The Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease

Abstract

Alternative splicing of mRNA is an essential mechanism to regulate and increase the diversity of the transcriptome and proteome. Alternative splicing frequently occurs in a tissue- or time-specific manner, contributing to differential gene expression between cell types during development. Neural tissues present extremely complex splicing programs and display the highest number of alternative splicing events. As an extension of the central nervous system, the retina constitutes an excellent system to illustrate the high diversity of neural transcripts. The retina expresses retinal specific splicing factors and produces a large number of alternative transcripts, including exclusive tissue-specific exons, which require an exquisite regulation. In fact, a current challenge in the genetic diagnosis of inherited retinal diseases stems from the lack of information regarding alternative splicing of retinal genes, as a considerable percentage of mutations alter splicing or the relative production of alternative transcripts. Modulation of alternative splicing in the retina is also instrumental in the design of novel therapeutic approaches for retinal dystrophies, since it enables precision medicine for specific mutations.

Keywords: alternative splicing; deep intronic variants; inherited retinal dystrophies; microexons; non-canonical splice site variants; retina; splicing factors.

Figure 1

Alternative splicing in the neural retina. (A) Common mechanisms of alternative splicing in the retina. Boxes represent exons, lines represent introns, promoters are represented with arrows and polyadenylation sites are indicated with -AAAA. Exon regions included in the alternative transcript are colored. (B) Microexons have a role in late neurogenesis and are relevant in neurological disorders. The reduced expression of neural-specific splicing factors that regulate the inclusion of microexons is linked to the altered splicing of microexons in patients with neurological disorders. (C) Novel alternative splicing events in the human retina detected by RNA sequencing (data from [35]).

Figure 2

Schematic representation of the splicing process. (A) Assembly of the spliceosome: U1 snRNP recognizes the splice donor site (SDS) and U2 snRNP recognizes the splice acceptor site (SAS) to generate complex E. (B) U2 recognizes the adenosine at the branch-site and forms complex A. (C) The U4/U6·U5 tri-snRNP joins the spliceosome to form complex B. (D) U4 and U1 are released, U6 replaces U1 recognizing the SDS and interacts with U2, generating complex C and catalyzing the splicing reaction. (E) Exons are ligated, and intronic pre-mRNA and spliceosomal snRNPs are liberated.

Figure 3

Quantification of alternative splicing events described in genes causing IRDs. (A) Percentage of IRD genes presenting alternative splicing events. Only 10% of the IRD genes produces a unique transcript, and most genes (54%) generate between 2 and 10 transcripts. (B) Percentage of IRD genes showing diverse protein-coding transcripts. Only 15% of the IRD genes produce one protein isoform. Most of the genes (53%) produce between 2 and 5 diverse protein isoforms. The list of genes was obtained from https://sph.uth.edu/retnet/ (accessed on 20 January 2021). Information about the number and type of the transcripts was obtained from https://www.ensembl.org/ (accessed on 20 January 2021).

Figure 4

Overview of cis acting mutations altering splicing: NCSS (A,B), deep intronic (C–E) and deep exonic variants (F). (A) ABCA4 exon 3 shows a weak natural exon skipping. The c.161G>A mutation increase exon 3 skipping (producing a truncated ABCA4 protein) and the p.Cys54Tyr amino acid substitution. (B) The ABCA4 c.4849-8C>G mutation lowers the value of the poly-Py tract, thus causing intron 34 retention and production of a truncated protein. (C) The CEP290 c.2991+1655A>G mutation creates a new SDS, that induce inclusion of a cryptic exon (exon X) encoding a premature stop codon. (D) The USH2A c.7595-2144A>G mutation creates a new SDS that causes pseudoexon inclusion (PE40) and introduces a premature stop codon. (E) The ABCA4 c.1938-619A>G mutation, located in a cryptic pseudoexon (PE), leads to the recognition of ESEs that promote PE inclusion, leading to the truncation of the protein. (F) The RHO c.620T>G mutation creates a strong splice donor site that results in a 30 amino acid in-frame deletion.