iCLIP Data 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 ).

Racoon_clip-a complete pipeline for single-nucleotide analyses of iCLIP and eCLIP data.

A vast variety of biological questions connected to RNA-binding proteins can be tackled with UV crosslinking and immunoprecipitation (CLIP) experiments. However, the processing and analysis of CLIP data are rather complex. Moreover, different types of CLIP experiments like iCLIP or eCLIP are often processed in different ways, reducing comparability between multiple experiments. Therefore, we aimed to build an easy-to-use computational tool for the processing of CLIP data that can be used for both iCLIP and eCLIP data, as well as data from other truncation-based CLIP methods. Here, we introduce racoon_clip, a sustainable and fully automated pipeline for the complete processing of iCLIP and eCLIP data to extract RNA binding signal at single-nucleotide resolution. racoon_clip is easy to install and execute, with multiple pre-settings and fully customizable parameters, and outputs a conclusive summary report with visualizations and statistics for all analysis steps. racoon_clip is implemented as a Snakemake-powered command line tool (Snakemake version ≥7.22, Python version ≥3.9). The latest release can be downloaded from GitHub (https://github.com/ZarnackGroup/racoon_clip/tree/main) and installed via pip. A detailed documentation, including installation, usage, and customization, can be found at https://racoon-clip.readthedocs.io/en/latest/. The example datasets can be downloaded from the Short Read Archive (SRA; iCLIP: SRR5646576, SRR5646577, SRR5646578) or the ENCODE Project (eCLIP: ENCSR202BFN).

Arabidopsis thaliana GLYCINE RICH RNA-BINDING PROTEIN 7 interaction with its iCLIP target LHCB1.1 correlates with changes in RNA stability and circadian oscillation.

The importance of RNA-binding proteins (RBPs) for plant responses to environmental stimuli and development is well documented. Insights into the portfolio of RNAs they recognize, however, clearly lack behind the understanding gathered in non-plant model organisms. Here, we characterize binding of the circadian clock-regulated Arabidopsis thaliana GLYCINE-RICH RNA-BINDING PROTEIN 7 (AtGRP7) to its target transcripts. We identified novel RNA targets from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) data using an improved bioinformatics pipeline that will be broadly applicable to plant RBP iCLIP data. 2705 transcripts with binding sites were identified in plants expressing AtGRP7-GFP that were not recovered in plants expressing an RNA-binding dead variant or GFP alone. A conserved RNA motif enriched in uridine residues was identified at the AtGRP7 binding sites. NMR titrations confirmed the preference of AtGRP7 for RNAs with a central U-rich motif. Among the bound RNAs, circadian clock-regulated transcripts were overrepresented. Peak abundance of the LHCB1.1 transcript encoding a chlorophyll-binding protein was reduced in plants overexpressing AtGRP7 whereas it was elevated in atgrp7 mutants, indicating that LHCB1.1 was regulated by AtGRP7 in a dose-dependent manner. In plants overexpressing AtGRP7, the LHCB1.1 half-life was shorter compared to wild-type plants whereas in atgrp7 mutant plants, the half-life was significantly longer. Thus, AtGRP7 modulates circadian oscillations of its in vivo binding target LHCB1.1 by affecting RNA stability.

A Pipeline for Analyzing eCLIP and iCLIP Data with Htseq-clip and DEWSeq.

Individual-nucleotide crosslinking and immunoprecipitation (iCLIP) sequencing and its derivative enhanced CLIP (eCLIP) sequencing are methods for the transcriptome-wide detection of binding sites of RNA-binding proteins (RBPs). This chapter provides a stepwise tutorial for analyzing iCLIP and eCLIP data with replicates and size-matched input (SMI) controls after read alignment using our open-source tools htseq-clip and DEWSeq. This includes the preparation of gene annotation, extraction, and preprocessing of truncation sites and the detection of significantly enriched binding sites using a sliding window based approach suitable for different binding modes of RBPs.

Galaxy CLIP-Explorer: a web server for CLIP-Seq data analysis.

BackgroundPost-transcriptional regulation via RNA-binding proteins plays a fundamental role in every organism, but the regulatory mechanisms lack important understanding. Nevertheless, they can be elucidated by cross-linking immunoprecipitation in combination with high-throughput sequencing (CLIP-Seq). CLIP-Seq answers questions about the functional role of an RNA-binding protein and its targets by determining binding sites on a nucleotide level and associated sequence and structural binding patterns. In recent years the amount of CLIP-Seq data skyrocketed, urging the need for an automatic data analysis that can deal with different experimental set-ups. However, noncanonical data, new protocols, and a huge variety of tools, especially for peak calling, made it difficult to define a standard.FindingsCLIP-Explorer is a flexible and reproducible data analysis pipeline for iCLIP data that supports for the first time eCLIP, FLASH, and uvCLAP data. Individual steps like peak calling can be changed to adapt to different experimental settings. We validate CLIP-Explorer on eCLIP data, finding similar or nearly identical motifs for various proteins in comparison with other databases. In addition, we detect new sequence motifs for PTBP1 and U2AF2. Finally, we optimize the peak calling with 3 different peak callers on RBFOX2 data, discuss the difficulty of the peak-calling step, and give advice for different experimental set-ups.ConclusionCLIP-Explorer finally fills the demand for a flexible CLIP-Seq data analysis pipeline that is applicable to the up-to-date CLIP protocols. The article further shows the limitations of current peak-calling algorithms and the importance of a robust peak detection.

SEQing: web-based visualization of iCLIP and RNA-seq data in an interactive python framework

BackgroundRNA-binding proteins interact with their target RNAs at specific sites. These binding sites can be determined genome-wide through individual nucleotide resolution crosslinking immunoprecipitation (iCLIP). Subsequently, the binding sites have to be visualized. So far, no visualization tool exists that is easily accessible but also supports restricted access so that data can be shared among collaborators.ResultsHere we present SEQing, a customizable interactive dashboard to visualize crosslink sites on target genes of RNA-binding proteins that have been obtained by iCLIP. Moreover, SEQing supports RNA-seq data that can be displayed in a different window tab. This allows, e.g. crossreferencing the iCLIP data with genes differentially expressed in mutants of the RBP and thus obtain some insights into a potential functional relevance of the binding sites. Additionally, detailed information on the target genes can be incorporated in another tab.ConclusionSEQing is written in Python3 and runs on Linux. The web-based access makes iCLIP data easily accessible, even with mobile devices. SEQing is customizable in many ways and has also the option to be secured by a password. The source code is available at https://github.com/malewins/SEQing.

ICLIP data analysis: A complete pipeline from sequencing reads to RBP binding sites.

Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.

Toward Identifying Subnetworks from FBF Binding Landscapes in Caenorhabditis Spermatogenic or Oogenic Germlines.

Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to “disentangle” these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25°; for comparison, wild-type oogenic hermaphrodites were also raised at 25°. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.

Abstract 4982: Oncogenic role of THOR, a conserved cancer/testis long noncoding RNA

Abstract Large scale transcriptome sequencing efforts have vastly expanded the catalog of long non-coding RNAs (lncRNAs) with varying evolutionary conservation, lineage expression, and cancer specificity. Through a recent large-scale transcriptomic analysis over 6,500 tumor RNA-seq samples, we discovered over 50,000 lncRNAs in the human genome, many of which exhibited highly evolutionarily conserved sequence patterns. Building upon the discovery of these highly conserved lncRNAs, we functionally characterized a novel ultraconserved lncRNA, THOR, which exhibits expression exclusively in testis and a broad range of human cancers. We establish THOR as the first discovered cancer/testis lncRNA, and further investigated its functional significance. THOR knockdown and overexpression in multiple cell lines and animal models alters cell or tumor growth supporting an oncogenic role. Namely, we generated CRISPR knockout cell line models, showing a definitive role for THOR in cancer progression. Additionally, given the sequence conservation of THOR through the mouse and zebrafish, we generated a zebrafish knockout model, and also a zebrafish overexpression model for THOR. Through RNA-pulldown followed by mass spectrometry, we discovered a conserved interaction of THOR with the RNA binding protein, IGF2BP1, in both human and zebrafish cells. We further show that THOR contributes to the mRNA stabilization activities of IGF2BP1. These findings are corroborated by iCLIP data for IGF2BP1. Notably, transgenic THOR knockout produced fertilization defects in zebrafish and also conferred a resistance to melanoma onset in an NRAS K61-induced zebrafish melanoma model. Likewise, ectopic expression of human THOR in zebrafish accelerated the onset of melanoma. THOR represents a novel class of functionally important cancer/testis lncRNAs whose structure and function have undergone positive evolutionary selection. Citation Format: Yashar Niknafs, Yasuyuki Hosono, Matthew K, John Prensner, Rohit Mehra, Sethuramasundaram Pitchiaya, Jean Tien, Rohit Malik, Weibin Zhao, Arul Chinnaiyan. Oncogenic role of THOR, a conserved cancer/testis long noncoding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4982.

Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNA-bound proteome. This protein constellation consists of many conserved RBPs, some of which are potential stage-specific mRNA interactors that likely reflect the dynamics of RNA–protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of these RBPs in embryogenesis, we focused on Hnrnpa1. iCLIP and subsequent mRNA reporter assays revealed a function for Hnrnpa1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3′ UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that Hnrnpa1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-mir-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of Hnrnpa1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA–protein interactions during vertebrate MZT and shows that Hnrnpa1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.

UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy.

Unique Molecular Identifiers (UMIs) are random oligonucleotide barcodes that are increasingly used in high-throughput sequencing experiments. Through a UMI, identical copies arising from distinct molecules can be distinguished from those arising through PCR amplification of the same molecule. However, bioinformatic methods to leverage the information from UMIs have yet to be formalized. In particular, sequencing errors in the UMI sequence are often ignored or else resolved in an ad hoc manner. We show that errors in the UMI sequence are common and introduce network-based methods to account for these errors when identifying PCR duplicates. Using these methods, we demonstrate improved quantification accuracy both under simulated conditions and real iCLIP and single-cell RNA-seq data sets. Reproducibility between iCLIP replicates and single-cell RNA-seq clustering are both improved using our proposed network-based method, demonstrating the value of properly accounting for errors in UMIs. These methods are implemented in the open source UMI-tools software package.

An IGF2BP3-Cdk9 Pathway Governs the Human Fetal-Adult Megakaryocyte Transition

Hematopoietic transitions that accompany fetal development, for example erythroid globin chain switching, play important roles in normal physiology and disease development. In the megakaryocyte lineage, human fetal progenitors show impaired execution of the morphogenesis program of enlargement, polyploidization, and proplatelet formation. Although these defects decline with gestational stage, they remain sufficiently severe at birth to predispose newborns to thrombocytopenia. These defects also contribute to inferior platelet recovery after cord blood stem cell transplantation and to inefficient platelet production by megakaryocytes (Mk) derived from pluripotent stem cells. In this study, comparison of neonatal versus adult human progenitors identified a blockade in the specialized positive transcription elongation factor b (P-TEFb) activation mechanism known to drive adult Mk morphogenesis. A central feature of this pathway is known to involve sustained high amplitude activation of the P-TEFb kinase (Cdk9/cyclin T1). This cascade is initiated by downregulation of core components of the repressive complex of P-TEFb: the 7SK snRNP consisting of the 7SK small nuclear RNA (7SK) and its stabilizing factors MePCE and LARP7. The resulting high amplitude activation of P-TEFb drives multiple features of Mk differentiation: induction of cytoskeletal morphogenetic factors (ACTN1, FLNA, MKL1, HIC5), silencing of erythroid genes, promotion of histone H2B K120 monoubiquitiniation (H2BUb1), and phosphorylation of Spt5 at T806 (pSpt5 T806). Critical, rate-limiting steps triggering this pathway comprise MePCE proteolysis by calpain 2 and downregulation of LARP7, both resulting in destabilization of 7SK.In comparison to Mk derived from adult peripheral blood stem cells (adult Mk), Mk derived from umbilical cord blood stem cells (neonatal Mk) showed evidence of decreased P-TEFb activation with decreases in: 1) the expression of the cytoskeletal morphogenetic factors, 2) global H2BUb1, and 3) pSpt5 T806. In addition, neonatal Mk retained expression of the erythroid marker glycophorin A (GPA). Surprisingly, neonatal Mk failed to downregulate 7SK despite the downregulation of its stabilizers MePCE and LARP7, suggesting the existence of alternative 7SK stabilizing factor/s unique to neonatal Mk. Our screening identified the oncofetal RNA-binding protein IGF2BP3 to be expressed in neonatal but not adult Mk, and ectopic expression of IGF2BP3 in adult Mk conferred neonatal phenotypic features including reduction in size, increased proliferation and leaky erythroid antigen expression. Immunoprecipitation and glycerol gradient studies indicated the participation of IGF2BP3 in the 7SK snRNP complex. Mining of endogenous IGF2BP3 iCLIP data indicated that 7SK is one of the top direct targets of IGF2BP3 and further mapped binding to the 7SK fourth hairpin, a critical stability determinant.In loss of function studies, the knockdown of IGF2BP3 in neonatal Mk resulted in destabilization of 7SK and upregulation of the Mk morphogenetic cytoskeletal factors, as well as increased levels of H2BUb1, pSpt5 T806, and hyperphosphorylated RNA Pol II. Phenotypically, the knockdown of IGF2BP3 in neonatal Mk elicited adult features including increased size, enhanced polyploidization, reduced proliferation and silencing of erythroid antigen expression. Collectively, these findings suggest that the block in P-TEFb activation in neonatal Mk results from ontogentic stage-specific expression of IGF2BP3 which prevents the 7SK destabilization normally associated with adult megakaryocytic P-TEFb activation. We also identified a pharmacologic approach to inhibit IGF2BP3 expression, through inhibition of bromodomain and extra- terminal (BET) proteins, which reproducibly promoted adult features in neonatal Mk including enlargement, inhibition of erythroid antigen expression, upregulation of morphogenetic cytoskeletal factors, and increased platelet formation in vitro. Enforced expression of IGF2BP3 in neonatal Mk significantly blunted the effects of BET inhibitors indicating the specificity of their action in downregulating IGF2BP3. These results identify IGF2BP3 as a human ontogenic masterswitch that restricts megakaryocyte development through modulating a lineage-specific P-TEFb activation mechanism, revealing new strategies toward enhancing platelet production. DisclosuresNo relevant conflicts of interest to declare.

Je, a versatile suite to handle multiplexed NGS libraries with unique molecular identifiers.

BackgroundThe yield obtained from next generation sequencers has increased almost exponentially in recent years, making sample multiplexing common practice. While barcodes (known sequences of fixed length) primarily encode the sample identity of sequenced DNA fragments, barcodes made of random sequences (Unique Molecular Identifier or UMIs) are often used to distinguish between PCR duplicates and transcript abundance in, for example, single-cell RNA sequencing (scRNA-seq). In paired-end sequencing, different barcodes can be inserted at each fragment end to either increase the number of multiplexed samples in the library or to use one of the barcodes as UMI. Alternatively, UMIs can be combined with the sample barcodes into composite barcodes, or with standard Illumina® indexing. Subsequent analysis must take read duplicates and sample identity into account, by identifying UMIs.ResultsExisting tools do not support these complex barcoding configurations and custom code development is frequently required. Here, we present Je, a suite of tools that accommodates complex barcoding strategies, extracts UMIs and filters read duplicates taking UMIs into account. Using Je on publicly available scRNA-seq and iCLIP data containing UMIs, the number of unique reads increased by up to 36 %, compared to when UMIs are ignored.ConclusionsJe is implemented in JAVA and uses the Picard API. Code, executables and documentation are freely available at http://gbcs.embl.de/Je. Je can also be easily installed in Galaxy through the Galaxy toolshed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1284-2) contains supplementary material, which is available to authorized users.

The PUF binding landscape in metazoan germ cells.

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their “binding landscape”). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF–RNA interactions. FBF-1 and FBF-2 can bind sites in the 5′UTR, coding region, or 3′UTR, but have a strong bias for the 3′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.

Modeling Protein-RNA Complexes

The advent of high-throughput technologies has led to the identification of many new in vivo protein-RNA interactions. While much work has been done to characterize these interactions based on secondary structures of RNA, less effort has been placed to identify three-dimensional (3D) motifs of RNA that are essential for protein-RNA recognition. This discrepancy in effort is not for lack of interest, but rather arises due to the significant challenges and complexities in modeling protein-RNA complexes in 3D. The challenges, in general, are twofold: i) sampling possible protein-RNA interactions and ii) ranking models based on their likelihood to exist in the cell; this process is also known as “scoring”. The former is often intractable - the flexibility of RNA adds significant complexity to the modeling process - but sampling search space can be reduced with experimental input of possible interacting regions (e.g. from HITS-CLIP, PAR-CLIP or iCLIP data). We designed a new 3D modeling protocol to generate reasonable models of protein-RNA complexes, by accounting for protein flexibility (using torsional angle sampling) and RNA flexibility (using previously developed “natural moves” for RNA). Using this protocol, we are able to obtain native-like protein-RNA interactions, even when modeling RNA structures starting from the unbound conformation. As expected, including experimental constraints significantly improves sampling efficiency. Because our protein-RNA models are robust and diverse, they serve as benchmark decoy datasets for evaluating protein-RNA scoring functions - the second aforementioned challenge. With our decoy set we are able to identify constructive features of different scoring functions, and also highlight limitations requiring further development. With improved scoring functions and known interacting regions probed from experiments, we foresee that our protocol can be used to effectively model novel protein-RNA complexes in 3D.

Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data.

Alternative splicing is a key mechanism for generating proteome diversity, however the mechanisms regulating alternative splicing are poorly understood. Using a genome-wide RNA interference screening strategy, we identified cleavage and polyadenylation specificity factor (CPSF) and symplekin (SYMPK) as cofactors of the well-known splicing regulator RBFOX2. To determine the role of CPSF in alternative splicing on a genome-wide level, we performed paired-end RNA sequencing (RNA-seq) to compare splicing events in control cells and RBFOX2 or CPSF2 knockdown cells. We also performed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify direct binding targets of RBFOX2 and CPSF2. Here, we describe the experimental design, and the quality control and data analyses that were performed on the dataset. The raw sequencing data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE60392.

A multiprotein occupancy map of the mRNP on the 3' end of histone mRNAs.

The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem–loop (SL) structure. This SL structure interacts with the stem–loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3′hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3′ end of RD-histone mRNAs together with SLBP.

Rbfox2-Coordinated Alternative Splicing of Mef2d and Rock2 Controls Myoblast Fusion during Myogenesis

Alternative splicing plays important regulatory roles during periods of physiological change. During development, a large number of genes coordinately express protein isoform transitions regulated by alternative splicing; however, the mechanisms that coordinate splicing and the functional integration of the resultant tissue-specific protein isoforms are typically unknown. Here we show that the conserved Rbfox2 RNA binding protein regulates 30% of the splicing transitions observed during myogenesis and is required for the specific step of myoblast fusion. Integration of Rbfox2-dependent splicing outcomes from RNA-seq with Rbfox2 iCLIP data identified Mef2d and Rock2 as Rbfox2 splicing targets. Restored activities of Mef2d and Rock2 rescued myoblast fusion in Rbfox2-depleted cultures, demonstrating functional cooperation of protein isoforms generated by coordinated alterative splicing. The results demonstrate that coordinated alternative splicing by a single RNA binding protein modulates transcription (Mef2d) and cell signaling (Rock2) programs to drive tissue-specific functions (cell fusion) to promote a developmental transition.

Direct Competition between hnRNP C and U2AF65 Protects the Transcriptome from the Exonization of Alu Elements

SummaryThere are ∼650,000 Alu elements in transcribed regions of the human genome. These elements contain cryptic splice sites, so they are in constant danger of aberrant incorporation into mature transcripts. Despite posing a major threat to transcriptome integrity, little is known about the molecular mechanisms preventing their inclusion. Here, we present a mechanism for protecting the human transcriptome from the aberrant exonization of transposable elements. Quantitative iCLIP data show that the RNA-binding protein hnRNP C competes with the splicing factor U2AF65 at many genuine and cryptic splice sites. Loss of hnRNP C leads to formation of previously suppressed Alu exons, which severely disrupt transcript function. Minigene experiments explain disease-associated mutations in Alu elements that hamper hnRNP C binding. Thus, by preventing U2AF65 binding to Alu elements, hnRNP C plays a critical role as a genome-wide sentinel protecting the transcriptome. The findings have important implications for human evolution and disease.

ICLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV-cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data demonstrate that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons, but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an ‘RNA map’ indicates how the positioning of hnRNP particles determines their effect on inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.