hiCLIP

hiCLIP

hiCLIP sequences RNA duplexes bound to RBPs in vivo. The unique cloning and linker-adapter system in hiCLIP identifies whether the RBP-bound duplex originates from the same RNA or different RNAs.

Similar to CLIP library preparation techniques, RNA and RBPs are UV-crosslinked, partially digested, and immunoprecipitated. The signature hiCLIP cloning and linker-adapters are ligated to both strands of the duplex. The 3' end of the linker-adapter is dephosphorylated and ligated to the 5' end of the other strand. The bound proteins are removed with proteinase K. The cDNA library is prepared in a similar dashion to iCLIP for high-throughput sequencing. The cloning and linker-adapters enable the mapping of the hybrid reads to the transcriptome and distinguish whether the duplex is formed by the same RNAs or different RNAs.

Pros:

• Maps RBP-bound RNA duplexes in vivo
• Linker-adapter remediates physical and computational challenges seen in crosslinking, ligation, and sequencing of hybrids (CLASH)
• Detects long-range RNA duplex interactions ¹

Cons:

• Does not identify non-RBP-bound RNA duplexes
• Technically complex procedure
• Uses radioactive labeling
• Artifacts may be introduced in the circularization step

• hiCLIP: HiClip: Sugimoto Y., Vigilante A., Darbo E., Zirra A., Militti C., et al. (2015) hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature 519: 491-494
• 1. Lu Z. and Chang H. Y. (2016) Decoding the RNA structurome. Curr Opin Struct Biol 36: 142-148

1. Lu Z. and Chang H. Y. Decoding the RNA structurome. Curr Opin Struct Biol. 2016;36:142-148
2. Weidmann C. A., Mustoe A. M. and Weeks K. M. Direct Duplex Detection: An Emerging Tool in the RNA Structure Analysis Toolbox. Trends Biochem Sci. 2016;41:734-736
3. Nawy T. Structural biology: RNA structure served in vivo. Nature Methods. 2015;12:383-383
4. Burgess D. J. RNA. Detailed probing of RNA structure in vivo. Nat Rev Genet. 2015;16:255

1. Sugimoto Y., Vigilante A., Darbo E., et al. hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature. 2015;519:491-494