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

Abstract

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

Figure 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. fHTT_n 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) cycles 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. See Fig. S4 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 (eWLC^MFE - black dashed line in B). Red line, sum of 6 Gaussians. Peak averages correlate with predicted structures: (i) reference MFE structure with one single-stranded CAG triplet (0.19 ± 0.06 nm), (ii) no unpaired CAG triplet (−1.76 ± 0.04 nm), (iii) three unpaired CAG triplets (3.87 ± 0.02 nm). D. Distributions of unfolding (black) and refolding (red) forces, from experiments as in B. (N = 286 FECs).

Figure 2:. Expanded HTT mRNA unravels non-cooperatively.

A. Three successive stretch (black) and relaxation (red) cycles for fHTT_12–40 mRNAs, fit to eWLC models as in Fig. 1. Cooperative transitions (asterisks) become less common as the repeat expands and are lost for ΔCCG (bottom). B. Probability density of unfolding (black) and refolding (red) forces for each fHTT mRNA. Average unfolding force for fHTT₁₂ was 12.4 pN, compared to 11.8 pN for fHTT₇. Shaded region indicates non-cooperative transitions.

Figure 3.. 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 L_c, before unfolding at 13.2 pN; see Supplementary Figs. 2–3 for analysis details. Dashed lines, eWLC fits to structures differing by 2 unpaired CAG triplets. Inset: Expansion of low force region. B. Distribution of ΔL_c^MFE for all fHTT mRNAs. Stretch, black; relax; red. C. Passive mode fluctuations in ΔL_c^MFE for fHTT₄₀ at ~ 6 pN pretension (red circle in A). Black dashed lines show states used to fit HMM model, red line shows HMM fit. Similar results were obtained when the HMM fit was unconstrained (Fig. S10). Hopping rates of 0.9–3 s⁻¹ are comparable to the folding kinetics of a stable 21 bp hairpin ^, . D. Transition density plot from the HMM model in C. See Fig. S11 for data at high force.

Figure 4.. Intermolecular association of partially unfolded expHTT RNA.

A. Tethered fHTT₄₀ mRNA is moved from a microfluidic channel containing buffer (i-iii) to one containing 500 nM free fHTT₄₀ mRNA (iv), where it can form intermolecular base pairs. B. Example stretch (black) and relax (red) FECs in buffer (left) and for the same molecule after exposure to free mRNA (right). C. Successive cycles showing (*) free RNA binding at high force; (**) release at low force; (***) unfolding and partial release at high force. D. Conversion to dsRNA after several cycles. Black, last full extension; red, relaxation; blue, converts to dsRNA at high force (inset) and remains base paired during subsequent cycles. Right, observed FEC compared with dsRNA eWLC model (blue dashed line).

Figure 5.. Stick-slip model for CAG repeat aggregation in HD.

Short CAG tracts form composite hairpins that unfold and refold cooperatively with limited slippage of base pairs. Expanded CAG repeats unravels and refolds non-cooperatively like an accordion, producing a mixture of secondary structures that include unstable, slippery CAG hairpins and stable, sticky CAG•CCG hairpins that unfold in small rips, producing single-stranded regions that nucleate base pairing with another RNA, producing a network that can lead to aggregation (bottom). Although the intermediate structures vary, the cartoons illustrate conformations that agree with the force extension results.