A Disease-Causing Intronic Point Mutation C19G Alters Tau Exon 10 Splicing via RNA Secondary Structure Rearrangement

Abstract

Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the ∼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5′ splice site (5′ss, located at the exon 10–intron 10 junction). These mutations tend to increase the 4R:3R ratio by destabilizing the hairpin, thereby improving 5′ss recognition by U1 snRNP. Interestingly, a single C-to-G mutation at the 19th nucleotide in intron 10 (C19G or +19G) decreases the level of exon 10 inclusion significantly from 56% to 1%, despite the disruption of a G-C base pair in the bottom stem of the hairpin. Here, we show by biophysical characterization, including thermal melting, fluorescence, and single-molecule mechanical unfolding using optical tweezers, that the +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability. The cell culture alternative splicing patterns of a series of minigenes reveal that the splicing activities of the mutants with destabilizing mutations on the top stem can be compensated in a position-dependent manner by the +19G mutation in the bottom stem. We observed an excellent correlation between the level of exon 10 inclusion and the rate of mechanical unfolding at 10 pN, indicating that the unfolding of the splice site hairpins (to facilitate subsequent binding of U1 snRNA) may be aided by helicases or other proteins.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing

“…Remarkably, asPNA(+8/+18) showed a weakened binding to the hairpin with a +19G mutation (tau-19G-Cy3, K d = 7.0 ± 1.5 nM) (Figure 3f, Supplementary Figure S2), even though the mutation was adjacent to, but not within, the recognition site of the asPNA. A C+19G mutation has been shown to cause RNA secondary structural rearrangement, resulting in the stabilization of the splice site hairpin (Figure 1e and Figure 2f) [29]. In addition, asPNA(+8/+18) may have invaded two and one base pairs below the A bulge and G bulge in the wild-type and +19G mutant, respectively (Figure 2).…”

“…The formation of this hairpin masks the 5′ss, thus inhibiting its recognition by U1 small nuclear ribonucleoprotein (U1 snRNP, Figure 1a), which is a key initial step in pre-mRNA splicing [25,26,27]. Point mutations in the hairpin region affect its stability by introducing mismatched base pairs or by structural rearrangement within the hairpin [15,18,19,20,21,22,23,28,29].…”

“…The hairpin has a single A bulge, which results in the formation of a top stem and a bottom stem above and below the A bulge, respectively (Figure 1b and Figure 2f). Mutations in the relatively more stable top stem often destabilize it by introducing a mismatched base pair or by local structural rearrangement (Figure 1c,d) and tend to increase the 4R/3R ratio [15,29]. On the other hand, a single C-to-G mutation in the relatively less stable bottom stem at the 19th nucleotide downstream of the 5′ss (C+19G or +19G) (Figure 1e) causes a significant decrease in the 4R/3R ratio [15,29,33].…”

“…Mutations in the relatively more stable top stem often destabilize it by introducing a mismatched base pair or by local structural rearrangement (Figure 1c,d) and tend to increase the 4R/3R ratio [15,29]. On the other hand, a single C-to-G mutation in the relatively less stable bottom stem at the 19th nucleotide downstream of the 5′ss (C+19G or +19G) (Figure 1e) causes a significant decrease in the 4R/3R ratio [15,29,33]. The +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability (Figure 1e and Figure 2g) [29].…”

“…On the other hand, a single C-to-G mutation in the relatively less stable bottom stem at the 19th nucleotide downstream of the 5′ss (C+19G or +19G) (Figure 1e) causes a significant decrease in the 4R/3R ratio [15,29,33]. The +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability (Figure 1e and Figure 2g) [29]. Abnormal 4R/3R ratios caused by these mutations lead to the pathogenesis of FTDP-17.…”