Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq

Abstract

RNA-binding proteins (RBPs) and noncoding RNAs orchestrate post-transcriptional processes through the recognition of specific sites on targeted transcripts. Thus, understanding the connection between binding to specific sites and active regulation of the whole transcript is essential. Many immunoprecipitation techniques have been developed that identify either whole transcripts or binding sites of RBPs on each transcript using cell lysates. However, none of these methods simultaneously measures the strength of each binding site and quantifies binding to whole transcripts. In this study, we compare current procedures and present digestion optimized (DO)-RIP-seq, a simple method that locates and quantifies RBP binding sites using a continuous metric. We have used the RBP HuR/ELAVL1 to demonstrate that DO-RIP-seq can quantify HuR binding sites with high coverage across the entire human transcriptome, thereby generating metrics of relative RNA binding strength. We demonstrate that this quantitative enrichment of binding sites is proportional to the relative in vitro binding strength for these sites. In addition, we used DO-RIP-seq to quantify and compare HuR's binding to whole transcripts, thus allowing for seamless integration of binding site data with whole-transcript measurements. Finally, we demonstrate that DO-RIP-seq is useful for identifying functional mRNA target sets and binding sites where combinatorial interactions between HuR and AGOmicroRNAs regulate the fate of the transcripts. Our data indicate that DO-RIP-seq will be useful for quantifying RBP binding events that regulate dynamic biological processes.

DO-RIP-seq to quantify RNA binding sites transcriptome-wide

“…This step is followed by immunoprecipitations with antibodies against the RBP of interest, while using non-specific antibodies in parallel to measure background and account for overall transcript abundance. As described here, DO-RIP-seq was developed using the RBP ELAVL1/HuR as a test case, and the experiments yielded probabilistic measures (log of odds scores, LOD) for binding sites in HEK293 cells [25], and for XIST long non-coding RNA in mouse trophoblasts [26]. HuR binding site LOD scores correlated with binding strength and motif preference [25].…”

“…As described here, DO-RIP-seq was developed using the RBP ELAVL1/HuR as a test case, and the experiments yielded probabilistic measures (log of odds scores, LOD) for binding sites in HEK293 cells [25], and for XIST long non-coding RNA in mouse trophoblasts [26]. HuR binding site LOD scores correlated with binding strength and motif preference [25]. Also DO-RIP-seq analysis of whole transcript association distinguished functional groups of messages and enriched gene sets [25].…”

“…HuR binding site LOD scores correlated with binding strength and motif preference [25]. Also DO-RIP-seq analysis of whole transcript association distinguished functional groups of messages and enriched gene sets [25]. In addition, we have used DORIP-seq to successfully identify the binding sites of other RBPs, e.g.…”

“…Here we describe d igestion o ptimized R NA i mmuno p recipitation with deep seq uencing (DO-RIP-seq) as a method that quantitates at the whole transcript target ( R IP- S eq- L ike or RSL) level and at the binding site level (BSL) using continuous metrics. DO-RIP-seq methodology was developed using the RBP HuR/ELAVL1 as a test case (Nicholson et al 2016). DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions.…”

“…DO-RIP-seq employs treatment of cell lysates with a nuclease under optimized conditions to yield partially digested RNA fragments bound by RNA binding proteins, followed by immunoprecipitations that capture the digested RNA-protein complexes and assess non-specific or background interactions. Analyses of sequenced cDNA libraries made from the bound RNA fragments yielded two types of enrichment scores; one for RSL binding events and the other for BSL events (Nicholson et al 2016). These analyses plus the extensive read coverage of DO-RIP-seq allows seamless integration of binding site and whole transcript information.…”