RNA Affinity Purification followed by sequencing (RAP-seq) allows identification of transcriptome-wide RBP binding sites using recombinant RBPs and purified total cellular RNA

RAPseq is a new in vitro method used to study RNA-protein interactions at scale. In RAPseq, a recombinant RBP is produced as fusion with a HaloTag which is used to recover and purify the RBP of interest. The RBP-Halo fusion is then incubated with fragmented native total RNA that retains endogenous sequence and modification features. Total RNA is derived from any given sample of interest. The bound RNA fragments are subsequently eluted and cloned using a small RNA library preparation protocol for sequencing the pool of bound molecules on an Illumina NGS platform. In this study, RAPseq was used to identify RNA interactomes of 26 novel RBPs (non-canonical RBPs) discovered through proteome-wide screens. RAPseq was applied to profile six vertebrate HUR orthologs, revealing both conserved and divergent binding preferences. Additionally, RAPseq was used to compare wild type and cancer-associated variants of IGF2BP1, IGF2BP2 and IGF2BP3 to uncover transcriptome-wide alterations in their RNA binding patterns. Furthermore, co-RAPseq enabled a transcriptome-wide assessment of cooperative RNA binding between HUR and PTBP1, providing insight into their combinatorial regulatory mechanisms. Lastly, we developed mod-RAPseq, using T7 RNA polymerase to in vitro transcribe RNA from cDNA without endogenous RNA modifications. By comparing binding to this modification-depleted RNA versus native RNA, we identified m6A- and m5C-dependent binding or YTHDF1 and YBX1, respectively.