Integrative analysis of RNA binding proteins identifies DDX55 as a novel regulator of 3’UTR isoform diversity

The 3’ untranslated regions (3’UTRs) of mRNAs play a critical role in controlling gene expression and function because they contain binding sites for microRNAs and RNA binding proteins (RBPs) that alter mRNA stability, localization, and translation. Most mRNA 3’ ends contain multiple polyadenylation sites (PAS) that can be utilized in condition-specific manners, a process known as alternative polyadenylation (APA), however the mechanisms driving the regulation of APA remain poorly characterized. By integrating a large set of over 500 RNA binding protein (RBP) depletion and binding experiments across two cell lines generated by the ENCODE consortium, we uncovered a number of RBPs in each cell type whose depletion leads to widespread alteration of 3’ UTR patterns. These include not only known regulators of APA, but also many putative novel regulators of 3’UTR isoform expression. We focused analysis on the largely unstudied DEAD box RNA helicase, DDX55, and validate its novel role in 3’UTR isoform regulation using molecular assays and targeted 3’ end sequencing experiments (QuantSeq REV V2, Lexogen). Our findings identify DDX55 as a new regulator of APA, particularly at PAS that contain features of RNA secondary structure. Our data also suggest additional previously unrecognized regulators of 3’ UTR processing and differential stability.