Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis
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The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ∼8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3-4 d.
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HITS-CLIP isolates RNA fragments directly bound by an RBP of interest.
“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 Moore et al., 2014
Additional support for this argument comes from predominant crosslinking to different nucleotides for several other RBPs with distinct binding specificities.
“Additional evidence supporting this argument comes from predominant crosslinking to different nucleotides for several other RBPs with distinct binding specificity (Moore et al., 2014).”
From HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism (2014) · cites this paper as Moore et al., 2014
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- Authors
- Moore, M. J., Zhang, C., Gantman, E. C., Mele, A., Darnell, J. C., & Darnell, R. B.
- Affiliations
- Rockefeller University (US)
- Corresponding author
- Darnell, Robert B
- Venue
- Nature Protocols
- Published
- 2014-01-09
- Volume
- 9
- Issue
- 2
- Pages
- 263-293
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC4156013
- OpenAlex
- W2021699617
- Cited by
- 251
- References
- 60
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 14 · 2023: 21 · 2022: 13 · 2021: 24 · 2020: 24 · 2019: 32 · 2018: 25 · 2017: 34 · 2016: 25 · 2015: 21 · 2014: 6
- FWCI
- 11.75
- Type
- journal article
- Language
- en
- Update notices
- erratum (2016-02-25)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (K99GM095713, R00GM095713, K99/R00GM95713)
- National Institute of Neurological Disorders and Stroke (R01NS081706, NS34389, NS081706, R01NS034389, R56NS034389)
- 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 › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- HITS-CLIP, Argonaute proteins, RNA-binding proteins, RNA-protein interactions, microRNA, small regulatory RNAs, RNA regulation
- MeSH terms
- Cross-Linking Reagents, RNA, Ultraviolet Rays, Mutagenesis, RNA-Binding Proteins, Protein Interaction Mapping, Immunoprecipitation, High-Throughput Nucleotide Sequencing, Argonaute Proteins
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