Structural determinants for alternative splicing regulation of the MAPT pre-mRNA

Abstract

Alternative splicing at the MAPT gene exon 10 yields similar levels of the 3R and 4R tau protein isoforms. (1) The presence of mutations, particularly in exon 10 and intron 10-11, changes the quantity of tau isoforms. Domination each of the isoform yields tau protein aggregation and frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Here, we report for the first time the secondary structure of the 194/195 nucleotide region for the wild type (WT) and 10 mutants of the MAPT gene pre-mRNA determined using both chemical and microarray mapping. Thermodynamic analyses indicate that single nucleotide mutations in the splicing regulatory element (SRE) that form a hairpin affect its stability by up to 4 and 7 kcal/mol. Moreover, binding the regulatory hairpin of small molecule ligands (neomycin, kanamycin, tobramycin and mitoxantrone) enhance its stability depending on the nature of the ligands and the RNA mutations. Experiments using the cos-7 cell line indicate that the presence of ligands and modified antisense oligonucleotides affect the quantity of 3R and 4R isoforms. This finding correlates with the thermodynamic stability of the regulatory hairpin. An alternative splicing regulation mechanism for exon 10 is postulated based on our experimental data and on published data.

Changes in the expression of splicing factor transcripts and variations in alternative splicing are associated with lifespan in mice and humans

“…Alternative splicing, the process by which single genes can make multiple gene products in an adaptive and reactive fashion is a key part of this process (Cartegni et al ., 2002). Indeed, breakdown in the regulation of mRNA splicing is a prominent feature in many age‐related diseases such as Alzheimer's disease, Parkinson's disease and several tumour types (Scuderi et al ., 2014; Danan‐Gotthold et al ., 2015; Lisowiec et al ., 2015; Lu et al ., 2015). This may indicate that defects in the splicing machinery may cause the cellular response to stress to be less specific, with effects on cellular resiliency and accumulation of DNA damage.…”

The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

“…This hairpin structure was also supported by SHAPE mapping. 55 62 These results indicate that htra2β1 and SRp54, which antagonizes its activity, bind to these regions. [59][60][61][62] On the other hand, the N279K mutation, which changes a U to a G, increases exon 10 inclusion by strengthening an AG-rich region that acts as a splicing enhancer.…”

“…13,53,54 Among 70 nt windows spanning the intron 9-exon 10 junction, the window with the lowest z-score (−1.05) contains a hairpin structure modeled using SHAPE mapping ( Figure 3B). 55 A G-to-U mutation at position E10−10 near this junction was observed to increase the 4R-to-3R tau ratio ( Figures 4D and 4E). 56 The previously validated hairpin and splice site regulator at the exon 10-intron 10 junction was predicted as part of a larger hairpin spanning nt 73 -93 of exon 10 and E10+1 -E10+31 of intron 10, which contains a U1 snRNA binding site ( Figure 3C).…”

“…A hairpin at the intron 9-exon 10 junction was predicted to form by SHAPEconstrained RNA folding algorithms ( Figure 4D). 55 A G-to-U mutation at position E10−10 of this splice junction ( Figure 4E) weakens a polypyridine tract and a 2 nt bulge loop was predicted to form as a result of the G-to-U mutation, which stabilizes the wild type hairpin by 0.4 kcal/mol. This mutation leads to increased selection of the splice site and exon 10 inclusion.…”

“…Splicing of exon 10 is regulated by cis-elements, such as exonic splicing enhancers, silencers (ESEs and ESSs, respectively) and structured RNAs, and trans-factors, such as serinearginine (SR) proteins that bind to ESEs adjacent to the 5′ and 3′ splice sites and promote association with U1 and U2 snRNP, respectively 51,55,57,58. These splice sites are weak, and their activities (and exon 10 inclusion) are dependent on the ESEs 58.…”