Finding signals that regulate alternative splicing in the post-genomic era

Abstract

Alternative splicing of pre-mRNAs is central to the generation of diversity from the relatively small number of genes in metazoan genomes. Auxiliary cis elements and trans-acting factors are required for the recognition of constitutive and alternatively spliced exons and their inclusion in pre-mRNA. Here, we discuss the regulatory elements that direct alternative splicing and how genome-wide analyses can aid in their identification.

Figure 1

Functionally significant examples of different types of alternative splicing. (a) Alternative inclusion of a cassette exon is very common. Neuron-specific inclusion of the N1 exon in the c-src proto-oncogene generates an insertion in the SH3 protein-protein interaction domain that alters its binding to other proteins [34]. (b) Alternative exons may be mutually exclusive, such as exons IIIb and IIIc in the fibroblast growth factor receptor 2 (FGFR-2) gene. Use of IIIb produces a receptor with high affinity for keratinocyte growth factor (KGF), whereas use of IIIc produces a high-affinity FGF receptor. Loss of the IIIb isoform is thought to be important in prostate cancer [35]. (c) The choice of an alternative 5' splice site in the Wilms' Tumor suppressor gene Wt1 results in the insertion of the three amino acids lysine, threonine, and serine (KTS). The +KTS and -KTS forms play distinct roles in kidney and gonad formation, and shift of the balance toward the -KTS form is associated with Frasier syndrome [36]. (d) In the transformer (tra) gene in Drosophila, selection of a female-specific alternative 3' splice site produces a single long open reading frame that gives rise to a regulatory protein that controls female somatic sexual differentiation. In male flies, tra mRNAs lack a long open reading frame, and no protein is made [37]. (e) Alternative terminal exons in the gene encoding calcitonin and calcitonin-gene-related peptide (CGRP) give rise to a hormone involved in calcium homeostasis in the thyroid gland, or a neuropeptide involved in vasodilation in the nervous system [38]. (f) Alternative promoter usage in the myosin light chain (MLC) gene leads to different first exons, which pair with mutually exclusive downstream exons to give rise to distinct protein isoforms, namely MLC1 and MLC3 [39]. This type of alternative splicing pattern results primarily from transcriptional regulation, not from the regulation of splice-site choice per se. (g) Intron retention is one of the rarest forms of alternative splicing in humans. Retention of intron 2 in the human muscle-specific chloride channel 1 (ClC-1) mRNA in myotonic dystrophy (DM) patients introduces a premature stop codon and leads to downregulation of ClC-1 expression, contributing to problems in muscle relaxation (myotonia) [2].

Figure 2

Typical features of alternative exons. Alternative exons are on average less than half the size of constitutive exons and have weak 5' and/or 3' splice sites. Auxiliary elements aid or prevent the recognition of these exons by binding trans-acting factors in different cellular contexts, and how often an exon is included in the mRNA depends on a balance between positive and negative regulation. Enhancer (+) and silencer (-) elements can be found within the alternative exon (yellow box in the center) or the flanking introns (lines). Splicing decisions are controlled by multiple elements, and for a given exon these can be different elements, multiple copies of the same element located at different sites, or a combination of the two (as indicated by the non-yellow colored boxes). Different alternative exons are regulated by different sets of auxiliary elements, but alternative exons that are regulated by the same trans-acting factors have some common elements. Intronic elements can be distal, but are more often located in the introns adjacent to the alternative exon (near the exon-intron boundary), and in some cases can overlap with, or be contained within, the consensus splice site sequences that are recognized by the basal spliceosomal machinery.