R01HG011864

Stamp Technology to Enable Single-Cell and Isoform-Sensitive Detection of RBP Sites - 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 states.

Despite their association with disease and the importance of regulating gene expression, 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, and 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, the 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 the single-cell level and at scale, opening up new paradigms of biological questions.

San Diego University Of California

NOT APPLICABLE

93.172 - Human Genome Research

National Human Genome Research Institute (NHGRI) [HHS - NIH]

La Jolla, California 920930695 United States

Single Zip Code

Novel Genomic Technology Development (R01 Clinical Trial Not Allowed) (PAR-18-777)

Amendment Since initial award the total obligations have increased 298% from $995,400 to $3,961,692.