Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

Kristopher W Brannan,Eric R Kofman,Brian A Yee,Kevin D Dong,Gene W Yeo,Pratibha Jagannatha,Ryan J Marina,Isaac A Chaim,Daniel A Lorenz,Jason G Underwood,Assael A Madrigal

doi:10.1038/s41592-021-01128-0

Kristopher W Brannan, Eric R Kofman + Show 9 more

Open Access

https://doi.org/10.1038/s41592-021-01128-0

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Abstract

RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on UV-crosslinking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP- and cell type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

Highlights

RNA-binding proteins (RBPs) interact with RNA molecules from synthesis to decay to affect their metabolism, localization, stability and translation [1, 2]
Cells expressing low (50ng/ml doxycycline) and higher (1μg/ml doxycycline) levels of RBFOX2-Surveying Targets by APOBEC Mediated Profiling (STAMP) for 72 hours had enriched C-to-U edit clusters on the 3’ untranslated region (3’UTR) of the known RBFOX2 target APP mRNA, and these edit clusters coincided with reproducible RBFOX2 binding sites as detected by enhanced Crosslinking Immunoprecipitation (CLIP) of either endogenous RBFOX2 [33] or the RBFOX2-APOBEC1 fusion (Figure 1B)
RBFOX2-STAMP induced edits within this APP 3’UTR target region were 10-fold to 25-fold more frequent than background controlSTAMP edits at 0.9 and 0.999 (SAILOR) confidence thresholds, respectively (Figure 1C, Table S1, Table S2). These results demonstrate that fusion of the APOBEC1 module to a well characterized RBP enriches for target-specific edits

Summary

Introduction

RNA-binding proteins (RBPs) interact with RNA molecules from synthesis to decay to affect their metabolism, localization, stability and translation [1, 2]. The eukaryotic ribosome is itself composed of a collection of RBPs that can interact directly with mRNA coding sequences [3, 8]. Ribosome profiling methods such as ribo-seq have become a mainstay in the evaluation of transcriptome-scale ribosome occupancy [9, 10]. While there has been rapid progress in single-cell measurements of chromatin accessibility [13], gene expression [14, 15], and surface protein-levels [16, 17], there is currently no available technology for measuring RBP- and ribosome-mRNA interactions at single-cell or isoform-aware resolution

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