Making alternative splicing decisions during epithelial-to-mesenchymal transition (EMT)

Abstract

Alternative splicing generates multiple mRNAs from a single transcript and is a major contributor to proteomic diversity and to the control of gene expression in complex organisms. Not surprisingly, this post-transcriptional event is tightly regulated in different tissues and developmental stages. An increasing body of evidences supports a causative role of aberrant alternative splicing in cancer. However, very little is known about its impact on cellular processes crucially involved in tumor progression. The aim of this review is to discuss the link between alternative splicing and the epithelial-to-mesenchymal transition (EMT), one of the major routes by which cancer cells acquire invasive capabilities and become metastatic. We begin with a brief overview of alternative splicing. Next, we discuss alternative splicing factors that regulate EMT. Finally, we provide examples of target genes presenting alternative splicing changes that contribute to the morphological conversions in the EMT process.

Fig. 1

Cis-acting sequences controlling splicing reaction and five different types of alternative splicing. a Within the intron, many cis-acting sequences are required for pre-mRNA splicing: the 5′ splice site (donor), the 3′ splice site (acceptor), the polypyrimidine tract and the branchpoint sequence, which includes an adenine nucleotide representing the nucleophile for the first step of splicing. b During transcription, through pre-mRNA splicing, introns are precisely removed and exons are joined together to reconstitute the reading frame and to generate translatable mRNAs. c–g Alternatively spliced mRNAs can result through the usage of various combinations of donor and acceptor sites from different exons. The five types of alternative splicing are therefore called: exon skipping (or cassette exon), intron retention, mutually exclusive exons, alternative 5′ splice sites and alternative 3′ splice sites. At the protein level, alternative splicing drastically affects the amino acid sequence by deletion or insertion of domains, frame-shifts, or stop codons. A single pre-mRNA can often exhibit multiple regions that undergo alternative splicing, giving rise to complex splicing patterns and, as a consequence, to many different final mRNAs

Fig. 2

Cis- and trans-acting elements controlling alternative splicing decisions. Alternatively spliced exons are usually characterized by weak splice sites (short and degenerate). Recognition of these sites is influenced by the presence of four types of regulatory RNA sequences: exonic splicing enhancers (ESE) and silencers (ESS) and intronic splicing enhancers (ISE) and silencers (ISS). Splicing enhancer elements are most commonly bound by splicing factors of the SR family, whereas among the proteins interacting with splicing silencers there are the heterogeneous nuclear ribonucleoproteins (hnRNPs). These ubiquitous splicing factors act by a enhancing or b preventing the binding of specific spliceosomal components to the pre-mRNA, such as snRNP Ul to the 5′ splice site, snRNP U2 to the branchpoint and the U2AF heterodimer that recognizes the polypyrimidine tract and the conserved dinucleotide AG at the 3′ splice site. c Specific splicing patterns result from cooperative as well as antagonistic effects (“combinatorial control”) of multiple RNA-binding proteins. Thus, exon inclusion or skipping is determined by balance of these competing activities, which in turn reflect the relative abundance and/or cellular localization of the cognate RNA-binding activator and repressor proteins

Fig. 3

Alternative splicing of Ron proto-oncogene during epithelial-to-mesenchymal transitions (EMT). Skipping of Ron exon 11 results in the production of ∆Ron and is controlled by a splicing silencer (–) and an enhancer (+) located in the constitutive exon 12 [10]. The splicing factor SRSF1, by promoting ∆Ron splicing, activates EMT, leading to acquisition of an invasive phenotype. Cells that undergo EMT (green) during tumor progression are characterized by the loss of cell–cell adhesion, cytoskeleton rearrangements, and increased cell motility. Interestingly, morphological and molecular hallmarks of EMT are transient and are restricted to the invasive front of metastasizing carcinomas. SRSF1 levels are regulated during EMT through alternative splicing associated with nonsense-mediated RNA decay (AS-NMD) by another splicing factor (Sam68) that binds within the 3′ UTR-intron of the SRSF1 transcript [11]. a In mesenchymal cells, Sam68 phosphorylation by the extracellular signal regulated kinase 1/2 (ERK1/2) is sufficient to increase the full-length transcript of SRSF1 thus increasing SRSF1 protein levels that in turn drive a ∆Ron-mediated EMT. b Diffusible factors specifically secreted by epithelial cells (blu) repress ERK 1/2 activity by this means inhibiting Sam68 phosphorylation, which reduces SRSF1 protein levels through increased NMD of its transcript