Stick-slip unfolding favors self-association of expanded HTT mRNA

Figures

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  Fig. 1. Normal HTT RNA with 7 CAG repeats unfolds cooperatively. A RNAs corresponding to the CAG repeat region of human HTT exon 1 were attached to optically trapped beads via dsDNA handles. fHTTn mRNAs contain n = 7, 12, 20, or 40 CAG triplets (red line) plus flanking CCGCAA(CCG)7 repeats (gold line). B Example stretch (black, S1) and relax (red, R1) FEC for fHTT7 at 90 nm/s show unfolding/refolding transitions near 11.5 pN. Inset: slip transitions at low tension. Dashed curves represent eWLC models for folded (black), unfolded (gray) and slipped (blue) states. The expected ∆Lc for transitioning from the fully folded to the fully unfolded state is 26.0 nm. See Supplementary Fig. 4 for details. S1 and R1 are plotted with an offset for clarity. C Contour lengths of slipped intermediates before the main unfolding transition from eWLC fits (blue dashed line in (B)) relative to the folded state (eWLCMFE—black dashed line in (B)). Red line, sum of 5 Gaussians; black dashed line, individual Gaussians. The histogram was fit using GraphPad Prism version 10.2.3 for Mac OS X. Labeled peak averages correlate with structures predicted by mfold54 and footprinting10: (i) no unpaired CAG triplet (–1.76…

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  Fig. 2. Expanded HTT mRNA unravels non-cooperatively. A Three successive cycles of stretching (black) and relaxation (red) for fHTT12-40 mRNAs, fit to eWLC models as in Fig. 1. Cooperative transitions (asterisks) become less common as the repeat expands and are lost for ∆CCG (bottom). Expected ∆Lc from the fully folded to the fully unfolded state is 36.58 nm for fHTT12; 50.74 nm for fHTT20; 86.14 nm for fHTT40; 68.44 nm for fHTT40∆CCG. B Probability density of unfolding and refolding forces for stretching (black) and relaxation (red) for each fHTT mRNA. Average unfolding force for fHTT12 was 12.4 pN, compared to 11.8 pN for fHTT7. Shaded region indicates the force range for non-cooperative stick-slip unfolding transitions.

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  Fig. 3. Applied force facilitates base pair exchange. A FECs and eWLC fits from two fHTT40 molecules after the first pull of a force-ramp cycle. Insets highlight conformational dynamics within the main transition, with many smaller transitions occurring as the RNA gradually extends (1). Occasionally, the tethered fHTT mRNA transitions to a much higher force (2) just after the start of a non-cooperative transition (1), reflecting temporary refolding to a structure with a shorter contour length. B Cartoon depicting how applied force induces base pair shuffling that exposes CAG and CCG triplets. Exposed single-stranded bases can participate in transient long-range base pairs with the flanking CCG repeats.

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  Fig. 4. Continuous stepwise slippage of expanded CAG tract. Slipped intermediates of fHTT40 analyzed as in Fig. 1. A Example FEC demonstrating transitions at low force that extend or shorten Lc, before unfolding at 13.2 pN; see Supplementary Figs. 2, 3 for analysis details. Black dashed line: eWLC model for fully folded fHTT40. Blue dashed lines, eWLC fits to structures differing by 2–4 unpaired CAG triplets. Inset: Expansion of low force region. B Distribution of ΔLcMFE for all fHTT mRNAs. Stretch, black; relax; red. C Passive mode fluctuations in ΔLcMFE for fHTT40 at ~6 pN pretension. Black dashed lines show HMM state, red line shows the HMM model. Similar results were obtained when the HMM fit was unconstrained (Supplementary Fig. 9). Hopping rates of 0.9–3 s−1 are comparable to the folding kinetics of a stable 21 bp hairpin33,34. D. Transition density plot from the HMM model in (C). See Supplementary Fig. 10 for data at high force.

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  Fig. 5. Intermolecular association of partially unfolded expHTT RNA. A Laminar flow microfluidics for intermolecular interactions. fHTT40 mRNA is tethered in buffer (channels i–iii) before exposure to 500 nM free fHTT40 mRNA (channel iv), where it can form intermolecular base pairs. B Example stretch (black) and relax (red) FECs in buffer (left, channel iii) and for the same molecule after exposure to free mRNA (right, channel iv). C Consecutive cycles of stretching (S1–S3, black) and relaxation (R1–R3, red) of a single fHTT40 molecule, showing (*) free RNA binding at high force; (**) release at low force; (***) unfolding and partial release at high force. D Similar experiment as in (C) with another fHTT40 molecule, showing conversion to dsRNA after (***) unfolding and full release of free mRNA; (*) binding and conversion to dsRNA during unfolding; (**) partial melting of the dsRNA at high force. Green dashed line, eWLC for double-stranded fHTT40 mRNA.

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  Fig. 6. Stick-slip model for CAG repeat aggregation in HD. A Short CAG tracts form composite hairpins that unfold and refold cooperatively with limited slippage of base pairs. B Expanded CAG repeats slip and rearrange non-cooperatively, producing a mixture of secondary structures that include unstable, slippery CAG hairpins (red) and stable, sticky CAG•CCG hairpins that unfold in small rips (red and gold). Single-stranded regions nucleate base pairing with another RNA, producing a network that can lead to aggregation (bottom). Although transitions between specific intermediate structures are not resolved in our experiments, the cartoons illustrate conformations consistent with the force-extension results.