Investigating RNA-RNA interactions through computational and biophysical analysis

Abstract

Numerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Following in vitro transcription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using size-exclusion chromatography coupled with multi-angle light scattering and analytical ultracentrifugation. Next, we demonstrate that the 5' and 3' terminal regions of JEV interact with nM affinity using microscale thermophoresis, and this affinity is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3D structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.

Graphical Abstract

Graphical abstract illustrating the complementary computational and biophysical pathway used to characterize the RNA-RNA interaction between JEV 5' and 3' terminal regions in vitro. The pathway can be easily adapted to other RNA-RNA interactions in solution.

Figure 1.

JEV structural organization and theoretical secondary structure prediction. Visual representation of JEV genome organization and different construct regions which were designed and studied. Predicted secondary structure of the cyclization between JEV 5′ TR and JEV 3′ TR from constraining co-folding, highlighted red portions represents the 11 conserved nucleotides which form a stable base pairing interaction to facilitate the long-range genome interaction.

Figure 2.

Consensus secondary structure of the 5′ TR/3′ TR long-range interaction, computed from a structural multiple sequence alignment of 20 mosquito-borne flaviviruses (Supplementary Figure S1). Coloring of base pairs follows the RNAalifold schema and indicates different covariation levels, ranging from red (no covariation, full primary sequence conservation) to violet (full covariation, all six possible combinations of base pairs) at corresponding columns of the underlying alignment. The duplex formed by the almost fully sequence-conserved 11 nt cyclization sequences (highlighted in gray) represents the only long-range interaction in the consensus structure. Canonical 5’TR elements (SLA, SLB, and cHP), as well as 3’TR elements (DB, sHP and 3’SL) are predicted to fold in the consensus structure, indicating that they are energetically more favorable than an extended long-range interaction duplex structure.

Figure 3.

Purification of JEV TR RNA. (A) Size-exclusion chromatogram representing the purification of both RNA. Shaded boxes represent the region which was collected for downstream experiments to avoid any potential oligomeric species. (B) Urea PAGE of associated size exclusion chromatography fractions showing a single size of RNA (∼220 nt) which is the correct size of the expected RNA.

Figure 4.

Light scattering analysis of JEV TR RNA cyclization. (A) Multiple size exclusion chromatography runs associated with SEC-MALS. (B) MALS traces of each peak from the 5′ TR + 3′ TR run, and the absolute molecular weight across them. (C) Sedimentation distribution profiles of JEV 5′ TR and 3′ TR obtained from sedimentation velocity-analytical ultracentrifugation. Sedimentation coefficient values are corrected to standard solvent conditions (20 °C, water)

Figure 5.

Affinity analysis of JEV TR RNA cyclization. (A) MST raw data traces for JEV 3′ TR + 5′ TR. Blue line represents the ‘cold’ time and the red line represent the ‘hot’ region, and the difference between the two is used to calculate the ΔF_norm. (B) Microscale thermophoresis measurements of different combinations of JEV TRs representing concentration versus fraction bound. Measurement ran on ‘high’ MST power.

Figure 6.

Energy landscape of the predicted 3′ TR and 5′ TR CS interaction. The energy landscape represents all possible substructures that can occur along a direct path from a single base pair interaction to the full CS interaction.

Figure 7.

Small-angle X-ray scattering ab inito model reconstructions of JEV RNA. (A) JEV 5’ TR RNA, rotations are 180° about both the X- and Y-axis and showing D_max. (B) JEV 3’ UTR RNA illustrating relative conformational fluidity through clustering analysis of 100 ab inito models. Shown models represent three of the largest clusters of models. (C) JEV 5′ TR + 3′ UTR RNA–RNA complex. JEV 5′ TR is represented in blue, while 3′ UTR is represented in shades of red/purple.