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 ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific 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.
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Raw and assembled sequencing data from this study have been deposited in NCBI’s Gene Expression Omnibus (GEO) under accession code GSE155729. Processed edit coordinates are available in Supplementary Tables 1, 2 and 4. Differential edit and gene expression data are available in Supplementary Tables 3 and 5–9. Published ribosome profiling data used in this study are deposited in the GEO under accession code GSE94460 and polysome sequencing data are deposited in the GEO under accession code GSE109423.
Source code and analysis scripts for edit quantification are available as Supplementary Software. Updated versions can be found at https://github.com/YeoLab/sailor/ and https://github.com/YeoLab/Yeo_STAMP_Nature_Methods/.
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We thank G. Viero at the Institute of Biophysics, CNR Unit at Trento for helpful advice concerning analysis of Ribo-seq data. We thank T. Michael at the J. Craig Venter Institute for use of the Oxford Nanopore PromethION system. We are grateful to K. D. Meyer for the gift of the YTH-APOBEC1 construct. We thank J. Rothstein at Johns Hopkins University School of Medicine for the gift of the NPC cell line. We are grateful to the La Jolla Institute’s Immunology Sequencing Core and the IGM Genomics Center, University of California San Diego, for use of the 10x Chromium and Illumina sequencing platforms. This work was partially supported by the National Institutes of Health (NIH; HG004659 and HG009889 to G.W.Y.). I.A.C. is a San Diego IRACDA Fellow supported by an NIH/NIGMS award (K12GM068524). R.J.M. was supported in part by an institutional award to the UCSD Genetics Training Program from the National Institute for General Medical Sciences, (T32GM008666) and a Ruth L. Kirschstein National Research Service award (F31NS111859). K.W.B is a University of California President’s Postdoctoral Fellow supported by an NIH/NINDS Career Transition Award (K22NS112678).
G.W.Y. is a cofounder, a member of the Board of Directors, on the Scientific Advisory Board, an equity holder and a paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interests have been reviewed and approved by the University of California San Diego, in accordance with its conflict-of-interest policies. The authors declare no other competing interests.
Peer review information Nature Methods thanks Alfredo Castello and the other, anonymous, reviewers for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Irreproducible Discovery Rate (IDR) analysis comparing ≥ 0.5 confidence edit windows for increasing levels of RBFOX2-STAMP at 24, 48 and 72 hours. b, Differential expression (DEseq2) analysis of RBFOX2-STAMP for increasing levels of RBFOX2-STAMP at 72 hours. c, Fraction of RBFOX2-APOBEC1 eCLIP peaks overlapping low and high induction RBFOX2-STAMP edit sites at increasing expression (TPM) thresholds. d, STAMP edit-site filtering and cluster-calling workflow. e, Number of control- and RBFOX2-STAMP edit sites and clusters retained after each filtering step in D. f, Cumulative distance measurement from RBFOX2-STAMP distal edit-clusters to eCLIP peaks on targets genes. g, Pie chart showing the proportion of N-terminally fused RBFOX2-APOBEC1 STAMP edit-clusters overlapping with either 1) RBFOX2-APOBEC1 N-terminal fusion high-confidence eCLIP peaks (l2fc>2 and l10p>3 over input) containing the conserved RBFOX2 binding motif (GCAUG), 2) equally stringent eCLIP peaks not containing the conserved motif, 3) the conserved motif falling outside of eCLIP peaks, or 4) neither eCLIP peaks nor conserved motifs. h, Quantification of expression from no dox (0ng/ml) low (50ng/ml) or high (1µg/ml) doxycycline induction of SLBP-APOBEC1 and TIA1-APOBEC1 fusions compared to endogenous expression. i, Irreproducible Discovery Rate (IDR) analysis comparing 0.5 ≥ confidence level edit windows for increasing levels of TIA1-STAMP at 72 hours. j, Fraction of SLBP eCLIP peaks (log2fc>2 and -log10p>3 over size-matched input, reproducible by IDR) with SLBP-STAMP edit-clusters, compared to size-matched shuffled regions, calculated at different edit site confidence levels before and after site filtering (see Materials and Methods for filtering procedure). Numbers atop bars are Z-scores computed comparing observed with the distribution from random shuffles. *** denotes statistical significance at p = 0, one-sided exact permutation test. k, Fraction of TIA1-APOBEC1 eCLIP peaks (log2fc>2 and -log10p>3 over size-matched input) with TIA1-STAMP edit-clusters, compared to size-matched shuffled regions, calculated at different edit site confidence levels before and after site filtering (see Materials and Methods for filtering procedure). Numbers atop bars are Z-scores computed comparing observed with the distribution from random shuffles. *** denotes statistical significance at p = 0, one-sided exact permutation test. l, Motif enrichment using HOMER and shuffled background on TIA1-STAMP edit-clusters.
Extended Data Fig. 2 Ribo-STAMP reproducibility and response to mTOR pathway perturbations, related to Figure 2.
a, Quantification of expression from no dox (0ng/ml) low (50ng/ml) or high (1µg/ml) doxycycline induction of RPS2-APOBEC1 fusion compared to endogenous expression. b–d, Scatterplot comparisons of CDS+3′UTR EPKM values from RPS2-STAMP replicate experiments showing high, dose-dependent correlation at 24 (B), 48 (C) and 72 hours (D). e, Scatterplot comparison of CDS EPKM values with CDS+3′UTR EPKM values for RPS2-STAMP. f, Pearson R2 values for low and high induction control- or RPS2-STAMP EPKM compared to poly-ribosome-enriched polysome-seq RPKM. g, Comparison of EPKM from vehicle treated 72-hour high-induction control-STAMP compared to Torin-1 treated 72-hour high-induction control-STAMP showing no significant signal reduction for top ribosome occupied quartile genes containing Torin-1 sensitive TOP genes as detected by ribo-seq (Q1 p = 1.0, n = 3589 genes, Wilcoxon rank-sum one-sided) and polysome profiling (Q1 p = 1.0, n = 3589 genes, Wilcoxon rank-sum one-sided). h, Scatterplot comparison of CDS+3′UTR EPKM values on ribo-seq top quartile genes (n = 3589) for Torin-1 treated and vehicle treated RPS2-STAMP 72-hour high (1µg/ml) doxycycline inductions as in Figure 2H. i, Scatterplot comparison of CDS+3′UTR RPKM values on ribo-seq quartile-1 genes (n = 3589) for Torin-1 treated and vehicle treated RPS2-STAMP 72-hour high (1µg/ml) doxycycline inductions.
Extended Data Fig. 3 Long-read STAMP reveals isoform specific binding profiles, related to Figure 3.
a, Heatmap of control- and RBFOX2-STAMP edit fractions calculated from the final exon of all detected primary and secondary alternative polyadenylation (APA) isoforms meeting coverage criteria (see materials and methods). b, IGV tracks showing RBFOX2-APOBEC1 eCLIP peaks, control- and RBFOX2-STAMP short-read edit clusters, compared to control- and RBFOX2-STAMP long-read (PB) alignments on long, middle and short APA isoforms of the target gene PIGN, with green colored C-to-U conversions on different isoforms.
a, Overlap between single-cell and bulk RBFOX2-STAMP target genes containing edit-clusters. b, Fraction of RBFOX2-APOBEC1 eCLIP peaks overlapping low and high induction single-cell RBFOX2-STAMP edit-clusters at increasing expression (TPM) thresholds.
Extended Data Fig. 5 Single-cell RBP-RNA interaction detection by STAMP for multiple RBPs and in multiple cell types, related to figure 5.
a, UMAP plot using ε score from RBFOX2-STAMP and TIA1-STAMP mixture with capture sequence RBFOX2-STAMP (blue, n = 844) and TIA1-STAMP cells (red, n = 527) highlighted. b, UMAP plot as in A color-coded by Louvain clustering into RBFOX2-cluster (blue), and TIA1-cluster (red), or background-cluster (gray) populations. c, UMAP plot of gene expression for ε score Louvain clusters defined in B. d, Motif enrichment using HOMER from ≥ 0.99 confidence edits from combined RBFOX2-cluster and control-STAMP cells. e, UMAP plot showing expression of neural precursor cell markers NES, PAX6, SOX2 and DCX. f, Motif enrichment using HOMER from ≥ 0.99 confidence edits from combined control- and RBFOX2-STAMP HEK293T and NPC cells.
Extended Data Fig. 6 Single Ribo-STAMP detects ribosome occupancy from individual cells, related to Figure 6.
a, Genome-wide comparison of CDS+3′UTR EPKM values for bulk and single-cell EPKM-derived RPS2-population. b, Comparison of EPKM-derived RPS2-population CDS and CDS+3′UTR EPKM values. c, Comparison of EPKM-derived RPS2-population total mRNA RPKM values with total mRNA RPKM values from polysome-seq input. d, Comparison of EPKM-derived RPS2-population CDS+3′UTR EPKM values with total mRNA RPKM values from polysome-seq input. e, UMAP analysis of ε score from merged 72-hour high-induction RPS2-STAMP (green), control-STAMP (orange) and mixed-cell RBFOX2:TIA1-STAMP (purple) single-cell experiments. f, UMAP plot as in E with only capture sequence RBFOX2-STAMP (blue, n = 844) and TIA1-STAMP cells (red, n = 527) highlighted. d, Individual cell barcode overlap for EPKM-derived and ε score-derived RPS2-populations.
All control–STAMP edit site coordinates and levels; related to Figs. 1 and 2 and Extended Data Figs. 1 and 2.
All RBP–STAMP (RBFOX2, SLBP and TIA1) edit site coordinates and levels; related to Fig. 1 and Extended Data Fig. 1.
Differential gene expression from RBFOX2–STAMP as evaluated by DEseq2; related to Extended Data Fig. 1.
All Ribo-STAMP (RPS2 and RPS3) edit site coordinates and levels; related to Fig. 2 and Extended Data Fig. 2.
All control–STAMP and RBFOX2–STAMP edit percentages on final exons of primary and secondary APA isoforms from PB long-read sequencing; related to Fig. 3 and Extended Data Fig. 3.
Differential gene editing between background population and RBFOX2 and TIA1 populations; related to Fig. 5.
Differential gene expression between HEK293T cells and NPCs; related to Fig. 5 and Extended Data Fig. 5.
Differential gene editing between HEK293T cells and NPCs for RBFOX2–STAMP; related to Fig. 5.
Differential gene editing between background population and RPS2, RBFOX2 and TIA1 populations; related to Fig. 6.
Oligonucleotides used in this study.
Source code and analysis scripts for edit quantification.
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Brannan, K.W., Chaim, I.A., Marina, R.J. et al. Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes. Nat Methods 18, 507–519 (2021). https://doi.org/10.1038/s41592-021-01128-0
Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing
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