Profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA substrates are still lacking, especially when the interaction is dynamic or materials are limited. Here we present an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq), which relies on in-situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet cross-linking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic binding of RBPs over a short period of time, as demonstrated by the discovery of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 min. Overall design: We first developed an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq) to identify PTBP1 binding sites in HepG2 cells. To validate the PTBP1 binding sites identified by ARTR-seq, we performed ARTR-seq for PTBP1 knocked down HepG2 cells. To determine the efficiency of ARTR-seq for small cell number inputs, we constructed ARTR-seq libraries using different numbers of HepG2. We next constructed ARTR-seq libraries to identify the binding sites for several RBPs, including RBFOX2 and HNRNPC in HepG2 cells, and YTHDF1, YTHDF2 and YTHDC1 in HeLa cells. We then generated ARTR-seq libraries in a section of OCT-embedded E11 mouse embryos to identify the RBFOX2 binding sites. To monitor the dynamic binding of G3BP1 during the stress granules assembly, we performed ARTR-seq for the HeLa cells treated with sodium arsenite for various time intervals.