STAMP technology to enable single-cell and isoform-sensitive detection of RBP sites

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PROJECT SUMMARY RNA-binding proteins (RBPs) interact with RNA molecules from synthesis to decay to control their metabolism, subcellular localization, stability and translation. Methods for transcriptome-wide detection of RBP-RNA interactions provide insights into how RBPs regulate gene expression programs and how RNA processing is disrupted in disease state. Despite their association with disease and although the importance of regulating gene expression is well appreciated, only a small fraction of the over 2,000 RBPs identified thus far have known RNA targets and molecular roles. Commonly, immunoprecipitation-based technologies coupled to high throughput (Illumina) sequencing, such as RNA immunoprecipitation (RIP) and Crosslinking Immunoprecipitation (CLIP), and ribosome profiling are used to identify RBP targets and binding sites across the transcriptome. However, these experimental protocols are labor-intensive, require large amounts of input material, are not adaptable to high-throughput workflows. To overcome these limitations, we develop a novel technology, reagent resource, experimental protocols and a computational framework, that we collectively term STAMP (Surveying Targets By APOBEC-Mediated Profiling), for detecting RBP-RNA targets and translation at the single-cell and single- molecule level. In preliminary data we demonstrate, for the first time in the field, discovery of RBP-RNA sites and translation states at single-cell resolution. We anticipate that STAMP can be used reliably to identify RNA targets, binding sites and even extract motifs from a few cells to a single cell, thus effectively increasing limits of detection over current methods by several orders of magnitude. Combined with simultaneous RNA-seq analyses, STAMP will enable the combined identification of RBP binding sites and global measurement of gene expression, a long- standing goal for the gene expression, genomics and RNA communities. As a corollary, even without single cell analyses, STAMP can accept ultra-low input material which enables rare cell-types to be collected and analyzed for RBP-interactomes. By applying STAMP to ribosomal proteins, we extend this approach for single-cell detection of ribosome association while simultaneously measuring gene expression. Our conceptual and technological innovations will, for the first time, enable translation efficiency and RBP-interactomes to be measured at single-cell level and at scale, opening up new paradigms of biological questions.