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Published January 10, 2024 | Version v1
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Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing

Creators

  • 1. University of Chicago

Description

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 targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site 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 crosslinking 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 RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.

Data availability

All the sequencing data generated in this study have been deposited in the NCBI's Gene Expression Omnibus (GEO) under the accession number GSE226161. Previously published data are available under accession numbers GSE42701 (CLIP-seq), ENCSR384KAN and ENCSR981WKN target="_blank"(eCLIP), E-MTAB-3108 (iCLIP), GSE78832 (irCLIP), GSE137925 (LACE-seq), GSE92995 (sCLIP), DRA005743 (tRIP-seq) and GSE195654 (RT&Tag). The data were downloaded and processed as described in the articles. The processed .bam files of RNA-seq data for knockdown HNRNPC, PTBP1 and RBFOX2, along with their corresponding control data, were downloaded from ENCODE portal under the accession numbers of ENCSR052IYH, ENCSR305XWT, ENCSR767LLP, ENCSR104ABF, ENCSR064DXG and ENCSR603TCV. The published PAR-CLIP data and the corresponding peaks for YTHDF2 are available under the GEO accession number GSE49339. The m6A modification sites identified by m6A-SAC-seq are available under the GEO accession number GSE198246. Source data are provided with this paper.

Codes for processing ARTR-seq data are available in the following GitHub repository: https://github.com/mingming-cgz/ARTR-seq.

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Additional details

Identifiers

DOI
10.1038/s41592-023-02146-w
Other
oai:uchicago.tind.io:10494

Funding

National Institutes of Health
RM1 HG008935

UChicago Information

Division(s)
Physical Sciences Division
Department(s)
Biochemistry and Molecular Biology, Chemistry
Center(s) or Institute(s)
Institute for Biophysical Dynamics