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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
In the citing paper
“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]
In the citing paper
“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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Citations
Cites
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“Importantly, microRNAs also regulate lncRNAs, as shown in recent global analysis of Argonaute (Ago)-bound transcripts through the HITS-CLIP technique (Chi et al., 2009; Licatalosi et al., 2008).”
BackgroundAlternative isoform regulation in human tissue transcriptomes
Wang, E. T., Sandberg, R., Luo, S., Khrebtukova, I., Zhang, L., Mayr, C., Kingsmore, S. F., Schroth, G. P., & Burge, C. B. (2008) · Nature · doi:10.1038/nature07509 · Cited by 5,181
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“Thus, unlike CLIP-Seq tag clusters from RNA-binding proteins that influence splicing (Licatalosi et al., 2008), mA peaks did not significantly coincide with exon-exon junctions, suggesting that mA is unlikely to primarily function to directly influence the binding of splicing factors.”
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Rinn, J. L., & Chang, H. Y. (2012) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-051410-092902 · Cited by 4,091
“These studies employ protein immunoprecipitation followed by microarray or deep-sequencing to enumerate all RNAs associated with a protein complex of interest (RIPseq) (108) (49, 109); often times crosslinking is performed to trap the relevant interactions in living cells (CLIP-seq) (110).”
Method
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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