The pathobiology of splicing

Abstract

Ninety-four percent of human genes are discontinuous, such that segments expressed as mRNA are contained within exons and separated by intervening segments, called introns. Following transcription, genes are expressed as precursor mRNAs (pre-mRNAs), which are spliced co-transcriptionally, and the flanking exons are joined together to form a continuous mRNA. One advantage of this architecture is that it allows alternative splicing by differential use of exons to generate multiple mRNAs from individual genes. Regulatory elements located within introns and exons guide the splicing complex, the spliceosome, and auxiliary RNA binding proteins to the correct sites for intron removal and exon joining. Misregulation of splicing and alternative splicing can result from mutations in cis-regulatory elements within the affected gene or from mutations that affect the activities of trans-acting factors that are components of the splicing machinery. Mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. An understanding of the role of splicing in disease expands potential opportunities for therapeutic intervention by either directly addressing the cause or by providing novel approaches to circumvent disease processes.

Figure 1. Alternative splicing patterns increase mRNA diversity

Through alternative use of exons, introns, promoters, and polyadenylation sites, alternative splicing acts to greatly increase the diversity of mRNA transcripts.

Figure 2. The combination of cis-acting elements and trans-acting factors determine splice site selection

A) Three core splicing sequences essential for splicing of all exons are the 5’ splice site beginning with an invariant GU dinucleotide, the 3’ splice site ending with an invariant AG dinucleotide, and the branch site sequence. These elements are recognized by components of the spliceosome, such as the U1 and U2 snRNPs and U2AF. B) Additional regulatory sequences located in the intron and exon are also required for exon recognition and to modulate splice site selection of alternatively used splice sites. Exonic splicing enhancers (ESE) and silencers (ESS) are bound by positive and negative splicing regulators, such as SR proteins and hnRNP proteins, respectively. Intronic splicing enhancers (ISE) and silencers (ISS) also recruit splicing regulatory complexes. C) Disease-causing mutations within cis-acting elements disrupt proper recognition by splicing components.

Figure 3. Antisense oligonucleotide (AON) based therapies

Modified AONs that avoid RNase H-mediated degradation of target pre-mRNAs can modulate splicing in several ways by A) binding to splice sites, B) splicing enhancer elements, or C) splicing silencer elements and therefore abolish control by the corresponding splicing regulatory proteins. D) AONs designed to bind and degrade the target pre-mRNA by endogenous RNase H can block expression of potentially harmful mutant mRNAs. E) AONs that bind pathogenic RNA can prevent interactions with RNA binding proteins causing release of both the proteins and the RNA.