Abstract
HITS-CLIP (High-Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation, CLIP-Seq) libraries contain fragments of the RNA sequences bound in vivo by an RNA binding protein (RBP). Such fragments, especially if they represent RNA duplexes bound in vivo by the RBP, can occasionally be ligated together to form chimeric CLIP tags. Chimeric CLIP tags from Argonaute CLIP libraries can provide the exact base pairing profiles of small RNAs with their target RNA sequences, thus solving a critical problem in the field of post-transcriptional regulation. We recently reported an analysis of chimeric reads from the Drosophila Piwi protein Aubergine, which revealed a novel mechanism for mRNA entrapment within germ RNP granules. We term this novel approach chimeric CLIP (cCLIP) and present here the main steps that a researcher can take after the acquisition of the deep sequencing data, for the identification of candidate chimeric reads in Piwi CLIP libraries. Extending the scope beyond small-RNA binding proteins, we believe that cCLIP can be utilized to elucidate the in vivo functions of RNA-binding proteins in general, and especially those that modulate RNA secondary structures. We, therefore, also describe aspects of the generalized chimeric read identification problem, which can find use in the analysis of the CLIP libraries of any RNA-binding protein.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Licatalosi DD et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456:464–469
Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479–486
Ule J et al (2003) CLIP identifies nova-regulated RNA networks in the brain. Science 302(5648):1212–1215
Kameswaran V et al (2013) Epigenetic regulation of the DLK1-MEG3 MicroRNA cluster in human type 2 diabetic islets. Cell Metab 19:135–145
Vourekas A, Alexiou P, Vrettos N, Maragkakis M, Mourelatos Z (2016) Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature 531:390–394
Grosswendt S et al (2014) Unambiguous identification of miRNA: target site interactions by different types of ligation reactions. Mol Cell 54:1042–1054
Sugimoto Y et al (2015) {hiCLIP} reveals the in vivo atlas of {mRNA} secondary structures recognized by Staufen 1. Nature 519:491–494
Helwak A, Kudla G, Dudnakova T, Tollervey D (2013) Mapping the human miRNA interactome by CLASH reveals frequent noncanonical binding. Cell 153:654–665
Moore MJ et al (2015) miRNA-target chimeras reveal miRNA 3′-end pairing as a major determinant of Argonaute target specificity. Nat Commun 6:8864
Vourekas A, Mourelatos Z (2014) HITS-CLIP (CLIP-Seq) for mouse piwi proteins. Methods Mol Biol 1093:73–95
Popow J et al (2011) HSPC117 is the essential subunit of a human tRNA splicing ligase complex. Science 331:760–764
Vourekas A et al (2015) The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing. Genes Dev 29:617–629
Yang Q, Fairman ME, Jankowsky E (2007) DEAD-box-protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values. J Mol Biol 368:1087–1100
Maragkakis M, Alexiou P, Mourelatos Z (2015) GenOO: a modern perl framework for high throughput sequencing analysis. bioRxiv. doi:10.1101/019265
Smith TF, Waterman MS (1981) Identification of common molecular subsequences. J Mol Biol 147:195–197
Acknowledgments
We thank members of the Mourelatos laboratory for discussions. This research was supported by NIH grant GM072777 and a grant from ALS Therapy Alliance to Z.M.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Alexiou, P., Maragkakis, M., Mourelatos, Z., Vourekas, A. (2018). cCLIP-Seq: Retrieval of Chimeric Reads from HITS-CLIP (CLIP-Seq) Libraries. In: Okamura, K., Nakanishi, K. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 1680. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7339-2_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7339-2_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7338-5
Online ISBN: 978-1-4939-7339-2
eBook Packages: Springer Protocols