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HITS-CLIP yields genome-wide insights into brain alternative RNA processing
Open accessAbstract
Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo.
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“crosslinking and immunoprecipitation followed by high-throughput sequencing (HITS-CLIP) has been developed to isolate RNA fragments directly bound by an RBP of interest (Darnell, 2010; Licatalosi et al., 2008; Moore et al., 2014; Ule et al., 2005a).”
From HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism (2014) · cites this paper as Licatalosi et al., 2008
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“Indeed, dual functions for splicing regulators in 3′-end poly-A site selection and mRNA transport have been described26,27”
From A compendium of RNA-binding motifs for decoding gene regulation (2013) · cites this paper as [26]
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“Alternative splicing (AS) is a highly tissue-specific process18 that greatly increases the complexity of the potential set of RNA molecules produced from multi-exon genes.”
From Decoding neuroproteomics: integrating the genome, translatome and functional anatomy (2014) · cites this paper as [18]
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Decrypting the genome's alternative messages
Hartmann, B., & Valcárcel, J. (2009) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2009.02.006 · Cited by 67
“Crosslinking / immunoprecipitation followed by deep RNA sequencing (HITS-CLIP) can yield this information [76 ,77 ].”
BackgroundMethodInfluentialDynamic recruitment of microRNAs to their mRNA targets in the regenerating liver
Schug, J., McKenna, L. B., Walton, G., Hand, N., Mukherjee, S., Essuman, K., Shi, Z., Gao, Y., Markley, K., Nakagawa, M., et al. (2013) · BMC Genomics · doi:10.1186/1471-2164-14-264 · Cited by 66
“To identify which miRNAs participate in the regenerative process and to identify their targets, we used a technique pioneered by the Darnell laboratory in which UV cross-linked miRNA: mRNA complexes are immunoprecipitated with an antibody to Argonaute, an essential component of the RISC, and then subjected to deep seq…”
Leggere, J. C., Saito, Y., Darnell, R. B., Tessier-Lavigne, M., Junge, H. J., & Chen, Z. (2016) · eLife · doi:10.7554/elife.14264 · Cited by 67
“fied many potential NOVA targets that are involved in various neural developmental processes (Licatalosi et al., 2008; Ule et al., 2003; Zhang et al., 2010).”
BackgroundRNA-binding proteins and translational regulation in axons and growth cones
Hörnberg, H., & Holt, C. E. (2013) · Frontiers in Neuroscience · doi:10.3389/fnins.2013.00081 · Cited by 67
“New techniques such as crosslinking immunoprecipitation (CLIP) (Ule et al., 2005) and high throughput sequencingCLIP (HITS-CLIP) (Licatalosi et al., 2008) will be valuable in future studies for identifying RBP-mRNA complexes in different axon populations and developmental time points.”
BackgroundTranslational control from head to tail
Groppo, R., & Richter, J. D. (2009) · Current Opinion in Cell Biology · doi:10.1016/j.ceb.2009.01.011 · Cited by 74
Deep-RBPPred: Predicting RNA binding proteins in the proteome scale based on deep learning
Zheng, J., Zhang, X., Zhao, X., Tong, X., Hong, X., Xie, J., & Liu, S. (2018) · Scientific Reports · doi:10.1038/s41598-018-33654-x · Cited by 66
Ravanidis, S., Kattan, F.-G., & Doxakis, E. (2018) · International Journal of Molecular Sciences · doi:10.3390/ijms19082280 · Cited by 70
“A high percentage of NOVA binding sites were also mapped near poly(A) sites in the 3′ UTRs of target mRNAs suggesting a role in alternative polyadenylation [234].”
BackgroundDecoding neuroproteomics: integrating the genome, translatome and functional anatomy
Kitchen, R. R., Rozowsky, J. S., Gerstein, M. B., & Nairn, A. C. (2014) · Nature Neuroscience · doi:10.1038/nn.3829 · Cited by 63
Alternative Splicing of Neuronal Differentiation Factor TRF2 Regulated by HNRNPH1/H2
Grammatikakis, I., Zhang, P., Panda, A. C., Kim, J., Maudsley, S. R., Abdelmohsen, K., Yang, X., Martindale, J. L., Motiño, O., Hutchison, E. R., et al. (2016) · Cell Reports · doi:10.1016/j.celrep.2016.03.080 · Cited by 67
“A characteristic example is the neuronal-specific RBP NOVA, which binds RNA (Licatalosi et al., 2008; Ule et al., 2006) and regulates splicing of genes encoding synaptic proteins (Irimia et al.”
BackgroundHayakawa‐Yano, Y., Suyama, S., Nogami, M., Yugami, M., Koya, I., Furukawa, T., Zhou, L., Abe, M., Sakimura, K., Takebayashi, H., et al. (2017) · Genes & Development · doi:10.1101/gad.300822.117 · Cited by 63
“5 wild-type mouse whole brains with three biological replicates, as described elsewhere with several modifications (Licatalosi et al. 2008; Weyn-Vanhen-tenryck et al. 2014).”
MicroRNA Target Prediction and Validation
Ritchie, W., Rasko, J. E. J., & Flamant, S. (2012) · Advances in experimental medicine and biology · doi:10.1007/978-94-007-5590-1_3 · Cited by 65
Activity-Dependent Alternative Splicing Increases Persistent Sodium Current and Promotes Seizure
Lin, W., Günay, C., Marley, R., Prinz, A. A., & Baines, R. (2012) · Journal of Neuroscience · doi:10.1523/jneurosci.6042-11.2012 · Cited by 64
“5 (Licatalosi et al., 2008; Brooks et al., 2011).”
Chang, X., Li, B., & Rao, A. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1422490112 · Cited by 63
Arabidopsis RNA immunoprecipitation
Terzi, L. C., & Simpson, G. G. (2009) · The Plant Journal · doi:10.1111/j.1365-313x.2009.03859.x · Cited by 62
Pedrotti, S., Giudice, J., Dagnino-Acosta, A., Knoblauch, M. A., Singh, R. K., Hanna, A. D., Mo, Q., Hicks, J. M., Hamilton, S. L., & Cooper, T. A. (2015) · Human Molecular Genetics · doi:10.1093/hmg/ddv003 · Cited by 66
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Big data and deep learning for RNA biology
Hwang, H., Jeon, H., Yeo, N., & Baek, D. (2024) · Experimental & Molecular Medicine · doi:10.1038/s12276-024-01243-w · Cited by 64
“To address this challenge, RBP binding prediction models have been commonly trained using experimental data from RNAcompete 168 and CLIP-based experiments 9 , such as HITS-CLIP 169 , PAR-CLIP 170 , and eCLIP 171 .”
MethodGenomic SELEX: A discovery tool for genomic aptamers
Zimmermann, B., Bilusic, I., Lorenz, C., & Schroeder, R. (2010) · Methods · doi:10.1016/j.ymeth.2010.06.004 · Cited by 62
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“Sam68-cross-linked in vivo immunoprecipitation (CLIP) was performed as previously described (33), with minor modifications.”
MethodRNA regulatory networks diversified through curvature of the PUF protein scaffold
Wilinski, D., Qiu, C., Lapointe, C. P., Nevil, M., Campbell, Z. T., Hall, T. M. T., & Wickens, M. (2015) · Nature Communications · doi:10.1038/ncomms9213 · Cited by 62
“For example, three other PUF proteins – human PUM2 14 , S. cerevisiae Puf4p 6 and C. elegans FBF-2 7 – show a single dominant length of site in vivo , measured either by CLIP methods 23 or inferred from RIP-Chip 24 (Fig.”
Tiny giants of gene regulation: experimental strategies for microRNA functional studies
Steinkraus, B. R., Tögel, M., & Fulga, T. A. (2016) · Wiley Interdisciplinary Reviews Developmental Biology · doi:10.1002/wdev.223 · Cited by 64
Towards understanding pre-mRNA splicing mechanisms and the role of SR proteins
Sahebi, M., Hanafi, M. M., van Wijnen, A. J., Azizi, P., Abiri, R., Ashkani, S., & Taheri, S. (2016) · Gene · doi:10.1016/j.gene.2016.04.057 · Cited by 64
“Most of these approaches are used to reveal binding patterns and consensus sites for RNA-binding proteins as well as to obtain the ‗RNA maps‘, or binding profiles, of certain splicing regulators (Licatalosi et al., 2008; Xue et al., 2009; Yeo et al., 2009; Witten and Ule, 2011).”
Crosslinking of DNA-linked ligands to target proteins for enrichment from DNA-encoded libraries
Denton, K. E., & Krusemark, C. J. (2016) · MedChemComm · doi:10.1039/c6md00288a · Cited by 62
Detection of N6‑methyladenosine modification residues (Review)
Zhu, W., Wang, J., Xu, Z., Cao, M., Hu, Q., Chen, P., Guo, M., Wei, J., & Yang, H. (2019) · International Journal of Molecular Medicine · doi:10.3892/ijmm.2019.4169 · Cited by 65
“Photo‐crosslinking technologies are diffusely applied to stabi‐ lize direct protein‐RNA interactions (63).”
BackgroundThe Future of Cross-Linking and Immunoprecipitation (CLIP)Preprint
Ule, J., Hwang, H., & Darnell, R. B. (2018) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a032243 · Cited by 65
“Although the success of this approach was shown by the very first studies of Nova, CLIP also led to an unexpected finding that nuclear RBPs have many thousands of high-affinity binding sites, most of which are far from exons (Ule et al. 2003; Licatalosi et al. 2008).”
InfluentialSelf-citationHuang, W., Xiong, T., Zhao, Y., Heng, J., Han, G., Wang, P., Zhao, Z., Shi, M., Li, J., Wang, J., et al. (2024) · Nature Genetics · doi:10.1038/s41588-023-01620-7 · Cited by 60
Corioni, M., Antih, N., Tanačković, G., Zavolan, M., & Krämer, A. (2010) · Nucleic Acids Research · doi:10.1093/nar/gkq1042 · Cited by 64
“Interestingly, the alternative splicing factor Nova also binds 30 UTRs and regulates alternative polyadenylation in the brain (38).”
BackgroundSpecific RNP capture with antisense LNA/DNA mixmers
Rogell, B., Fischer, B., Rettel, M., Krijgsveld, J., Castelló, A., & Hentze, M. W. (2017) · RNA · doi:10.1261/rna.060798.117 · Cited by 59
“After immunoprecipitation, RNAs bound to the targeted RBP can be identified by next-generation sequencing (crosslinking, immunoprecipitation and sequencing, CLIP-seq) (Licatalosi et al. 2008; Milek et al. 2012).”
BackgroundToward a Genome-Wide Landscape of Translational ControlPreprint
Larsson, O. S., Tian, B., & Sonenberg, N. (2012) · Cold Spring Harbor Perspectives in Biology · doi:10.1101/cshperspect.a012302 · Cited by 67
“Moreover, there is a possibility that some regulation involves several players, including RBPs, microRNAs, and RNA elements, with linear or nonlinear interactions.”
MethodInfluentialBiogenesis, Functions, Interactions, and Resources of Non-Coding RNAs in Plants
Chao, H., Hu, Y., Zhao, L., Xin, S., Ni, Q., Zhang, P., & Chen, M. (2022) · International Journal of Molecular Sciences · doi:10.3390/ijms23073695 · Cited by 58
“Therefore, a series of approaches were also developed to reveal how and where ncRNAs interacted with corresponding proteins [110,130].”
BackgroundMHCLMDA: multihypergraph contrastive learning for miRNA–disease association prediction
Peng, W., He, Z., Dai, W., & Lan, W. (2023) · Briefings in Bioinformatics · doi:10.1093/bib/bbad524 · Cited by 60
Genetic variation of pre‐mRNA alternative splicing in human populations
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“We expect that availability of transcriptomewide assays for RNA binding proteins (which have ribonuclease activity) like high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) [40] or Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PARCLIP) [41] would…”
BackgroundRBP-Var: a database of functional variants involved in regulation mediated by RNA-binding proteins
Mao, F., Xiao, L., Li, X., Liang, J., Teng, H., Cai, W., & Sun, Z. S. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv1308 · Cited by 58
“CLIP-seq that combines high-throughput sequencing (HITS)-CLIP (16), photoactivatable ribonucleosideenhanced (PAR)-CLIP (17) and individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) (18), is a powerful tool for investigating the in vivo binding sites of RBPs on a transcriptome-wide scale, providing…”
Tissue‐specific mechanisms of alternative polyadenylation: Testis, brain, and beyond (2018 update)
MacDonald, C. C. (2019) · Wiley Interdisciplinary Reviews - RNA · doi:10.1002/wrna.1526 · Cited by 57
On the function and relevance of alternative 3′‐ UTRs in gene expression regulation
Pereira‐Castro, I., & Moreira, A. (2021) · Wiley Interdisciplinary Reviews - RNA · doi:10.1002/wrna.1653 · Cited by 60
“Using HITS-CLIP, it was shown that NOVA associates with YCAY elements near poly(A) sites determining poly(A) site selection in the brain (Licatalosi et al., 2008).”
MethodGenetically encoded chemical crosslinking of RNA in vivo
Sun, W., Wang, N., Liu, H., Yu, B., Jin, L., Ren, X., Shen, Y., & Wang, L. (2022) · Nature Chemistry · doi:10.1038/s41557-022-01038-4 · Cited by 60
RNA-binding protein CPEB1 remodels host and viral RNA landscapes
Batra, R., Stark, T. J., Clark, A. E., Belzile, J.-P., Wheeler, E. C., Yee, B. A., Huang, H., Gelboin-Burkhart, C. A., Huelga, S. C., Aigner, S., et al. (2016) · Nature Structural & Molecular Biology · doi:10.1038/nsmb.3310 · Cited by 59
“Families of RBPs such as Muscleblind-like (MBNL), NOVA, RBFOX, and HNRNPs have been shown to control both AS and APA 17,19,26–29 .”
Hook, L., Hancock, M. H., Landais, I., Grabski, R., Britt, W., & Nelson, J. (2014) · Current Opinion in Virology · doi:10.1016/j.coviro.2014.03.015 · Cited by 66
“Recently, biochemical approaches based on RISC immunoprecipitation (RIP), including RIP-Chip, PAR-CLIP, and HITS-CLIP, have been developed to directly identify targeted transcripts incorporated into the RISC complex [2 ,13 ,19 ,20 ,21,22 ,23,24,25 ,26].”
MethodDeciphering the impact of genetic variation on human polyadenylation using APARENT2
Linder, J., Koplik, S. E., Kundaje, A., & Seelig, G. (2022) · Genome Biology · doi:10.1186/s13059-022-02799-4 · Cited by 49
Transcriptome-wide analysis of protein–RNA interactions using high-throughput sequencing
Milek, M., Wyler, E., & Landthaler, M. (2011) · Seminars in Cell and Developmental Biology · doi:10.1016/j.semcdb.2011.12.001 · Cited by 64
Quaking Regulates Hnrnpa1 Expression through Its 3′ UTR in Oligodendrocyte Precursor Cells
Zearfoss, N. R., Clingman, C. C., Farley, B. M., McCoig, L. M., & Ryder, S. (2011) · PLoS Genetics · doi:10.1371/journal.pgen.1001269 · Cited by 60
“Use of the cross-linked immunoprecipitation technique [53–56] should greatly facilitate such an analysis.”
MethodIntegrated structural biology to unravel molecular mechanisms of protein-RNA recognition
Schlundt, A., Tants, J., & Sattler, M. (2017) · Methods · doi:10.1016/j.ymeth.2017.03.015 · Cited by 59
RNAmotifs: prediction of multivalent RNA motifs that control alternative splicing
Cereda, M., Pozzoli, U., Rot, G., Juvan, P., Schweitzer, A. C., Clark, T. A., & Ule, J. (2014) · Genome biology · doi:10.1186/gb-2014-15-1-r20 · Cited by 58
“Moreover, crosslinking-based methods are affected by mild sequence biases [6]; thus, it is important to develop methods that can derive the regulatory motifs independently of the CLIP data.”
InfluentialSelf-citationThe physiological impact of microRNA gene regulation in the retina
Sundermeier, T. R., & Palczewski, K. (2012) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-012-0976-7 · Cited by 58
Kuroyanagi, H., Watanabe, Y., Suzuki, Y., & Hagiwara, M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt097 · Cited by 55
“The reporters also revealed position-dependent regulation of the UNC-75-target exons by the (G/U)UGUUGUG elements (Figure 2 and Supplementary Figures S2–S5), similar to tissue-specific splicing factors Nova (51,52), RBFOX family (53,54), PTBP1 (55), TIA proteins (56) and TDP-43 (57) in mammals.”
BackgroundThe RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid
York, A., Kutluay, S. B., Errando, M., & Bieniasz, P. D. (2016) · PLoS Pathogens · doi:10.1371/journal.ppat.1005833 · Cited by 62
“The CLIP method that was used in this study has been described previously [42] and was adapted from previously reported HITS-CLIP and PAR-CLIP protocols [43, 44], and is described briefly here.”
MethodA novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing
Angiolini, F., Belloni, E., Giordano, M., Campioni, M., Forneris, F., Paronetto, M. P., Lupia, M., Brandas, C., Pradella, D., Matteo, A. D., et al. (2019) · eLife · doi:10.7554/elife.44305 · Cited by 58
Global Approaches in Studying RNA-Binding Protein Interaction Networks
Sternburg, E. L., & Karginov, F. V. (2020) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2020.03.005 · Cited by 57
Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding
Hacisuleyman, E., Hale, C. R., Noble, N., Luo, J., Fak, J. J., Saito, M., Chen, J., Weissman, J. S., & Darnell, R. B. (2024) · Nature Neuroscience · doi:10.1038/s41593-024-01615-5 · Cited by 56
“To isolate local dendritic mRNAs enriched before and after depolarization, we extended previous CLIP studies 14,26,27 by integrating TurboID proximity ligation (PL) and termed this strategy PL-CLIP.”
MethodSelf-citationCompareads: comparing huge metagenomic experiments
Maillet, N., Lemaitre, C., Chikhi, R., Lavenier, D. D., & Peterlongo, P. (2012) · BMC Bioinformatics · doi:10.1186/1471-2105-13-s19-s10 · Cited by 56
“It also opened the doors to new techniques such as ChipSeq [2], ClipSeq [3], RadSeq [4] and the topic of this work, metagenomics [5].”
MethodAbasic pivot substitution harnesses target specificity of RNA interference
Lee, H.-S., Seok, H., Lee, D. H., Ham, J., Lee, W., Youm, E. M., Yoo, J. S., Lee, Y.-S., Jang, E.-S., & Chi, S. W. (2015) · Nature Communications · doi:10.1038/ncomms10154 · Cited by 58
Self-citationKargapolova, Y., Levin, M., Lackner, K. J., & Danckwardt, S. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx152 · Cited by 55
“Additionally, sCLIP exploits the benefits of an experimental barcoding approach (14,42) and thus allows multiplexing of several samples.”
BackgroundMethodFinding MicroRNA Targets in Plants: Current Status and Perspectives
Ding, J., Zhou, S., & Guan, J. (2012) · Genomics Proteomics & Bioinformatics · doi:10.1016/j.gpb.2012.09.003 · Cited by 57
“At the end, we also present our thinking on the outlook for future directions in the development of plant miRNA target finding methods.”
BackgroundAlternative Polyadenylation: Another Foe in Cancer
Erson-Bensan, A. E., & Can, T. (2016) · Molecular Cancer Research · doi:10.1158/1541-7786.mcr-15-0489 · Cited by 57
“However, the repressive function of NOVA2 is not effective if the poly(A) site is distant from the NOVA2-binding site; hence, polyadenylation of a distant site can be possible (44).”
BackgroundWachter, A., Rühl, C., & Stauffer, E. (2012) · Frontiers in Plant Science · doi:10.3389/fpls.2012.00081 · Cited by 54
“…for instance, revealed that binding around the alternative 5′ or constitutive 3′ splice site typically induces exon inclusion, whereas exon skipping is often associated with binding sites around the constitutive 5′ splice site or the alternative exon (Ule et al., 2006; Licatalosi et al., 2008).”
BackgroundTranscriptional profiling analysis and functional prediction of long noncoding RNAs in cancer
Yuan, J., Yue, H., Zhang, M., Luo, J., Liu, L., Wu, W., Xiao, T., Chen, X., Chen, X., Zhang, D., et al. (2016) · Oncotarget · doi:10.18632/oncotarget.6993 · Cited by 53
“Meanwhile, an integrative application of high-throughput sequencing data beyond level of gene expression, such as PARS-Seq [52] which enables genome-scale reconstruction of RNA secondary structure and CLIP-Seq [53] which allows detection of massive interacting RNAs for a specific protein, will contribute to better und…”
BackgroundConrad, N. K. (2015) · Virus Research · doi:10.1016/j.virusres.2015.06.012 · Cited by 54
Bioinformatics tools for lncRNA research
Iwakiri, J., Hamada, M., & Asai, K. (2015) · Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms · doi:10.1016/j.bbagrm.2015.07.014 · Cited by 55
Connections between chromatin signatures and splicing
Acuña, L. I. G., Fiszbein, A., Alló, M., Schor, I. E., & Kornblihtt, A. R. (2012) · Wiley Interdisciplinary Reviews - RNA · doi:10.1002/wrna.1142 · Cited by 56
Alternative Polyadenylation of mRNAs: 3'-Untranslated Region Matters in Gene Expression
Yeh, H.-S., & Yong, J. (2016) · Molecules and Cells · doi:10.14348/molcells.2016.0035 · Cited by 56
“For example, it has been shown that mRNA transcripts in brain and nervous tissues are prone to usage of distal poly(A) sites, leading to 3’ UTR lengthening, while transcripts in tissues a such as retina and placenta tend to use proximal poly(A) sites (Licatalosi et al., 2008; Zhang et al., 2005).”
BackgroundA Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons
Vidaki, M., Drees, F., Saxena, T., Lanslots, E., Taliaferro, J. M., Tatarakis, A., Burge, C. B., Wang, E. T., & Gertler, F. B. (2017) · Neuron · doi:10.1016/j.neuron.2017.06.048 · Cited by 56
“A modified CLIP protocol with mild lysis and IP conditions improved mRNA recovery in the Mena IP, allowing us to sequence the associated mRNA (Figure 1C: modified High-throughput RNA Sequencing after CLIP; HITS-CLIP, see Star Methods) (Darnell, 2010; Licatalosi et al., 2008).”
RNA-binding proteins and their role in kidney disease
Seufert, L., Benzing, T. R., Ignarski, M., & Müller, R. (2021) · Nature Reviews Nephrology · doi:10.1038/s41581-021-00497-1 · Cited by 57
Hale, C. R., Sawicka, K., Mora, K., Fak, J. J., Kang, J. J., Cutrim, P., Cialowicz, K. I., Carroll, T. S., & Darnell, R. B. (2021) · eLife · doi:10.7554/elife.71892 · Cited by 51
Self-citationThe RNA-Binding Protein Rrm4 is Essential for Efficient Secretion of Endochitinase Cts1
Koepke, J., Kaffarnik, F., Haag, C., Zarnack, K., Luscombe, N. M., König, J., Ule, J., Kellner, R., Begerow, D., & Feldbrügge, M. (2011) · Molecular & Cellular Proteomics · doi:10.1074/mcp.m111.011213 · Cited by 52
“To overcome this limitation, we subjected the CLIP libraries (18) to high-throughput sequencing (39), generating a total of 1,290,949 and 7,594,055 sequence reads (see Experimental procedures).”
MethodResultSelf-citationAsahara, H. (2016) · Journal of Bone Metabolism · doi:10.11005/jbm.2016.23.3.121 · Cited by 53
“High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) is a powerful strategy to determine the direct targets of each miRNA.[44] In HITS-CLIP, chondrocytes are UV cross-linked and an enriched mRNA–miRNA–Ago2 complex obtained by Ago2 immunoprecipitation is treated with RNase to compl…”
BackgroundZhang, Z., & Xing, Y. (2017) · Nucleic Acids Research · doi:10.1093/nar/gkx646 · Cited by 51
“Two popular approaches are CLIP-seq (crosslinking with immunoprecipitation followed by sequencing) (8–10) and RIP-seq (RNA immunoprecipitation followed by sequencing) (11).”
MethodSlutskin, I. V., Weingarten-Gabbay, S., Nir, R., Weinberger, A., & Segal, E. (2018) · Nature Communications · doi:10.1038/s41467-018-02980-z · Cited by 56
“RNA immunoprecipitation-based methods reveal genome-wide physical interactions between the miRISC complex and the target mRNA, but they cannot elucidate which of these interactions have regulatory outcomes 12,13 .”
BackgroundAlexandrov, I. M., Ivshina, M. P., Jung, D. Y., Friedline, R. H., Ko, H. J., Xu, M. F., O’Sullivan-Murphy, B., Bortell, R., Huang, Y., Urano, F., et al. (2012) · PLoS Genetics · doi:10.1371/journal.pgen.1002457 · Cited by 52
“response [15], and neurologic disease [19,20]; they may do so by, for example, controlling RNA decay [15], among other events.”
Evolution of alternative splicing in primate brain transcriptomes
Lin, L., Shen, S., Jiang, P., Sato, S., Davidson, B. L., & Xing, Y. (2010) · Human Molecular Genetics · doi:10.1093/hmg/ddq201 · Cited by 53
High-throughput discovery of post-transcriptional cis-regulatory elements
Wissink, E. M., Fogarty, E. A., & Grimson, A. (2016) · BMC Genomics · doi:10.1186/s12864-016-2479-7 · Cited by 52
“Further screens and other high-throughput approaches, including CLIP-seq [35, 36], could be used to directly test the importance of sequence context by systematically examining the influence of local sequence context on cis -regulatory elements.”
MethodThe PUF binding landscape in metazoan germ cells
Prasad, A. K., Porter, D. F., Kroll-Conner, P., Mohanty, I., Ryan, A. R., Crittenden, S. L., Wickens, M., & Kimble, J. E. (2016) · RNA · doi:10.1261/rna.055871.116 · Cited by 50
“Other CLIP studies have also found crosslink enrichment in regions adjacent to binding elements: for example, PARCLIP of human PUM2 in cell culture (Hafner et al. 2010) and HITS-CLIP of Nova in mouse brain (Licatalosi et al. 2008).”
BackgroundYan, Z., Hamilton, W. L., & Blanchette, M. (2020) · Bioinformatics · doi:10.1093/bioinformatics/btaa456 · Cited by 53
A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol
Anastasakis, D. G., Jacob, A., Κωνσταντινίδου, Π., Meguro, K., Claypool, D. J., Čekan, P., Haase, A. D., & Hafner, M. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab011 · Cited by 47
“Most of the established CLIP protocols that include on-beads adapter ligation steps, including HiTS-CLIP (23), iCLIP (24), irCLIP (25) and eCLIP (26), circumvent this problem by isolating a larger area above the unligated RBP (27), which may include nonspecific RNAs or RNAs interacting with additional, copurified RBPs…”
BackgroundMethodMicroRNA Target Identification—Experimental Approaches
Martínez-Sánchez, A., & Murphy, C. L. (2013) · Biology · doi:10.3390/biology2010189 · Cited by 56
“The same group had previously used this technique for the identification of protein-RNA interactions in living tissues in a genome-wide manner [82].”
Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases
Jazurek, M., Ciesiołka, A., Staręga‐Rosłan, J., Bilińska, K., & Krzyżosiak, W. J. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkw803 · Cited by 51
“However, due to the post-lysis reorganization of co-immunoprecipitated complexes (112) and the inability to distinguish direct from indirect RNA–protein interactions, several improvements in RIP analysis, such as CLIP (113,114), HiTS CLIP (115,116), PAR-CLIP (18,117), iCLIP (118,119) and eCLIP (120,121), have been dev…”
MethodThe RNA exosome nuclease complex regulates human embryonic stem cell differentiation
Belair, C., Sim, S., Kim, K., Tanaka, Y., Park, I., and, & Wolin, S. L. (2019) · The Journal of Cell Biology · doi:10.1083/jcb.201811148 · Cited by 51
“We cross-linked proteins to their targets with UV (254 nm) followed by immunoprecipitation and HITSCLIP (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation; Licatalosi et al., 2008).”
MethodHigh-throughput approaches to profile RNA-protein interactions
Nechay, M., & Kleiner, R. E. (2019) · Current Opinion in Chemical Biology · doi:10.1016/j.cbpa.2019.11.002 · Cited by 49
A large‐scale functional screen identifies Nova1 and Ncoa3 as regulators of neuronal miRNA function
Störchel, P. H., Thümmler, J., Siegel, G., Aksoy‐Aksel, A., Zampa, F., Sumer, S., & Schratt, G. M. (2015) · The EMBO Journal · doi:10.15252/embj.201490643 · Cited by 50
“The previously reported Nova1 target Rgs4 served as a positive control (Licatalosi et al , 2008).”
Computational analysis of CLIP-seq data
Uhl, M., Houwaart, T., Corrado, G., Wright, P. R., & Backofen, R. (2017) · Methods · doi:10.1016/j.ymeth.2017.02.006 · Cited by 50
“Particularly, CLIP-seq (cross-linking and immunoprecipitation followed by next generation sequencing) [14] has become the standard experimental procedure for studying transcriptome-wide RBP binding.”
BackgroundMethodBaumgarten, S., Cziesielski, M. J., Thomas, L. A., Michell, C., Esherick, L. Y., Pringle, J. R., Aranda, M., & Voolstra, C. R. (2017) · Molecular Ecology · doi:10.1111/mec.14452 · Cited by 48
“…(€Ank€o & Neugebauer, 2012; Licatalosi & Darnell, 2010) including specific interactions in cancer cells (van Kouwenhove, Kedde, & Agami, 2011), neurons (Darnell, 2013; Licatalosi et al., 2008; Ule et al., 2003) and endoparasite pathogenesis (Vembar, Macpherson, Sismeiro, Copp ee, & Scherf, 2015).”
Gámez‐Valero, A., & Beyer, K. (2018) · Genes · doi:10.3390/genes9020063 · Cited by 51
“Consensus sequences and binding motifs for both are significantly enriched in 3’UTR regions [163].”
BackgroundCBRPP: a new RNA-centric method to study RNA–protein interactions
Li, Y., Liu, S., Cao, L., Luo, Y., Du, H., Li, S., Zhang, Z., Guo, X., Tian, W., Wong, C. C. L., et al. (2021) · RNA Biology · doi:10.1080/15476286.2021.1873620 · Cited by 49
“CLIP-seq is very similar to RIP-seq, except for an additional crosslinking step [13].”
MethodSong, C., & He, C. (2011) · Accounts of Chemical Research · doi:10.1021/ar2000502 · Cited by 49
Global Promotion of Alternative Internal Exon Usage by mRNA 3′ End Formation Factors
Misra, A., Ou, J., Zhu, L. J., & Green, M. R. (2015) · Molecular Cell · doi:10.1016/j.molcel.2015.03.016 · Cited by 50
“Consistent with this idea, genome-wide studies have identified a large number of RBFOX- and NOVA-binding sites in 30 UTRs, implicating these proteins as regulators of alternative polyadenylation (Licatalosi et al., 2008; Wang et al., 2008).”
Rbfox3 controls the biogenesis of a subset of microRNAs
Kim, K. K., Yang, Y., Zhu, J., Adelstein, R. S., & Kawamoto, S. (2014) · Nature Structural & Molecular Biology · doi:10.1038/nsmb.2892 · Cited by 54
Yoon, J., & Gorospe, M. (2015) · Methods in molecular biology · doi:10.1007/978-1-4939-3378-5_2 · Cited by 48
“Subsequently, RBP cross-linking using UVC light (254 nm) and immunoprecipitation (CLIP) was developed, initially combined with Sanger Sequencing method [11] and later with highthroughput sequencing (RNA-seq) of the CLIP cDNA library [12, 13].”
MethodBorkiewicz, L., Kałafut, J., Dudziak, K., Przybyszewska-Podstawka, A., & Telejko, I. (2021) · Cancers · doi:10.3390/cancers13112643 · Cited by 48
Xing, D., Zhao, H., & Li, Q. Q. (2008) · PLANT PHYSIOLOGY · doi:10.1104/pp.108.129817 · Cited by 48
Shen, T., Li, H., Song, Y., Li, L., Lin, J., Wei, G., & Ni, T. (2019) · Aging · doi:10.18632/aging.101836 · Cited by 48
Noncoding RNA crosstalk in brain health and diseases
Mehta, S. L., Chokkalla, A. K., & Vemuganti, R. (2021) · Neurochemistry International · doi:10.1016/j.neuint.2021.105139 · Cited by 47
Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation
Ye, R., Hu, N., Cao, C., Su, R., Xu, S., Chen, Y., Zhou, X., & Xue, Y. (2023) · Molecular Cell · doi:10.1016/j.molcel.2023.03.001 · Cited by 52
Zhu, H., Yang, Y., Wang, Y., Wang, F., Huang, Y., Chang, Y., Wong, K., & Li, X. (2023) · Nature Communications · doi:10.1038/s41467-023-42547-1 · Cited by 48
“Moreover, HDRNet had better performance for RBPs with high and low expression levels and for target RNA events with high and low expression levels in different cellular contexts, as illustrated in Supplementary Note 7 and Supplementary Figs.”
InfluentialRNA Stimulates Aurora B Kinase Activity during Mitosis
Jambhekar, A., Emerman, A. B., Schweidenback, C. T. H., & Blower, M. D. (2014) · PLoS ONE · doi:10.1371/journal.pone.0100748 · Cited by 48
Allen, S. E., Darnell, R. B., & Lipscombe, D. (2010) · Channels · doi:10.4161/chan.4.6.12868 · Cited by 48
“Splicing regulation of e24a by Nova is conserved in the homologous Cacna1b genes of zebrafish and chicken, strongly suggesting that this AS exon plays a critical role in optimizing neuronal function in specific regions of the nervous system.(28) Additional Nova-2 binding sites have been identified in Cacna1b, motivati…”
BackgroundMethodSelf-citationMulti-disciplinary methods to define RNA–protein interactions and regulatory networks
Ascano, M., Gerstberger, S. C., & Tuschl, T. (2013) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2013.01.003 · Cited by 48
“The majority of methods for identification of binding sites of RBPs employ the use of an enrichment protocol, either by immunoprecipitation of the RBP of interest [1,2 ,3 ] or affinity purification of an ectopically expressed tagged form of the RBP, for example, by a His-tag [4,5].”
MethodResultNon-coding RNA and pseudogenes in neurodegenerative diseases: “The (un)Usual Suspects”
Costa, V., Esposito, R., Aprile, M., & Ciccodicola, A. (2012) · Frontiers in Genetics · doi:10.3389/fgene.2012.00231 · Cited by 49
“…et al., 2011; Tay et al., 2011), proposes that virtually all types of RNA can communicate each other through a new fascinating“biological alphabet,” in which MREs are the “letters” whose different combinations may form an entire universe of “words” (Licatalosi et al., 2008; Chi et al., 2009).”
BackgroundKapeli, K., & Yeo, G. W.-M. (2012) · Frontiers in Neuroscience · doi:10.3389/fnins.2012.00144 · Cited by 50
“The application of HITS-CLIP in heterogenic mouse brain tissue is standard (Licatalosi et al., 2008; Polymenidou et al., 2011), and in theory the technology required to perform HITS-CLIP in a cell type specific manner in vivo is obvious.”
MethodAlterations in Polyadenylation and Its Implications for Endocrine Disease
Rehfeld, A., Plass, M., Krogh, A., & Friis-Hansen, L. J. (2013) · Frontiers in Endocrinology · doi:10.3389/fendo.2013.00053 · Cited by 52
Metadata
- Authors
- Licatalosi, D. D., Mele, A., Fak, J. J., Ule, J., Kayikci, M., Chi, S. W., Clark, T. A., Schweitzer, A. C., Blume, J. E., Wang, X., Darnell, J. C., & Darnell, R. B.
- Affiliations
- Howard Hughes Medical Institute (US)
- MRC Laboratory of Molecular Biology (GB)
- Affymetrix (United States) (US)
- Rockefeller University (US)
- Corresponding author
- Darnell, Robert B.
- ORCID
- Venue
- Nature
- Published
- 2008-11
- Volume
- 456
- Issue
- 7221
- Pages
- 464-469
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC2597294
- OpenAlex
- W2044117548
- Cited by
- 1,352
- References
- 51
- Citations by year
- 2026: 37 · 2025: 53 · 2024: 46 · 2023: 44 · 2022: 72 · 2021: 81 · 2020: 59 · 2019: 77 · 2018: 85 · 2017: 89 · 2016: 95 · 2015: 97 · 2014: 103 · 2013: 89 · 2012: 111
- FWCI
- 29.24
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- UCL Discovery (University College London) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of Neurological Disorders and Stroke (R01NS040955, R01 NS34389, R01NS034389)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD040647)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
- Keywords
- HITS-CLIP, RNA-protein interactions, alternative splicing, nova, alternative polyadenylation, 3' untranslated region, RNA binding sites, brain, neurons, splicing factors, genome-wide mapping
- MeSH terms
- Neuro-Oncological Ventral Antigen, Animals, Antigens, Neoplasm, Cell Line, Cross-Linking Reagents, Exons, Humans, Neurons, Organ Specificity, RNA, Messenger, RNA-Binding Proteins, Genome, Alternative Splicing, Neocortex, Genomics, Polyadenylation, Immunoprecipitation, Mice
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