ENCODE

Characterization of the largest number of human RBPs to date

A Tool for Studying RBP-RNA Interactomes

Despite the growing numbers of candidate RBPs and the recognition that disrupted RBP-RNA interactions underlie many human diseases, a systematic understanding of which RBPs interact with what RNA(s) to control cellular homeostasis is lacking. In the previous period of review, studies by our lab (Brannan et al., Molecular Cell 2016) and others predicted that the human genome contained far more RBPs (over 2,500) than ever imagined. We developed enhanced cross-linking immunoprecipitation (eCLIP) featuring 1,000-fold higher library yields and drastically improved signal-to-noise in identifying true RBP binding sites (Van Nostrand et al., Nature Methods 2016). Excitingly, its high robustness, reproducibility and ease of use has led to rapid adoption in the field (cited >720 times). We since demonstrated the utility of eCLIP for its use with RBPs expressed as epitope-tagged proteins from endogenous human loci (REF). We also characterized commonly used reverse transcriptase enzymes with respect to their propensity to terminate at or near cross-linked nucleotides — important for identifying RBP binding sites at single-nucleotide resolution (REF). In 2022, we published an updated version of our original eCLIP protocol that enables Illumina sequencing to be performed in single-end mode.

As part of the NIH-funded ENCODE efforts, our lab also screened more than 700 commercially available antibodies against >500 RBPs for their ability to selectively immunoprecipitate the target protein. We found antibodies that are effective against >250 RBPs where all this information is publicly available to the community. With these antibodies, my lab is in the process of generating a large RBP-RNA interactome dataset in the world to-date, consisting of 250 RBPs in two human cell-lines. Our improvements in the cross-linking and immunoprecipitation (CLIP) protocol have enabled us to operate at this scale in a highly reproducible fashion, setting the standard for such analyses in the field. My lab also previously led an ambitious effort to validate 438 antibodies interrogating 365 human RBPs (Sundararaman et al., Molecular Cell 2016). We have continued our efforts and recently doubled the number of IP-grade antibodies characterized in the field.

Team Members

Learn more about ENCODE and eCLIP

We have continued to improve eCLIP computationally (Yee et al., RNA 20197) and experimentally, introducing non- radioactive visualization of protein-RNA complexes for their precise isolation (Van Nostrand et al., Genome Biology 20208). Using our antibody resource, we published the characterization of the largest number of human RBPs to date (Van Nostrand et al., Nature 20209), revealing RBP binding sites and their function, and binding preferences and subcellular localization of RBPs in vivo and in vitro. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation, and localization. By developing computational methods to analyze our eCLIP datasets, my lab resolved that the AQR protein associates after intronic lariat formation, clarified a branch point-based scanning model for 3’ splice site recognition, and found novel ribosomal RNA processing factors and RBPs that control retrotransposable elements in the human genome (Van Nostrand et al., Genome Biology 20208). My postdoc Eric Van Nostrand obtained his independent faculty position at Baylor College of Medicine and immediately competed successfully for the NHGRI Genomic Innovator award (1 of 11 awardees in 2011). Postdoc Meredith Corley in my lab also developed footprinting SHAPE-eCLIP which combines icSHAPE to identify RNA structural landscapes and eCLIP to identify RBP interactions at this structured sites (Corley et al., Molecular Cell 202010). This technology allowed us to resolve where RBPs form hydrogen bonds to RNA at single nucleotide resolution. With the growing importance of mRNA medicines, many biotech companies require the mapping of RNA structural landscapes in synthetic mRNA designs. We have also partnered with other labs to best utilize our eCLIP datasets to identify RNA editing regulation by RBPs in human cells (Quinones-Valdez et al. Commun Biol 201911), to identify functional genetic variants in RNA (Yang E-W et al., Nature Communications 201912) to identify chromatin-RNA binding protein interactions (Xiao et al., Cell 201913) and to identify novel ribosomal RNA regulators (Kaiser et al., Sci Rep 201914).

In my lab we have also leveraged eCLIP to continue to ask deeper, mechanistic questions about RBP function using LIN28 as an exemplar protein. LIN28 is a centrally important post-transcriptional regulator of embryonic development and changes in its expression levels are thought to maintain gene expression programs that promote tissue growth and morphogenesis. LIN28 is best known for its ability to regulate miRNA biogenesis but it also has the capacity to broadly bind other mammalian transcripts, as my lab was the first to show (Wilbert et al., Molecular