PARIS: Psoralen Analysis of RNA Interactions and Structures with High Throughput and Resolution

Abstract

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.

Keywords: 2D gel electrophoresis; AMT (4′-aminomethyltrioxsalen hydrochloride); Cross-linking; High-throughput sequencing; Proximity ligation; Psoralen; RNA structure; RNA–RNA interaction.

Fig. 1

Outline of the PARIS experimental strategy. Major steps are explained on the right side

Fig. 2

Example results of AMT cross-linking and RNA fragmentation. (a) AMT cross-linked cell pellets (right) have a darker color than the non-cross-linked ones (left). (b) After S1/PK digestion, adding TRIzol and chloroform, phase separation is faster for the non-cross-linked cells (left) than the cross-linked cells (right), so the cross-linked samples have a milky appearance. (c) S1/PK extraction produces a characteristic broad peak between 2000 and 4000 nt in the Bioanalyzer electrophoretic trace of cross-linked cells. The height of the broad peak is variable among cell types and different batches of experiments. (d) ShortCut RNase III digestion reduces RNA to a smaller size (usually below 150 nt) to facilitate 2D gel purification and library preparation

Fig. 3

Diagram and example result for 2D gel purification of cross-linked and digested RNA. (a) First dimension native gel. Gel slices containing RNA around 30–150 bp are usually cut out from the first dimension for the second dimension. (b) Second dimension denatured gel. Gel slices are aligned between the glass plates before pouring the urea denatured gel solution at the bottom of the gel plates (as indicated by the arrow in the middle). (c) An example second dimension gel. The yellow-boxed area indicate the cross-linked RNA to be extracted for library preparation

Fig. 4

The PARIS analysis pipeline, modified from the PARIS paper [21]. Major analyses outlined here include DG and NG assembly, visualization of RNA structure data and models in IGV, two approaches of phylogenetic analysis, analysis of alternative structures, identification and visualization of RNA–RNA interactions

Fig. 5

An example visualization of RNA structures in IGV. The structure model is in the linear format (quasi-concentric arcs for an RNA duplex). A subset of gapped reads is assembled into two DGs. The two DGs can be assembled into one NG, since they do not overlap with each other. For more examples, see the PARIS paper [21]

Fig. 6

Analysis and visualization of a new interaction between SNORD16 (or U16) and U6 in human HEK 293T cells and mouse ES cells. (a) The blue highlighted regions are the sequences involved in base pairing. (b) The interaction model. Box D is a characteristic sequence motif in the C/D box snoRNAs. SNORD16 is known to guide modification of 18S rRNA (snoRNABase) [30]. Here, we show that it may also guide the modification of U6 snRNA at C77, the same site as the SNORD10 target (snoRNABase) [31], but the base pairing is less perfect compared to the SNORD10–U6 interaction. For more examples, see Fig. 5 and Fig. S6 the PARIS paper [21]