3'READS+RIP defines differential Staufen1 binding to alternative 3'UTR isoforms and reveals structures and sequence motifs influencing binding and polysome association

Using 3ʹ region extraction and deep sequencing coupled to ribonucleoprotein immunoprecipitation (3’READS+RIP), together with reanalyzing previous STAU1 binding and RNA structure data, we delineate STAU1 interactions transcriptome-wide, including binding differences between alternative polyadenylation (APA) isoforms. Consistent with previous reports, RNA structures are dominant features for STAU1 binding to CDSs and 3ʹUTRs. Overall, relative to short 3ʹUTR counterparts, longer 3ʹUTR isoforms of genes have stronger STAU1 binding, most likely due to a higher frequency of RNA structures, including specialized IRAlus. However, a sizable fraction of genes express transcripts showing the opposite trend, attributable to AU-rich sequences in their alternative 3'UTRs, possibly recruiting antagonistic RBPs and/or destabilizing STAU1-binding RNA structures. Using STAU1-knockout cells, we show that strong STAU1 binding to mRNA 3'UTRs generally enhances polysome association. However, IRAlus have little impact on STAU1-mediated polysome association despite having strong interactions with the protein. 8 3'READS+RIP libraries from STAU1-KO cells (STAU1_IP, GFP_IP, STAU1_Input, GFP_Input in two replicates); 4 3'READS+ libraries from cell frationated samples (KO_C, WT_C, KO_P, WT_P in one replicate)