What’s Wrong in a Jump? Prediction and Validation of Splice Site Variants

Abstract

Alternative splicing (AS) is a crucial process to enhance gene expression driving organism development. Interestingly, more than 95% of human genes undergo AS, producing multiple protein isoforms from the same transcript. Any alteration (e.g., nucleotide substitutions, insertions, and deletions) involving consensus splicing regulatory sequences in a specific gene may result in the production of aberrant and not properly working proteins. In this review, we introduce the key steps of splicing mechanism and describe all different types of genomic variants affecting this process (splicing variants in acceptor/donor sites or branch point or polypyrimidine tract, exonic, and deep intronic changes). Then, we provide an updated approach to improve splice variants detection. First, we review the main computational tools, including the recent Machine Learning-based algorithms, for the prediction of splice site variants, in order to characterize how a genomic variant interferes with splicing process. Next, we report the experimental methods to validate the predictive analyses are defined, distinguishing between methods testing RNA (transcriptomics analysis) or proteins (proteomics experiments). For both prediction and validation steps, benefits and weaknesses of each tool/procedure are accurately reported, as well as suggestions on which approaches are more suitable in diagnostic rather than in clinical research.

Selection of Diagnostically Significant Regions of the SLC26A4 Gene Involved in Hearing Loss

“…The “classical” splice acceptor and splice donor variants disrupting canonical splice sites at positions -1, -2 (3’ acceptor splice site) or +1, +2 (5’ donor splice site) are the most important for proper splicing and can lead to exon skipping or the emergence of an alternative splicing motif. The variants located in intronic regions near splice junctions (outside the canonical splice sites) appear to be also important for splicing, but studies that evaluated their pathogenic effects in detail on splicing are still limited [ 49 , 52 , 53 , 54 ]. In addition, mutations in deep intronic regions have been documented in multiple diseases.…”

VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms using long-read sequencing

“…Up to 15% of the pathogenic variants affect RNA splicing not only by involving canonical splicing sites but also by exonic and intronic non-canonical sites in increasing cases (Riolo et al, 2021). Therefore, they can be missed during conventional DNA screening or misinterpreted by in silico splicing predictions.…”

Characterization of peripheral white blood cells transcriptome to unravel the regulatory signatures of bovine subclinical mastitis resistance

“…The splicing factor PRPF8 is critical for breast cell survival and has potential prognostic value in breast cancer ( Cao et al, 2022 ). What is more, these two SNPs are variants of spliced polypyrimidine fragments ( Supplementary Table S5 ), which are recognized by the polypyrimidine fragment binding protein of the spliceosome complex and are essential for the initial recognition of introns in mammals ( Riolo et al, 2021 ), and the expansion of the polypyrimidine fragment enhances the efficiency of splicing ( Romfo and Wise, 1997 ). Therefore, we hypothesized that the SNPs rs42705933 and rs133847062 may act as pathogenic mutations that cause the genes PIK3C2B and PRPF8 to exhibit exon skipping, which in turn affects the resistance of cows to subclinical mastitis, but the detailed regulatory mechanism remains to be verified in subsequent future studies.…”