Quantitative prediction of variant effects on alternative splicing in MAPT using endogenous pre-messenger RNA structure probing

Abstract

Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor messenger RNA (mRNA) structure and downstream function is particularly challenging. Here we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on precursor mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the Bact complex with precursor mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a b-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and six newly discovered mutations on inclusion of exon 10 of MAPT. This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.

Lineage-specific splicing regulation of MAPT gene in the primate brain

“…Examining MAPT exon 10 splicing-regulatory mechanisms from the evolutionary perspective allowed us to identify previously uncharacterized splicing-regulatory elements. It is clear that the previously identified splicing-regulatory elements, including those in exon 10 recognized by multiple SR proteins and hnRNPs or the stem-loop structure at the border of exon 10 and intron 10, 25 , 40 , 41 , 42 , 57 are insufficient to explain the developmental-stage- and lineage-specific splicing switch of this critical exon. While we and others have previously demonstrated that MBNL regulates exon 10 splicing, 44 , 45 the extent to which such regulation affects the developmental splicing switch is unclear.…”

RNA structure: implications in viral infections and neurodegenerative diseases

“…Another example is Alzheimer's disease (AD), which is associated with the dysregulation of the tau protein variants ratio (4R:3R ratio) (encoded by the MART gene). The role of RNA secondary structure in the splicing regulation of the MART gene has been investigated (Strang et al 2019;Kumar, et al 2022). In one of the studies, DMS-Map was used to examine the structures of pre-mRNA and mature MART-encoded mRNA, and data were collected for predicting alternative splicing outcomes (Kumar, et al 2022).…”

“…The role of RNA secondary structure in the splicing regulation of the MART gene has been investigated (Strang et al 2019;Kumar, et al 2022). In one of the studies, DMS-Map was used to examine the structures of pre-mRNA and mature MART-encoded mRNA, and data were collected for predicting alternative splicing outcomes (Kumar, et al 2022). They found that the mutations can change the structural ensembles in the exon10-intron10 junction, differentiating alternative splicing events.…”

How does precursor RNA structure influence RNA processing and gene expression?

“…A hairpin at the MAPT exon10–intron10 junction directly overlaps with the 5′ splice site and can be disrupted by disease-associated variants [ 49 , 50 ]. The MAPT hairpin blocks normal spliceosomal recognition by the U1 snRNP at the 5′ splice site and causes exon skipping and formation of the shorter 3R MAPT mature transcript [ 51 ] ( Figure 1 A). RNA structure in pre-processed transcripts has been shown to block U1 interactions and alter splicing in other precursor RNAs in addition to MAPT , including SMN2, VWF, ATM and BCL2L1 [ 52–56 ] ( Table 2 ).…”