Identifying the interactions conferring functional mechanical rigidity on RNase-resistant RNA from Zika virus

Figures

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  Fig. 1.. ZIKV xrRNA structure and mutations. (A) Crystal structure of ZIKV xrRNA (PDB ID 5TPY) and (B) secondary structure, showing color-coded helices P1-P4, PK, and 5′-TC. Mutations studied here are noted in the secondary structure. (C) Cartoon of SMFS experiment: A single xrRNA is held by duplex handles between two beads trapped in laser beams. (D) Sample FEC of wild-type ZIKV xrRNA showing unfolding (black) and refolding (red) of the native fold. Unfolding occurs at high force coincident with overstretching of the duplex handles (cyan). The intermediates in refolding (I1-4) allow the formation of individual helices to be identified.

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  Fig. 2.. Force spectroscopy of 5′-TC mutants. (A–C) U4C mutant lacking the base triple U4⋅A24:U42. (A) Most (~52%) unfolding FECs started in ring-knotted state Nm1, unfolding P4 first and then intermediate Im1. (B) Unfolding force distribution for Im1. (C) Other types of FECs: (i) ~1% started in a structure with 5′ end threaded (held by the G3:C44 pair) but ring still open; (ii) ~44% showed the presence of stem-loops P1–P4 only. (D–F) G3C mutant lacking the pseudoknotted base pair G3:C44. (D) Half of unfolding FECs started in ring-knotted state Nm2, unfolding P4 first before intermediate Im2. (E) Unfolding force distribution for Im2. (F) Other types of FECs: (i) ~38% started with 5′ end threaded (held by the U4⋅A24:U42 triple) but ring still open; (ii) ~7% showed the presence of stem-loops P1–P4 only; and (iii) ~5% started in a structure with an alternate 5′-TC (G2:C44) but unfolded P1 and PK.

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  Fig. 3.. Force spectroscopy of PK mutants. (A–C) A37G/U51G lacks one base pair in PK. (A) ~40% of unfolding FECs started in a ring-knot with an alternative PK (Irm3) that was still mechanically resistant (cyan: overstretching transition). (B) Unfolding force distribution for Irm3. Only a few FECs of the ring-knotted mutant showed unfolding below the overstretching transition (grey box). (C) Other types of FECs: (i) ~21% started in a structure with both 5′-TC but lacking P1 and PK; (ii) ~39% involved P1–P4 only. (D–F) G34C/A37G/U51G mutant lacks two base pairs in PK. (D) ~35% of unfolding FECs started in a ring-knot with a shorter alternate PK (Im4). (E) Unfolding force distribution for Im4 showing average force of 50 pN. (F) Other types of FECs were the same as for A37G/U51G: (i) ~10% had both 5′-TC but lacked P1 and PK; (ii) ~55% involved P1–P4 only.

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  Fig. 4.. Load-bearing residues in WT-ZIKV xrRNA from MD simulations. Plot of the ratio of pairwise force magnitudes with vs. without external load applied to 5′ end, for every pair of nucleotides. Boxes: pairwise interactions with the nucleotides changed in the mutants (red: 5′-TC mutations, brown: PK mutations).

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  Fig. 5.. Correlation of extreme mechanical resistance and XR activity. (A) Representative gel of xrRNA digestion by Xrn1. Adding Xrn1 to the undigested RNA sample (inset, Top Left) produces a fragment with the length of the xrRNA in the case of RNase resistance (inset, Bottom Left), otherwise the RNA is fully digested. XR activity is given by the fraction of the RNA that remains in the partially digested fragment. (B) XR activity is uncorrelated with the prevalence of ring-knot folds (Left), but varies linearly with the prevalence of extreme mechanical resistance (Right). Dashed line: linear fit [y = (1.01 ± 0.04)x – (1 ± 1), R2 = 0.994].