Large-scale identification of RBP-RNA interactions by RAPseq refines essentials of post-transcriptional gene regulation
Over the past decade, thousands of putative human RNA binding proteins (RBPs) have been identified and increased the demand for specifying RNA binding capacities. Here, we developed RNA affinity purification followed by sequencing (RAPseq) that enables in vitro large-scale profiling of RBP binding to native RNAs. First, by employing RAPseq, we found that vertebrate HURs recognize a conserved RNA binding motif and bind predominantly to introns in zebrafish compared to 3UTRs in human RNAs. Second, our dual RBP assays (co-RAPseq) uncovered cooperative RNA binding of HUR and PTBP1 within an optimal distance of 27 nucleotides. Third, we developed T7-RAPseq to discern m6A-dependent and - independent RNA binding sites of YTHDF1. Fourth, RAPseq of 26 novel non-canonical RBPs revealed specialized moonlighting interactions. Last, five pathological IGF2BP family variants exhibited different RNA binding patterns. Overall, our simple, scalable and versatile method enables to fast-forward RBP-related questions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/467743v1_ufig1.gif" ALT="Figure 1"> View larger version (106K): org.highwire.dtl.DTLVardef@1f77ce0org.highwire.dtl.DTLVardef@12778fcorg.highwire.dtl.DTLVardef@77ff8corg.highwire.dtl.DTLVardef@2a7f24_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIRAPseq reveals in vitro-derived RBP-RNA interactomes C_LIO_LIthe vertebrate-conserved HUR binding motif adapted to species-unique RNA features C_LIO_LIco-RAPseq and T7-RAPseq uncover binding cooperativity and modification dependencies C_LIO_LInon-canonical RBPs have specialized RNA interactomes C_LI