Alzheimer’s disease and other neurodegenerative disorders have stumped the medical community for decades, but now with advanced computational methods researchers are uncovering molecular mechanisms behind these devastating diseases. In a study published in bioRxiv on March 17, scientists at Scripps Research Institute reveal how disease-associated mutations disrupt pre-mRNA splicing of the Tau gene, which aggregates in diseased neurons and kills the cell. They further demonstrate that this mis-regulation in gene-splicing generates abnormal forms and amounts of Tau contribute to disease-pathology, providing novel targets for therapeutic intervention.
“Mutations that disrupt RNA structure can be deleterious and causes disease. Therefore, knowledge of RNA structure can provide insights into disease mechanism and how to affect protein production for therapeutic benefit,” says Matthew Disney, lead author of the study. Tau is ripe with opportunities here, as a gene that undergoes extensive alternative splicing. The gene’s 16 exons are alternatively spliced into at least 6 different isoforms with different lengths and numbers of microtubule binding domains (MTBs). “Mutations within sequences near the exon-intron junctions that affect RNA structure may disrupt normal splicing and cause disease,” says Disney.
Indeed, it is already known that the ratio of Tau isoforms is disrupted in “Taupathies,” neurodegenerative diseases characterized by Tau aggregates like Alzheimer’s and Parkinson’s disease. “For example, exon 10 encodes an MBD and is alternatively spliced, resulting in protein isoforms with four (4R) or three (3R) MBDs. Deregulation of exon 10 alternative splicing due to mutation manifests in various diseases, including frontotemporal dementia with parkinsonism-17 (FTDP),… where the 4R isoform is expressed at aberrantly high levels,” Disney explains.
Disney and his team systematically examine the sequence of the Tau pre-mRNA transcript to identify these dysregulated splice sites. “There has been little done to systematically analyze and study the structures within Tau’s encoding RNA and their connection to disease pathology,” he says. The team deep dives into RNA structure at intron-exon junctions, where RNA stem-loop and hairpin structures recruit splicing machinery. With advanced thermodynamic folding algorithms they predict the formation of these structures, and examine how mutations associated with taupathies alter these structures to impact Tau splicing.
In total, they identify 19 RNA structures at exon-intron junctions, including five adjacent to alternatively spliced exons that encode MBDs. In some cases, mutations destabilize the RNA structures, eliminating splice sites. “A structured region was predicted at the exon 6- intron 6 junction where deletion… within the predicted hairpin structure, decreased exon 6 inclusion, indicating that the region acts as a strong splicing enhancer,” says Disney. In other cases, mutations created additional cryptic splice sites.
By altering RNA structures, the mutations dictate whether MTB-encoding exons are included or excluded, and thereby influencing the number of MTB repeats which impacts the function and aggregation of Tau within the cell. In particular, the team finds a number of mutations that influence exon 10 inclusion, which shift the balance of the 4R vs. 3R isoform from 1:1 to as much as 30:1. “The G(+3) A and C(+14) T mutations associated with (FTDP) diseases destabilize the hairpin structure at the exon 10-intron 10 junction… and are verified by cell-based assay to upregulate exon 10 inclusion,” Disney reports.
In addition the team identifies additional RNA structures in 5’ and 3’ untranslated regions of the Tau transcript, known to regulate gene expression. “These studies define novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases,” says Disney. Indeed, scientists have recently developed a powerful toolbox of methods to correct aberrant gene-splicing in the context of disease. These include antisense oligonucleotides, modified small nuclear RNAs, trans-splicing and small molecular compounds, which have proven promising in pre-clinical testing of various splicing diseases including cystic fibrosis, hemophilia B, muscular dystrophy and many other thus far incurable genetic disorders.