Cross-linking And Immunoprecipitation Research Articles

Mapping protein-RNA binding in plants with individual-nucleotide-resolution UV cross-linking and immunoprecipitation (plant iCLIP2).

Despite crucial roles of RNA-binding proteins (RBPs) in plant physiology and development, methods for determining their transcriptome-wide binding landscape are less developed than those used in other model organisms. Cross-linking and immunoprecipitation (CLIP) methods (based on UV-mediated generation of covalent bonds between RNAs and cognate RBPs in vivo, purification of the cross-linked complexes and identification of the co-purified RNAs by high-throughput sequencing) have been applied mainly in mammalian cells growing in monolayers or in translucent tissue. We have developed plant iCLIP2, an efficient protocol for performing individual-nucleotide-resolution CLIP (iCLIP) in plants, tailored to overcome the experimental hurdles posed by plant tissue. We optimized the UV dosage to efficiently cross-link RNA and proteins in plants and expressed epitope-tagged RBPs under the control of their native promoters in loss-of-function mutants. We select epitopes for which nanobodies are available, allowing stringent conditions for immunopurification of the RNA-protein complexes to be established. To overcome the inherently high RNase content of plant cells, RNase inhibitors are added and the limited RNA fragmentation step is modified. We combine the optimized isolation of RBP-bound RNAs with iCLIP2, a streamlined protocol that greatly enhances the efficiency of library preparation for high-throughput sequencing. Plant researchers with experience in molecular biology and handling of RNA can complete this iCLIP2 protocol in ~5 d. Finally, we describe a bioinformatics workflow to determine targets of Arabidopsis RBPs from iCLIP data, covering all steps from downloading sequencing reads to identifying cross-linking events ( https://github.com/malewins/Plant-iCLIPseq ), and present the R/Bioconductor package BindingSiteFinder to extract reproducible binding sites ( https://bioconductor.org/packages/release/bioc/html/BindingSiteFinder.html ).

MiR-204 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) replication via inhibiting LC3B-mediated autophagy

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been regarded as a persistent challenge for the swine farms worldwide. microRNAs (miRNAs) play key roles in regulating almost every important biological process, including virus-host interaction. In this study, we found that miR-204 was highly expressed in cells that were not permissive to PRRSV infection compared with cells susceptible to PRRSV infection. Subsequently, we demonstrated that overexpression of miR-204 significantly inhibited PRRSV replication in porcine alveolar macrophages (PAMs). Through bioinformatic analysis, we found that there existed a potential binding site of miR-204 on the 3′UTR of microtubule associated protein 1 light chain 3B (MAP1LC3B, LC3B), a hallmark of autophagy. Applying experiments including luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay, we demonstrated that miR-204 directly targeted LC3B, thereby downregulating autophagy. Meanwhile, we investigated the interplay between autophagy and PRRSV replication in PAMs, confirming that PRRSV infection induces autophagy, which in turn facilitates viral replication. Overall, we verify that miR-204 suppresses PRRSV replication via inhibiting LC3B-mediated autophagy in PAMs. These findings will provide a novel potential approach for us to develop antiviral therapeutic agents and controlling measures for future PRRSV outbreaks.

Transcriptome-wide RNA binding analysis of C9orf72 poly(PR) dipeptides.

An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.

Nf-core/clipseq - a robust Nextflow pipeline for comprehensive CLIP data analysis.

Crosslinking and immunoprecipitation (CLIP) technologies have become a central component of the molecular biologists' toolkit to study protein-RNA interactions and thus to uncover core principles of RNA biology. There has been a proliferation of CLIP-based experimental protocols, as well as computational tools, especially for peak-calling. Consequently, there is an urgent need for a well-documented bioinformatic pipeline that enshrines the principles of robustness, reproducibility, scalability, portability and flexibility while embracing the diversity of experimental and computational CLIP tools. To address this, we present nf-core/clipseq - a robust Nextflow pipeline for quality control and analysis of CLIP sequencing data. It is part of the international nf-core community effort to develop and curate a best-practice, gold-standard set of pipelines for data analysis. The standards enabled by Nextflow and nf-core, including workflow management, version control, continuous integration and containerisation ensure that these key needs are met. Furthermore, multiple tools are implemented ( e.g. for peak-calling), alongside visualisation of quality control metrics to empower the user to make their own informed decisions based on their data. nf-core/clipseq remains under active development, with plans to incorporate newly released tools to ensure that pipeline remains up-to-date and relevant for the community. Engagement with users and developers is encouraged through the nf-core GitHub repository and Slack channel to promote collaboration. It is available at https://nf-co.re/clipseq.

RBP-Tar – a searchable database for experimental RBP binding sites

Background: RNA-binding proteins (RBPs) play a critical role in regulating gene expression by binding to specific sites on RNA molecules. Identifying these binding sites is crucial for understanding the many functions of RBPs in cellular function, development and disease. Current experimental methods for identifying RBP binding sites, such as ultra-violet (UV) crosslinking and immunoprecipitation (CLIP), and especially the enhanced CLIP (eCLIP) protocol, were developed to identify authentic RBP binding sites experimentally. Methods: To make this data more accessible to the scientific community, we have developed RBP-Tar (https://ncbr.muni.cz/RBP-Tar), a web server and database that utilises eCLIP data for 167 RBPs mapped on the human genome. The web server allows researchers to easily search and retrieve binding site information by genomic location and RBP name. Use case: Researchers can produce lists of all known RBP binding sites on a gene of interest, or produce lists of binding sites for one RBP on different genomic loci. Conclusions: Our future goal is to continue to populate the web server with additional experimental datasets from CLIP experiments as they become available and processed, making it an increasingly valuable resource for the scientific community.

In vivo PAR-CLIP (viP-CLIP) of liver TIAL1 unveils targets regulating cholesterol synthesis and secretion

System-wide cross-linking and immunoprecipitation (CLIP) approaches have unveiled regulatory mechanisms of RNA-binding proteins (RBPs) mainly in cultured cells due to limitations in the cross-linking efficiency of tissues. Here, we describe viP-CLIP (in vivo PAR-CLIP), a method capable of identifying RBP targets in mammalian tissues, thereby facilitating the functional analysis of RBP-regulatory networks in vivo. We applied viP-CLIP to mouse livers and identified Insig2 and ApoB as prominent TIAL1 target transcripts, indicating an important role of TIAL1 in cholesterol synthesis and secretion. The functional relevance of these targets was confirmed by showing that TIAL1 influences their translation in hepatocytes. Mutant Tial1 mice exhibit altered cholesterol synthesis, APOB secretion and plasma cholesterol levels. Our results demonstrate that viP-CLIP can identify physiologically relevant RBP targets by finding a factor implicated in the negative feedback regulation of cholesterol biosynthesis.

Efficient and sensitive profiling of RNA-protein interactions using TLC-CLIP.

RNA-binding proteins are instrumental for post-transcriptional gene regulation, controlling all aspects throughout the lifecycle of RNA molecules. However, transcriptome-wide methods to profile RNA-protein interactions in vivo remain technically challenging and require large amounts of starting material. Herein, we present an improved library preparation strategy for crosslinking and immunoprecipitation (CLIP) that is based on tailing and ligation of cDNA molecules (TLC). TLC involves the generation of solid-phase cDNA, followed by ribotailing to significantly enhance the efficiency of subsequent adapter ligation. These modifications result in a streamlined, fully bead-based library preparation strategy, which eliminates time-consuming purification procedures and drastically reduces sample loss. As a result, TLC-CLIP displays unparalleled sensitivity, enabling the profiling of RNA-protein interactions from as few as 1000 cells. To demonstrate the effectiveness of TLC-CLIP, we profiled four endogenous RNA-binding proteins, showcasing its reproducibility and improved precision resulting from a higher occurrence of crosslinking-induced deletions. These deletions serve as an intrinsic quality metric and increase both specificity and nucleotide-resolution.

Skipper analysis of eCLIP datasets enables sensitive detection of constrained translation factor binding sites

Technology for crosslinking and immunoprecipitation (CLIP) followed by sequencing (CLIP-seq) has identified the transcriptomic targets of hundreds of RNA-binding proteins in cells. To increase the power of existing and future CLIP-seq datasets, we introduce Skipper, an end-to-end workflow that converts unprocessed reads into annotated binding sites using an improved statistical framework. Compared with existing methods, Skipper on average calls 210%-320% more transcriptomic binding sites and sometimes >1,000% more sites, providing deeper insight into post-transcriptional gene regulation. Skipper also calls binding to annotated repetitive elements and identifies bound elements for 99% of enhanced CLIP experiments. We perform nine translation factor enhanced CLIPs and apply Skipper to learn determinants of translation factor occupancy, including transcript region, sequence, and subcellular localization. Furthermore, we observe depletion of genetic variation in occupied sites and nominate transcripts subject to selective constraint because of translation factor occupancy. Skipper offers fast, easy, customizable, and state-of-the-art analysis of CLIP-seq data.

Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.

Studying miRNA-mRNA Interactions: An Optimized CLIP-Protocol for Endogenous Ago2-Protein.

Transcriptome-wide analysis of RNA-binding partners is commonly achieved using UV crosslinking and immunoprecipitation (CLIP). Individual-nucleotide-resolution CLIP (iCLIP)enables identification of the specific position of the protein-RNA interaction. In addition to RNA-binding proteins (RBPs), microRNA (miRNA)-mRNA interactions also play a crucial role in the regulation of gene expression. Argonaute-2 (Ago2) mediates miRNA binding to a multitude of mRNA target sites, enabling the identification of miRNA-mRNA interactions by employing modified Ago2-CLIP protocols. Here, we describe an Ago2-specific CLIP protocol optimized for the use of small quantities of cell material, targeting endogenous Ago2 while avoiding possible methodological biases such as metabolic labeling or Ago2 overexpression and applying the latest advances in CLIP library preparation, the iCLIP2 protocol. In particular, we focus on the optimization of lysis conditions and improved radioactive labeling of the 5' end of the miRNA.

Identification of Global miRNA Targetomes within Human Hematopoietic Progenitors and Acute Myeloid Leukemia Uncovers Unique Molecular Networks Centered on Mitochondria Function in Leukemic Stem Cells

Previous studies comparing transcriptome and proteome profiles of the cells that constitute both the normal and malignant hematopoietic hierarchies have revealed substantial uncoupling of transcript and protein expression levels. Despite the widely established role of miRNAs in regulating many cellular functions, the precise contribution of miRNA-mediated post-transcriptional regulation linking gene expression and proteome signatures of hematopoietic stem cells (HSC) and leukemic stem cells (LSC) remains largely unknown. Previous efforts to characterize miRNA targets have largely relied on computational prediction algorithms, which suffer from high rates of false-positive and negative target predictions and ultimately preclude discovery of non-canonical miRNA-target interactions. In this study, we perform Argonaute 2 (AGO2) crosslinking and immunoprecipitation (CLIP)-seq to experimentally map endogenous miRNA-target interactions in human CD34+ hematopoietic stem and progenitor cells (HSPC) from cord blood and six primary AML patient samples in an unbiased, transcriptome-wide manner (Manakov et al. 2022). Using this approach, we identify hundreds of miRNAs directly ligated to thousands of unique targets from miRNA-target chimeric sequencing reads. We find limited overlap (~30%) between miRNA targets captured by CLIP-seq and computationally predicted targets, underscoring the importance of functional validation of miRNA targets in specific cell types. Surprisingly, we find remarkable similarity between abundant miRNAs from HSPC and AML samples, including miR-142 and miRNAs encoded by the miR-17-92 cluster. However, the targets and the gene regulatory networks controlled by these highly abundant miRNAs are vastly different between HSPC and AML. In HSPC, we find that targets of abundant miRNAs are enriched in cell cycle regulation, histone modification and megakaryocyte/myeloid differentiation. In contrast, targets of the same miRNAs are enriched in protein localization/translation, neutrophil function, regulation of apoptosis and electron transport chain (ETC) pathways in AML. Collectively, we find that the composition of the abundant miRNAs is similar in normal HSPC and AML tumors; however they control divergent gene regulatory networks. To identify miRNA-target networks that play functional roles in LSC, we performed small RNA sequencing in 23 AML patient samples where each sorted fraction was assessed for LSC content by xenotransplantation. We derived an LSC-specific miRNA signature consisting of 5 miRNAs (LSC miR-5 score) predictive of overall survival and capable of stratifying AML patients independent of mRNA expression and other established indicators of outcome. Comparison of CLIP-seq targets of the LSC-specific miRNAs from HSPC and AML patient blasts revealed the existence of AML-specific gene regulatory networks centred on oxidative phosphorylation, ETC and cell-matrix adhesion. We further interrogated the functional significance of one miRNA from the LSC miR-5 score, miR-let-7c. We find that miR-let-7c is highly expressed in LSC-enriched populations of two AML in vitro models that recapitulate the leukemic hierarchy. Enforced expression of miR-let-7c in OCI-AML22 cells resulted in expansion of the LSC-enriched CD34+CD38- population and reduction in the CD34- terminally differentiated blast population. Comparison of miR-let-7c targets from HSPC and AML patient samples revealed the existence of distinct gene regulatory networks. In HSPC, miR-let-7c targets are enriched in cell cycle and regulation of transcription, whereas in AML, mitochondrial ETC, oxidative phosphorylation, and cell-matrix adhesion were the most enriched pathways. Moreover, analysis of miR-let-7c targets identified a high prevalence of transcripts encoded by the mitochondrial (mt) DNA (MT-ATP6, MT-ATP8, MT-ND1, -2, -4, -5, MT-CO1), pointing towards a largely uncharacterized function of miRNAs in regulating transcripts encoded by mtDNA in AML. Collectively, miRNA sequencing in LSC-enriched AML samples and identification of miRNA targetomes in HSPC and AML patient samples have revealed unexpected mechanisms of post-transcriptional regulation in normal and malignant hematopoietic stem cells.

Alternative splicing of HSPA12A pre-RNA by SRSF11 contributes to metastasis potential of colorectal cancer.

Dysregulation of alternative splicing (AS) induced by serine/arginine-rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine-rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown. The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA-seq technology was used to analyze and screen SRSF11-triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini-gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co-IP) and Phospho-tag SDS-PAGE experiments were used to investigate PAK5-mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction. In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro-metastatic role in vitro and in vivo. By screening SRSF11-regulated AS events, we identified the binding motif of SRSF11-triggered splicing-switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N-cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation. SRSF11 exerts pro-metastatic effects in CRC by inhibiting the AS of HSPA12A pre-RNA. Our findings point to SRSF11-regulated HSPA12A splicing as a novel relationship between SRSF11-regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC.

Nucleotide-amino acid π-stacking interactions initiate photo cross-linking in RNA-protein complexes

Photo-induced cross-linking is a mainstay technique to characterize RNA-protein interactions. However, UV-induced cross-linking between RNA and proteins at “zero-distance” is poorly understood. Here, we investigate cross-linking of the RBFOX alternative splicing factor with its hepta-ribonucleotide binding element as a model system. We examine the influence of nucleobase, nucleotide position and amino acid composition using CLIR-MS technology (crosslinking-of-isotope-labelled-RNA-and-tandem-mass-spectrometry), that locates cross-links on RNA and protein with site-specific resolution. Surprisingly, cross-linking occurs only at nucleotides that are π-stacked to phenylalanines. Notably, this π-stacking interaction is also necessary for the amino-acids flanking phenylalanines to partake in UV-cross-linking. We confirmed these observations in several published datasets where cross-linking sites could be mapped to a high resolution structure. We hypothesize that π-stacking to aromatic amino acids activates cross-linking in RNA-protein complexes, whereafter nucleotide and peptide radicals recombine. These findings will facilitate interpretation of cross-linking data from structural studies and from genome-wide datasets generated using CLIP (cross-linking-and-immunoprecipitation) methods.

MiR-142-3p suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by directly targeting Rac1

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) has been recognized as one of the severest epidemics in pigs worldwide. microRNAs (miRNAs) play important roles in a variety of biological processes, including cell differentiation, proliferation and death, as well as viral infections and antiviral immune responses. In this study, we found that miR-142–3p was expressed lower in cells susceptible to PRRSV infection than in cells less or no permissive to PRRSV infection. Subsequently, we showed that overexpression of miR-142–3p remarkably inhibited PRRSV infection in PAMs, while blockage of endogenous miR-142–3p significantly enhanced PRRSV replication. Then, we demonstrated that miR-142–3p directly targeted Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of Rho GTPases family, by using luciferase reporter assay and UV cross-linking and immunoprecipitation (CLIP) assay. Importantly, we verified that miR-142–3p inhibited PRRSV entry into PAMs and accordingly suppressed PRRSV infection by downregulating Rac1 expression. These findings reveal an important role of miR-142–3p in modulating PRRSV infection and provide us with some ideas for developing novel antiviral therapy against PRRSV infection.

Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq.

Discovery of interaction sites between RNA-binding proteins (RBPs) and their RNA targets plays a critical role in enabling our understanding of how these RBPs control RNA processing and regulation. Cross-linking and immunoprecipitation (CLIP) provides a generalizable, transcriptome-wide method by which RBP/RNA complexes are purified and sequenced to identify sites of intermolecular contact. By simplifying technical challenges in prior CLIP methods and incorporating the generation of and quantitative comparison against size-matched input controls, the single-end enhanced CLIP (seCLIP) protocol allows for the profiling of these interactions with high resolution, efficiency and scalability. Here, we present a step-by-step guide to the seCLIP method, detailing critical steps and offering insights regarding troubleshooting and expected results while carrying out the ~4-d protocol. Furthermore, we describe a comprehensive bioinformatics pipeline that offers users the tools necessary to process two replicate datasets and identify reproducible and significant peaks for an RBP of interest in ~2 d.

Identification of the global miR-130a targetome reveals a role for TBL1XR1 in hematopoietic stem cell self-renewal and t(8;21) AML.

Gene expression profiling and proteome analysis of normal and malignant hematopoietic stem cells (HSCs) point to shared core stemness properties. However, discordance between mRNA and protein signatures highlights an important role for post-transcriptional regulation by microRNAs (miRNAs) in governing this critical nexus. Here, we identify miR-130a as a regulator of HSC self-renewal and differentiation. Enforced expression of miR-130a impairs B lymphoid differentiation and expands long-term HSCs. Integration of protein mass spectrometry and chimeric AGO2 crosslinking and immunoprecipitation (CLIP) identifies TBL1XR1 as a primary miR-130a target, whose loss of function phenocopies miR-130a overexpression. Moreover, we report that miR-130a is highly expressed in t(8;21) acute myeloid leukemia (AML), where it is critical for maintaining the oncogenic molecular program mediated by the AML1-ETO complex. Our study establishes that identification of the comprehensive miRNA targetome within primary cells enables discovery of genes and molecular networks underpinning stemness properties of normal and leukemic cells.

Metadensity: a background-aware python pipeline for summarizing CLIP signals on various transcriptomic sites.

Cross-linking and immunoprecipitation (CLIP) is a technology to map the binding sites of RNA-binding proteins (RBPs). The region where an RBP binds within RNA is often indicative of its molecular function in RNA processing. As an example, the binding sites of splicing factors are found within or proximal to alternatively spliced exons. To better reveal the function of RBPs, we developed a tool to visualize the distribution of CLIP signals around various transcript features. Here, we present Metadensity (https://github.com/YeoLab/Metadensity), a software that allows users to generate metagene plots. Metadensity allows users to input features such as branchpoints and preserves the near-nucleotide resolution of CLIP technologies by not scaling the features by length. Metadensity normalizes immunoprecipitated libraries with background controls, such as size-matched inputs, then windowing in various user-defined features. Finally, the signals are averaged across a provided set of transcripts. Metadensity is available at https://github.com/YeoLab/Metadensity, with example notebooks at https://metadensity.readthedocs.io/en/latest/tutorial.html. Supplementary data are available at Bioinformatics Advances online.

Corrigendum: CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein.

Corrigendum: CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein.

Multi-resBind: a residual network-based multi-label classifier for in vivo RNA binding prediction and preference visualization

BackgroundProtein-RNA interactions play key roles in many processes regulating gene expression. To understand the underlying binding preference, ultraviolet cross-linking and immunoprecipitation (CLIP)-based methods have been used to identify the binding sites for hundreds of RNA-binding proteins (RBPs) in vivo. Using these large-scale experimental data to infer RNA binding preference and predict missing binding sites has become a great challenge. Some existing deep-learning models have demonstrated high prediction accuracy for individual RBPs. However, it remains difficult to avoid significant bias due to the experimental protocol. The DeepRiPe method was recently developed to solve this problem via introducing multi-task or multi-label learning into this field. However, this method has not reached an ideal level of prediction power due to the weak neural network architecture.ResultsCompared to the DeepRiPe approach, our Multi-resBind method demonstrated substantial improvements using the same large-scale PAR-CLIP dataset with respect to an increase in the area under the receiver operating characteristic curve and average precision. We conducted extensive experiments to evaluate the impact of various types of input data on the final prediction accuracy. The same approach was used to evaluate the effect of loss functions. Finally, a modified integrated gradient was employed to generate attribution maps. The patterns disentangled from relative contributions according to context offer biological insights into the underlying mechanism of protein-RNA interactions.ConclusionsHere, we propose Multi-resBind as a new multi-label deep-learning approach to infer protein-RNA binding preferences and predict novel interactions. The results clearly demonstrate that Multi-resBind is a promising tool to predict unknown binding sites in vivo and gain biology insights into why the neural network makes a given prediction.

Modified Cross-Linking, Ligation, and Sequencing of Hybrids (qCLASH) to Identify MicroRNA Targets.

This protocol was designed to identify microRNA (miRNA) targetomes from smaller‐input samples by performing a simplified workflow of the Cross‐Linking and Sequencing of Hybrids (CLASH) technique developed in the Tollervey group. In this ribonomics‐based technique, Cross‐Linking and Immunoprecipitation (CLIP) of Argonaute (Ago) is combined with an RNA ligase reaction that yields covalently bound “hybrids” between miRNAs and their target RNAs. While this iteration of CLIP identifies “high‐confidence” or “unambiguous” miRNA targets, the added ligation step is highly inefficient and therefore requires large numbers of cultured cells. To make this powerful approach applicable to smaller cell numbers, we created qCLASH, incorporating a workflow that performs all enzymatic reactions on bead‐bound complexes and omits gel purification of immunoprecipitated Ago complexes associated with major loss of RNA. At a sequencing depth of 100 million reads per library, which is highly feasible with rapidly decreasing sequencing costs, qCLASH, when used with three biological replicates, results in thousands of high‐confidence miRNA targets. qCLASH was first developed to identify viral miRNA targetomes of endothelial cells infected with Kaposi's sarcoma−associated herpesvirus. Since then, qCLASH has been applied to Epstein‐Barr virus− and MHV68‐infected cells, and more recently to metastatic melanoma and breast cancer cells. Currently, protocols are under development to apply qCLASH to human solid tumor specimens. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Quick Cross‐Linking and Sequencing of Hybrids (qCLASH) Support Protocol: Optimization of Ago immunoprecipitation