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Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).Nature Methods Jun 2016As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and...
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.
Topics: Binding Sites; Cross-Linking Reagents; Gene Expression Profiling; Hep G2 Cells; Humans; Immunoprecipitation; K562 Cells; Photochemical Processes; RNA-Binding Proteins; Transcriptome; Ultraviolet Rays
PubMed: 27018577
DOI: 10.1038/nmeth.3810 -
Nature Protocols Jun 2019R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear...
R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.
Topics: Animals; Antibodies; Antibodies, Monoclonal; DNA; High-Throughput Nucleotide Sequencing; Humans; Immunoprecipitation; Mice; Nucleic Acid Conformation; Nucleic Acid Hybridization; Polymerase Chain Reaction; RNA
PubMed: 31053798
DOI: 10.1038/s41596-019-0159-1 -
Cold Spring Harbor Perspectives in... Aug 2018To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and... (Review)
Review
To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Cross-linking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA cross-linking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare complementary DNA (cDNA) libraries for sequencing. Here we describe the core steps of CLIP, its primary variations, and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice and discuss the mechanistic and physiologic insights that have already been gained from studies using CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms while providing important biologic and clinically relevant insights.
Topics: Animals; Cross-Linking Reagents; DNA, Complementary; Gene Expression Regulation; Humans; Immunoprecipitation; RNA
PubMed: 30068528
DOI: 10.1101/cshperspect.a032243 -
Analytical and Bioanalytical Chemistry Sep 2021The analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based...
The analysis of protein-protein interactions (PPIs) is essential for the understanding of cellular signaling. Besides probing PPIs with immunoprecipitation-based techniques, peptide pull-downs are an alternative tool specifically useful to study interactome changes induced by post-translational modifications. Peptides for pull-downs can be chemically synthesized and thus offer the possibility to include amino acid exchanges and post-translational modifications (PTMs) in the pull-down reaction. The combination of peptide pull-down and analysis of the binding partners with mass spectrometry offers the direct measurement of interactome changes induced by PTMs or by amino acid exchanges in the interaction site. The possibility of large-scale peptide synthesis on a membrane surface opened the possibility to systematically analyze interactome changes for mutations of many proteins at the same time. Short linear motifs (SLiMs) are amino acid patterns that can mediate protein binding. A significant number of SLiMs are located in regions of proteins, which are lacking a secondary structure, making the interaction motifs readily available for binding reactions. Peptides are particularly well suited to study protein interactions, which are based on SLiM-mediated binding. New technologies using arrayed peptides for interaction studies are able to identify SLIM-based interaction and identify the interaction motifs.
Topics: Immunoprecipitation; Mass Spectrometry; Peptides; Protein Interaction Mapping
PubMed: 33942139
DOI: 10.1007/s00216-021-03367-8 -
BioTechniques Dec 2004Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation,... (Review)
Review
Association between proteins and DNA is crucial for many vital cellular functions such as gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. It is important to know the genomic targets of DNA-binding proteins and the mechanisms by which they control and guide gene regulation pathways and cellular proliferation. Chromatin immunoprecipitation (ChIP) is an important technique in the study of protein-gene interactions. Using ChIP, DNA-protein interactions are studied within the context of the cell. The basic steps in this technique are fixation, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. Although ChIP is a very versatile tool, the procedure requires the optimization of reaction conditions. Several modifications to the original ChIP technique have been published to improve the success and to enhance the utility of this procedure. This review addresses the critical parameters and the variants of ChiP as well as the different analytical tools that can be combined with ChIP to enable better understanding of DNA-protein interactions in vivo.
Topics: Chromatin Immunoprecipitation; DNA; DNA-Binding Proteins
PubMed: 15597545
DOI: 10.2144/04376RV01 -
Molecular Cell Feb 2018RNA binding proteins (RBPs) regulate all aspects in the life cycle of RNA molecules. To elucidate the elements that guide RNA specificity, regulatory mechanisms, and... (Review)
Review
RNA binding proteins (RBPs) regulate all aspects in the life cycle of RNA molecules. To elucidate the elements that guide RNA specificity, regulatory mechanisms, and functions of RBPs, methods that identify direct endogenous protein-RNA interactions are particularly valuable. UV crosslinking and immunoprecipitation (CLIP) purifies short RNA fragments that crosslink to a specific protein and then identifies these fragments by sequencing. When combined with high-throughput sequencing, CLIP can produce transcriptome-wide maps of RNA crosslink sites. The protocol is comprised of several dozen biochemical steps, and improvements made over the last 15 years have increased its resolution, sensitivity, and convenience. Adaptations of CLIP are also emerging in the epitranscriptomic field to map the positions of RNA modifications accurately. Here, we describe the rationale for each step in the protocol and discuss the impact of variations to help users determine the most suitable option.
Topics: Binding Sites; High-Throughput Nucleotide Sequencing; Immunoprecipitation; Protein Binding; RNA; RNA Recognition Motif Proteins; RNA-Binding Proteins; Sequence Analysis, RNA; Transcriptome
PubMed: 29395060
DOI: 10.1016/j.molcel.2018.01.005 -
Current Protocols Sep 2022Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that...
Kinases are responsible for phosphorylation of proteins and are involved in many biological processes, including cell signaling. Identifying the kinases that phosphorylate specific phosphoproteins is critical to augment the current understanding of cellular events. Herein, we report a general protocol to study the kinases of a target substrate phosphoprotein using kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP). K-CLIP uses a photocrosslinking γ-phosphoryl-modified ATP analog, such as ATP-arylazide, to covalently crosslink substrates to kinases with UV irradiation. Crosslinked kinase-substrate complexes can then be enriched by immunoprecipitating the target substrate phosphoprotein, with bound kinase(s) identified using Western blot or mass spectrometry analysis. K-CLIP is an adaptable chemical tool to investigate and discover kinase-substrate pairs, which will promote characterization of complex phosphorylation-mediated cell biology. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Kinase-catalyzed crosslinking of lysates Basic Protocol 2: Kinase-catalyzed crosslinking and immunoprecipitation (K-CLIP).
Topics: Adenosine Triphosphate; Catalysis; Immunoprecipitation; Phosphoproteins; Phosphorylation
PubMed: 36135312
DOI: 10.1002/cpz1.539 -
Bioscience Reports Apr 2017G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now... (Review)
Review
G-protein-coupled receptors (GPCRs), which constitute the largest family of cell surface receptors, were originally thought to function as monomers, but are now recognized as being able to act in a wide range of oligomeric states and indeed, it is known that the oligomerization state of a GPCR can modulate its pharmacology and function. A number of experimental techniques have been devised to study GPCR oligomerization including those based upon traditional biochemistry such as blue-native PAGE (BN-PAGE), co-immunoprecipitation (Co-IP) and protein-fragment complementation assays (PCAs), those based upon resonance energy transfer, FRET, time-resolved FRET (TR-FRET), FRET spectrometry and bioluminescence resonance energy transfer (BRET). Those based upon microscopy such as FRAP, total internal reflection fluorescence microscopy (TIRFM), spatial intensity distribution analysis (SpIDA) and various single molecule imaging techniques. Finally with the solution of a growing number of crystal structures, X-ray crystallography must be acknowledged as an important source of discovery in this field. A different, but in many ways complementary approach to the use of more traditional experimental techniques, are those involving computational methods that possess obvious merit in the study of the dynamics of oligomer formation and function. Here, we summarize the latest developments that have been made in the methods used to study GPCR oligomerization and give an overview of their application.
Topics: Animals; Crystallography, X-Ray; Fluorescence Resonance Energy Transfer; Humans; Immunoprecipitation; Protein Multimerization; Receptors, G-Protein-Coupled
PubMed: 28062602
DOI: 10.1042/BSR20160547 -
Wiley Interdisciplinary Reviews. RNA Jan 2018RNA binding proteins (RBPs) play key roles in determining cellular behavior by manipulating the processing of target RNAs. Robust methods are required to detect the... (Review)
Review
RNA binding proteins (RBPs) play key roles in determining cellular behavior by manipulating the processing of target RNAs. Robust methods are required to detect the numerous binding sites of RBPs across the transcriptome. RNA-immunoprecipitation followed by sequencing (RIP-seq) and crosslinking followed by immunoprecipitation and sequencing (CLIP-seq) are state-of-the-art methods used to identify the RNA targets and specific binding sites of RBPs. Historically, CLIP methods have been confounded with challenges such as the requirement for tens of millions of cells per experiment, low RNA yields resulting in libraries that contain a high number of polymerase chain reaction duplicated reads, and technical inconveniences such as radioactive labeling of RNAs. However, recent improvements in the recovery of bound RNAs and the efficiency of converting isolated RNAs into a library for sequencing have enhanced our ability to perform the experiment at scale, from less starting material than has previously been possible, and resulting in high quality datasets for the confident identification of protein binding sites. These, along with additional improvements to protein capture, removal of nonspecific signals, and methods to isolate noncanonical RBP targets have revolutionized the study of RNA processing regulation, and reveal a promising future for mapping the human protein-RNA regulatory network. WIREs RNA 2018, 9:e1436. doi: 10.1002/wrna.1436 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Methods > RNA Analyses in Cells.
Topics: Gene Expression Regulation; Immunoprecipitation; Molecular Biology; RNA, Messenger; RNA-Binding Proteins; Sequence Analysis, RNA
PubMed: 28853213
DOI: 10.1002/wrna.1436 -
STAR Protocols Mar 2022Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from...
Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT and OGA activities before HCF-1 immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Daou et al. (2011).
Topics: Blotting, Western; HEK293 Cells; HeLa Cells; Humans; Immunoprecipitation; N-Acetylglucosaminyltransferases
PubMed: 35106498
DOI: 10.1016/j.xpro.2021.101108