A large-scale binding and functional map of human RNA-binding proteins.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome1. Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. 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 RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

RBP Image Database: A resource for the systematic characterization of the subcellular distribution properties of human RNA binding proteins.

RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with ∼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.

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/).

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.

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins.

BACKGROUND: A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. RESULTS: Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3' splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs. CONCLUSIONS: This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

ICOSL-augmented adenoviral-based vaccination induces a bipolar Th17/Th1 T cell response against unglycosylated MUC1 antigen.

Cellular immunity established via immunotherapy holds the potential to eliminate solid tumors. Yet, cancer vaccines have failed to induce tumor-reactive T cells of sufficient quality to control disease. The inducible T cell costimulator (ICOS) pathway has been implicated in both the selective induction of immunity over tolerance as well as licensing of IL-17-polarized cellular immunity. Herein, we evaluated the ability of ICOS ligand (ICOSL) to augment the immunogenicity of adenoviral-based vaccination targeting the unglycosylated MUC1 peptide antigen. Vaccination disrupted immunotolerance in a transgenic mouse model recognizing human MUC1 as a self-antigen, inducing robust MUC1-specific immunity. Augmenting vaccination with ICOSL induced a bipolar Th17/Th1 effector profile, marked by increased MUC1-specific IL-17A production and RORγt expression in CD4+ but not CD8+ T cells which predominantly expressed IFNγ/IL-2 and T-bet. The polarization and maintenance of Th17 cells established following ICOSL augmented vaccination was highly durable, with elevated IL-17A and RORγt levels detected in CD4+ T cells up to 10 months after initial immunization. Furthermore, provision of ICOSL significantly enhanced MUC1-specific IgG antibody in response to immunization. ICOSL signaling dramatically influenced CD4+ T cell phenotype, altering gene expression of transcription factors and regulators of effector function following immunization. Interestingly, ICOSL augmentation failed to alter the transcriptional profile of CD8+ T cells following immunization, affecting the magnitude, but not distribution, of gene expression. Collectively, ICOSL supports the induction of durable, antigen-specific Th17/Th1-mediated immunity in vivo, establishing a vaccination platform to enhance CD4+ T cell-mediated antitumor immunity and providing a crucial component of an effective cancer vaccine.

Hip fracture in a patient with severe pulmonary hypertension.

This case presents a discussion of an 80-year-old woman with severe pulmonary hypertension (PH) on chronic intravenous treprostinil infusion and oxygen therapy who presents with a subcapital hip fracture. Care is closely coordinated by an interdisciplinary team, including her PH specialist, in order to optimize her outcome.

Thrombospondin-1 type 1 repeats in a model of inflammatory bowel disease: transcript profile and therapeutic effects.

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease.