Elucidating the pathobiology of Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS) with its expanded RNA structure formation and proteinopathy

Abstract

Numerous neurological disorders are linked to sequences rich in guanine repeats found in introns, exons, and regulatory regions of genes. These sequences have been observed to form stable G-quadruplex (GQ) structures both in vitro and in vitro. Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS), a slowly progressive neurodegenerative disorder, is associated with the biallelic expansion of (AAGGG)_n pathogenic repeats in the second intron of the RFC1 gene. Though these G-rich pathogenic repeats in other neurological diseases are associated with protein loss of function, RNA gain of function, and/or protein gain of function, not much is known about the pathological mechanism associated with CANVAS. Herein, we report the formation of stable GQ conformations in the CANVAS-associated repeats i.e., r(AAGGG)_n, where 'r' stands for RNA. These GQs are critical regulators in neurological disorders leading to RNA foci formation and RNA binding protein sequestration. They also alter other causative processes like intron retention, which leads us to hypothesize a toxic Proteinopathy mechanism in CANVAS. Various biophysical and biomolecular assays characterized the interactions of three aggregation-prone RNA-binding proteins (RBPs): heterogeneous nuclear ribonucleoprotein H1/F (hnRNP H1/F), and DGCR8 with different pathogenic repeats [(AAGGG)₉] in vitro, further affirming the hypothesis. The biophysical observations are further supported by molecular dynamics analysis and cell-based studies, putting us a step closer to elucidating the pathological mechanism(s) in CANVAS neuropathy, paving the way for the development of innovative therapeutic interventions.

Keywords: CANVAS; Proteinopathy; RFC1; RNA r(AAGGG)n; Repeat expansions.

Fig. 1

The region of the RFC1 gene harbors an AluSx3 in its intron 2, which is in the opposite direction to that of the RFC1 exon. The normal RFC1 gene harbors (AAAAG)₁₁ repeats located at the terminus of the AluSx3 element, but in CANVAS, this is replaced by a variable length (AAGGG)_400−2000 repetition.

Fig. 2

CD spectroscopic analysis of CANVAS-associated repeat RNA. (a) CD spectra of RNA r(AAGGG)₄ repeats in the presence of various cations (K⁺, Na⁺, Li⁺ and Mg⁺⁺). (b) Change in ellipticity of r(AAGGG)₄ repeats with the increasing concentration of K⁺ cation (0–75 mM). (c) CD spectra of r(AAGGG)₄ RNA with the increasing temperature (25 °C to 75 °C). (d) CD spectra of the RNA r(AAAAG)₄ in different K⁺ concentrations. (e) The normalized thermal melting curve of r(AAGGG)₄ in varying concentrations of K⁺ cation. (f) The Bar graph depicting the T_m obtained in varying concentrations of K⁺ cation.

Fig. 3

The NMR spectra analysis and gel mobility shift in the CANVAS-associated RNAs. (a) 1D-¹H NMR spectra of r(AAGGG)₄ in varying concentrations of K⁺ ion (0–75 mM). (b) The NMR spectra of non-pathogenic RNA r(AAAAG)₄(c) The NMR spectra of r(AAGGG)₄ with the increasing temperature (20 °C to 70 °C). (d) Gel image of the shift in mobility of r(AAGGG)₄ with the varying K⁺ concentration.

Fig. 4

Interaction analysis of BRACO-19 with CANVAS repeats. (a) The isothermal titration thermogram of r(AAGGG)₄ and r(AAAAG)₄ titrated with BRACO-19. (b) Bar graph depicting the K_d value of BRACO-19 with r(AAGGG)₄ and r(AAAAG)₄. (c) Thermal melting plots of r(AAGGG)₄ with BRACO-19 (D/N = 0–1). (d) EMSA analysis of r(AAGGG)₄ and r(AAAAG)₄ with the increasing concentration of BRACO-19 (0–25 µM).

Fig. 5

The NMR broadening of BRACO-19 peaks. The upper panel illustrates the structure of BRACO-19 with the assigned positions of its aromatic protons. In the 1D-¹H NMR spectra below, asterisks highlight changes in proton peaks upon titration with increasing concentrations of r(AAGGG)₄ and r(AAAAG)₄. These spectra reveal how BRACO-19 interacts differently with these nucleic acid sequences, providing insights into their binding preferences and structural dynamics.

Fig. 6

mTFP-based reporter assay. (a) The confocal images of HEK293T cells transfected with (AAGGG)₄ and (AAAAG)₄ repeat in the presence of varying concentrations of BRACO-19. The experimental findings demonstrated a notable decrease in mTFP protein production as the concentration of BRACO-19 increased in cells transfected with pathogenic (AAGGG)₄ repeats. (b) The normalized fluorescence intensity (in %) of the pathogenic and non-pathogenic repeat harboring plasmid in the absence or presence of BRACO-19. Statistical significance was assessed using the student’s t-test. Asterisks denote significance compared to the control (untreated 0.0 µM) sample: *P < 0.05, **P < 0.01, ***P < 0.001. Values of *P = 0.0263 and **P = 0.0092 are indicated.

Fig. 7

(a) String Analysis. Network of protein-protein interactions for the eleven aggregation-prone RBPs in STRING. The 3D model is generated using version 12.0 of STRING-DB software (https://string-db.org/). (b) EMSA with CANVAS-associated pathogenic RNA repeats [r(AAGGG)₉] in the presence of increasing concentrations of hnRNP H1, hnRNP F, and DGCR8 (RRM) RBPs. Experiments were performed in duplicate, and the gels shown are representative results from one experiment.

Fig. 8

CD spectroscopic analysis of CANVAS-associated RNA repeats [r(AAGGG)₉] in presence of increasing concentrations (0–15 µM) of proteins (a) hnRNP H1, (b) hnRNP F, and (c) DGCR8 (RRM). The spectra illustrate changes in RNA secondary structure induced by protein binding, providing insights into the interactions between hnRNP H1, hnRNP F, DGCR8 (RRM), and r(AAGGG)₉ RNA.

Fig. 9

Fluorescence Spectroscopy. Fluorescence emission spectra of (a, c, e) hnRNP H1, hnRNP F, and DGCR8 along with (b, d, f) the linear Stern-Volmer plot of fluorescence quenching with the increasing concentration of CANVAS [r(AAGGG)₉] repeats RNA in 5 mM K₂HPO₄ buffer (pH 6.8).

Fig. 10

The isotherm of r(AAGGG)₉ titrated with (a) hnRNP H1, (b) hnRNP F, and (c) DGCR8 (RRM) RBPs. (d) Bar graph depicting the K_d value of three proteins - hnRNP H1, hnRNP F, DGCR8 (RRM) with r(AAGGG)₉ repeats RNA.

Fig. 11

Protein - RNA interaction using Molecular docking and MD simulation: (a) Docked complex & interacting residues of HnRNP F and r(AAGGG)₄, (b) RMSD plot of complex (HnRNP F and r(AAGGG)₄). (c) Docked complex & interacting residues of DGCR8 and r(AAGGG)₄ , (d) RMSD plot of the complex (DGCR8 and r(AAGGG)₄). (e) Docked complex & interacting residues of HnRNP H1 and r(AAGGG)₄ , (f) RMSD plot of the complex (HnRNP H1 and r(AAGGG)₄).