Gabriel A. Pratt

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)

As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges.

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

The Ro60 autoantigen binds endogenous retroelements and regulates inflammatory gene expression.

An Aluring new autoantibody target Autoimmunity is the immune systems ultimate act of betrayal. Cells designed to protect against invading microbes suddenly target the host instead. In the autoimmune disease systemic lupus erythematosus, antibodies target DNA and host proteins, including the RNA binding protein Ro60. Hung et al. discovered that Ro60 bound to endogenous Alu retroelements. They detected antibody-Ro60-Alu RNA immune complexes in the blood of individuals with lupus and an enrichment of Alu transcripts. Ro60 bound to Alu probably primes RNA-binding innate immune receptors within B cells, leading these cells to make antibodies that target Ro60-Alu RNA and drive disease-causing inflammation. Science, this issue p. 455 A lupus-associated autoantigen binds Alu endogenous retroelements and regulates type I interferon cytokine production. Autoantibodies target the RNA binding protein Ro60 in systemic lupus erythematosus (SLE) and Sjögren’s syndrome. However, it is unclear whether Ro60 and its associated RNAs contribute to disease pathogenesis. We catalogued the Ro60-associated RNAs in human cell lines and found that among other RNAs, Ro60 bound an RNA motif derived from endogenous Alu retroelements. Alu transcripts were induced by type I interferon and stimulated proinflammatory cytokine secretion by human peripheral blood cells. Ro60 deletion resulted in enhanced expression of Alu RNAs and interferon-regulated genes. Anti-Ro60–positive SLE immune complexes contained Alu RNAs, and Alu transcripts were up-regulated in SLE whole blood samples relative to controls. These findings establish a link among the lupus autoantigen Ro60, Alu retroelements, and type I interferon.

Target Discrimination in Nonsense-Mediated mRNA Decay Requires Upf1 ATPase Activity

RNA quality-control pathways get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the adenosine triphosphatase (ATPase) cycle of the SF1 helicase Upf1 is required for mRNA discrimination during nonsense-mediated decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD-translation and termination codon-proximal poly(A) binding protein-depend on the ATPase activity of Upf1 to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be a widely used activity in target RNA discrimination.

Musashi-2 attenuates AHR signalling to expand human haematopoietic stem cells

Umbilical cord blood-derived haematopoietic stem cells (HSCs) are essential for many life-saving regenerative therapies. However, despite their advantages for transplantation, their clinical use is restricted because HSCs in cord blood are found only in small numbers. Small molecules that enhance haematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified, but in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular circuitry that underpins the self-renewal of human HSCs will facilitate the development of targeted strategies that expand HSCs for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs, the post-transcriptional mechanisms that guide HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2–RNA interactions, we show that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signalling through post-transcriptional downregulation of canonical AHR pathway components in cord blood HSPCs. Our study gives mechanistic insight into RNA networks controlled by RNA-binding proteins that underlie self-renewal and provides evidence that manipulating such networks ex vivo can enhance the regenerative potential of human HSCs.

Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.

Resources for the Comprehensive Discovery of Functional RNA Elements

Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/).

A Large-Scale Binding and Functional Map of Human RNA Binding Proteins

Genomes encompass all the information necessary to specify the development and function of an organism. In addition to genes, genomes also contain a myriad of functional elements that control various steps in gene expression. A major class of these elements function only when transcribed into RNA as they serve as the binding sites for RNA binding proteins (RBPs) which act to control post-transcriptional processes including splicing, cleavage and polyadenylation, RNA editing, RNA localization, translation, and RNA stability. Despite the importance of these functional RNA elements encoded in the genome, they have been much less studied than genes and DNA elements. Here, we describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. These data expand the catalog of functional elements encoded in the human genome by addition of a large set of elements that function at the RNA level through interaction with RBPs.

CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins.

Identification of in vivo direct RNA targets for RNA binding proteins (RBPs) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA interaction landscape.

Variation in single-nucleotide sensitivity of eCLIP derived from reverse transcription conditions

Crosslinking and immunoprecipitation (CLIP) followed by high-throughput sequencing identifies the binding sites of RNA binding proteins on RNAs. The covalent RNA-amino acid adducts produced by UV irradiation can cause premature reverse transcription termination and deletions (referred to as crosslink-induced mutation sites (CIMS)), which may decrease overall cDNA yield but are exploited in state-of-the-art CLIP methods to identify these crosslink sites at single-nucleotide resolution. Here, we show the ratio of both crosslinked base deletions and read-through versus termination are highly dependent on the identity of the reverse transcriptase enzyme as well as on buffer conditions used. AffinityScript and TGIRT showed a lack of deletion of the crosslinked base with other enzymes showing variable rates, indicating that utilization and interpretation of CIMS analysis requires knowledge of the reverse transcriptase enzyme used. Commonly used enzymes, including Superscript III and AffinityScript, show high termination rates in standard magnesium buffer conditions, but show a single base difference in the position of termination for TARDBP motifs. In contrast, manganese-containing buffer promoted read-through at the adduct site. These results validate the use of standard enzymes and also propose alternative enzyme and buffer choices for particularly challenging samples that contain extensive RNA adducts or other modifications that inhibit standard reverse transcription.