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.
HITS-CLIP CLIP-Seq RNA-IP Argonaute Piwi Next generation sequencing Illumina cDNA Immunoprecipitation Chimeric reads RNA-binding protein Ribonucleoprotein complexes In vivo Transcriptomic analysis Base-paired RNA miRNA target sequences piRNA target sequences Posttranscriptional RNA processing Gene silencing
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
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.
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 531:390–394CrossRefPubMedPubMedCentralGoogle Scholar
Yang Q, Fairman ME, Jankowsky E (2007) DEAD-box-protein-assisted RNA structure conversion towards and against thermodynamic equilibrium values. J Mol Biol 368:1087–1100CrossRefPubMedPubMedCentralGoogle Scholar
Maragkakis M, Alexiou P, Mourelatos Z (2015) GenOO: a modern perl framework for high throughput sequencing analysis. bioRxiv. doi:10.1101/019265