupled with pooled single guide RNA (sgRNA) libraries targeting human protein-coding genes. The functional genetic screens and systematic analysis of RNA-seq data revealed POLDIP2 as the potential novel regulator of APA that has global impact on APA regulation. These findings suggest that the study of novel identified APA regulators could lead to a deeper understanding of detailed molecular mechanisms governing APA under physiological and pathological conditions.