Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and parkinsonism linked to chromosome 17 (alternative mRNA splicingyintronic mutationsystem-loop RNA structure)

Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17. Intronic mutations and some missense mutations increase splicing in of exon 10, leading to an increased ratio of four-repeat to three-repeat tau isoforms. Secondary structure predictions have led to the proposal that intronic mutations and one missense mutation destabilize a putative RNA stem-loop structure located close to the splicedonor site of the intron after exon 10. We have determined the three-dimensional structure of this tau exon 10 splicing regulatory element RNA by NMR spectroscopy. We show that it forms a stable, folded stem-loop structure whose thermodynamic stability is reduced by frontotemporal dementia and parkinsonism linked to chromosome 17 mutations and increased by compensatory mutations. By exon trapping, the reduction in thermodynamic stability is correlated with increased splicing in of exon 10. Abundant neurofibrillary lesions made of the microtubuleassociated protein tau constitute a defining neuropathological characteristic of Alzheimer’s disease (1). Filamentous tau protein deposits are also the defining characteristic of other neurodegenerative diseases, many of which are frontotemporal dementias or movement disorders that have been subsumed under the heading of ‘‘Pick complex’’ (1, 2). Recent work has shown that mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP17) (3–13). Known mutations are either coding region or intronic mutations located close to the splice-donor site of the intron after exon 10 of the tau gene. Many coding region mutations produce a reduced ability of tau to interact with microtubules, thus probably setting in motion the mechanisms that lead to the formation of tau filaments (13–16). Four mutations have been described at positions 13, 113, 114, and 116 of the intron after exon 10 (with the first nucleotide of the splice-donor site taken as 11) (4, 5, 10, 11). The S305N mutation in exon 10 also is located close to this splice-donor site (9). Six tau isoforms are produced in adult human brain by alternative mRNA splicing from a single gene (17–19). They differ from each other by the presence or absence of 29-aa or 58-aa inserts located in the amino-terminal half and a 31-aa repeat located in the carboxyl-terminal half. Inclusion of the latter, which is encoded by exon 10 of the tau gene, gives rise to the three tau isoforms with four repeats each (17, 18); the other three isoforms have three repeats each (18). The repeats and some adjoining sequences constitute the microtubulebinding domains of tau (20, 21). Similar levels of three-repeat and four-repeat tau isoforms are found in normal cerebral cortex (22), and the tau filaments from Alzheimer’s disease brain contain all six tau isoforms in a hyperphosphorylated state (23). Three of the intronic mutations (at positions 113, 114, and 116) and the S305N mutation at position 21 have been shown to increase splicing in of exon 10 (4, 24). Increased production of tau isoforms with four repeats has been described in brain tissue from individuals with three of the intronic mutations (5, 11, 15). The overproduction of four-repeat tau thus is sufficient to lead to a dementing disease (25). A potential mechanism underlying the overproduction of four-repeat tau isoforms was proposed, based on the prediction that a putative RNA stem-loop structure at the boundary between exon 10 and the intron after exon 10 would be destabilized by the intronic mutations (4, 5). We have investigated this proposal by determining the three-dimensional structure of the tau exon 10 splicing regulatory element RNA by using NMR spectroscopy. We show here that it does indeed form a stable, folded stem-loop structure that differs, however, in several respects from previous predictions. We also show that the intronic FTDP-17 mutations destabilize the stem-loop structure and that this destabilization is correlated with increased splicing in of exon 10.