PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution
RNA has the intrinsic propensity to form base pairs, leading to complex intramolecular and intermolecular helices. Direct measurement of base pairing interactions in living cells is critical to solving transcriptome structure and interactions, and investigating their functions (Lu and Chang, Curr Opin Struct Biol 36:142–148, 2016). Toward this goal, we developed an experimental method, PARIS (Psoralen Analysis of RNA Interactions and Structures), to directly determine transcriptome-wide base pairing interactions (Lu et al., Cell 165(5):1267–1279, 2016). PARIS combines four critical steps, in vivo cross-linking, 2D gel purification, proximity ligation, and high-throughput sequencing to achieve high-throughput and near-base pair resolution determination of the RNA structurome and interactome in living cells. In this chapter, we aim to provide a comprehensive discussion on the principles behind the experimental and computational strategies, and a step-by-step description of the experiment and analysis.
Key wordsPsoralen Cross-linking AMT (4′-aminomethyltrioxsalen hydrochloride) RNA structure RNA–RNA interaction 2D gel electrophoresis Proximity ligation High-throughput sequencing
The authors wish to thank Dr. Howard Chang at Stanford University for his generous support and encouragement. Z.L. is a Layton Family Fellow of the Damon Runyon-Sohn Foundation Pediatric Cancer Fellowship Award (DRSG-14-15). This work was supported by the National Natural Science Foundation of China (31671355) and the National Thousand Young Talents Program of China to Q.C.Z., and by the Jump Start Award for Excellence in Research to Z.L. (Stanford University, award No. 106).
- 2.Mattick JS, Makunin IV (2006) Non-coding RNA. Human molecular genetics 15 Spec No 1:R17-29. doi: 10.1093/hmg/ddl046
- 21.Lu Z, Zhang QC, Lee B, Flynn RA, Smith MA, Robinson JT, Davidovich C, Gooding AR, Goodrich KJ, Mattick JS, Mesirov JP, Cech TR, Chang HY (2016) RNA duplex map in living cells reveals higher-order transcriptome structure. Cell 165(5):1267–1279. doi: 10.1016/j.cell.2016.04.028 CrossRefPubMedPubMedCentralGoogle Scholar