Increased noncanonical splicing of autoantigen transcripts provides the structural basis for expression of untolerized epitopes

Abstract

Background: Alternative splicing is important for increasing the complexity of the human proteome from a limited genome. Previous studies have shown that for some autoantigens, there is differential immunogenicity among alternatively spliced isoforms.

Objectives: Herein, we tested the hypothesis that alternative splicing is a common feature for transcripts of autologous proteins that are autoantigens. The corollary hypothesis tested was that nonautoantigen transcripts have a lower frequency of alternative splicing.

Methods: The extent of alternative splicing within 45 randomly selected self-proteins associated with autoimmune diseases was compared with 9554 randomly selected proteins in the human genome by using bioinformatics analyses. Isoform-specific regions that resulted from alternative splicing were studied for their potential to be epitopes for antibodies or T-cell receptors.

Results: Alternative splicing occurred in 100% of the autoantigen transcripts. This was significantly higher than the approximately 42% rate of alternative splicing observed in the 9554 randomly selected human gene transcripts ( P < .001). Within the isoform-specific regions of the autoantigens, 92% and 88% encoded MHC class I and class II-restricted T-cell antigen epitopes, respectively, and 70% encoded antibody binding domains. Furthermore, 80% of the autoantigen transcripts underwent noncanonical alternative splicing, which is also significantly higher than the less than 1% rate in randomly selected gene transcripts ( P < .001).

Conclusion: These studies suggest that noncanonical alternative splicing may be an important mechanism for the generation of untolerized epitopes that may lead to autoimmunity. Furthermore, the product of a transcript that does not undergo alternative splicing is unlikely to be a target antigen in autoimmunity.

FIG 1

A, The 5′ and 3′ splice sites (SS) of introns typically occur at GT-AG flanking sequences. This type of splicing is termed canonical splicing, which accounts for >99% of splicing for randomly selected transcripts. B, Noncanonical splicing occurs when the intronic flanking sequences do not follow this GT-AG rule and accounts for <1% of splicing.

FIG 2

Diagrammatic representation of an autoantigen undergoing alternative splicing, resulting in at least 2 possible mRNA isoforms, which are then translated into 2 distinct protein isoforms. Isoform specific regions can result from inclusion of extra exons (red) or at the junctional region where an exon is spliced out (zigzag). These potentially encode the untolerized epitopes that may lead to autoimmune response (see Fig E1 in the Journal’s Online Repository at www.mosby.com/jaci for a more detailed diagrammatic representation of this figure).

FIG 3

Permissive splicing in autoimmunity working model. Autoimmunity depends on host susceptibility (eg, autoimmune diseases–associated HLA), and environmental insults (eg, viruses) as well as the frequency of splicing. The model predicts that for a protein to be a pathogenic autoantigen, it must have a high frequency of alternative splicing, which can be modulated by inflammatory cytokines. Of the 6 possible scenarios, model B fulfills these criteria.