Exonic and intronic splicing enhancers and silencers. (a) Pre-mRNA splicing is a highly complex process that is orchestrated by the spliceosome, and involves hundreds of different proteins and small nuclear RNAs (snRNAs). The start and the end of introns are recognized by the splicing machinery as these so-called splice sites contain consensus sequences. However, not all exons have strong splice sites, and consensus-like sequences are also present in introns. A family of splicing factors facilitates the recognition of proper exons through binding to exonic/intronic splicing enhancer sequences (E/ISEs) and impedes the inclusion of pseudoexons by binding to intronic/exonic splicing silencers (I/ESSs). The splicing factors involved are SR proteins (eg, SF2/ASF and Tra-2β) and hnRNPs, respectively. Generally, splicing enhancers are located in exons, and silencers in introns, but this is not always the case. Some exons contain both enhancer and silencer elements, and, depending on the tissue-specific levels of SR and hnRNP proteins, the exon is included or excluded from the transcript. (b) In exon 7 of SMN2, a translationally silent mutation disrupts an ESE while it creates an ESS. As a consequence, the splicing enhancing SF2/ASF can no longer bind, whereas the splicing silencing hnRNP A protein can now bind. The exon is no longer properly recognized and skipped in most transcripts.

Overview of the pathology of myotonic dystrophy. Expanded repeat units eventually lead to a misregulation of alternative splicing (the ClC-1 gene is shown as an example). Therapeutic approaches can either target the expanded transcripts or try to restore normal splicing.

Antisense-mediated exon skipping for DMD. DMD is caused by mutations (in this example, a deletion of exons 49–50, upper panel) in the DMD gene that disrupt the open reading frame. Consequently, protein translation is truncated prematurely and the resulting dystrophin cannot fulfill its normal function (ie, to connect the extracellular matrix and the cytoskeleton in muscle fibers). AONs complementary to an exon (exon 51 in this example, lower panel) can hide this exon from the splicing machinery during pre-mRNA splicing, resulting in the skipping of the targeted exon. By strategically targeting specific exons in DMD patients, the reading frame can be restored through exon skipping, allowing the synthesis of an internally truncated protein that is partially functional.

Splicing enhancers and silencers in and flanking SMN2 exon 7. SMN exon 7 splicing is regulated by many enhancing (blue) and silencing (yellow) elements located in intron 6, the exon itself and intron 7, and the exon has a weak 3′ splice site. An intronic splicing silencer, called element 1, is located in intron 6. The C to T transition in the proximal part of the exon disrupts an exonic splicing enhancer that normally recruits SF2/ASF and creates a new splicing silencer that binds hnRNP A1 (extinct). A Tra-2β binding site is located downstream of the extinct in the so-called conserved region that acts as an ESE. An additional silencer is located at the end of the exon in the 3′ cluster. Two intronic splicing silencers are located in intron 7, interspersed by an intronic splicing enhancer. Together, these sequences make up ‘element 2.' The first silencer is called ‘N1.' The more recently discovered second silencer has been shown to bind to hnRNP A1.