RNA splicing factors as oncoproteins and tumour suppressors

Abstract

The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these 'spliceosomal mutations' suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic and biological effects of spliceosomal mutations are crucial for the development of cancer therapies targeted at these mutations.

Figure 1. Simplified model of constitutive and alternative splicing

(a) The key sequence features that govern splicing are shown, including consensus sequences of the 5′ and 3′ splice sites and sequence motifs bound by trans-acting splicing factors (serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs) and others). Sequence elements required for assembly of the spliceosome onto the pre-mRNA, including the splice sites themselves, polypyrimidine (poly(Y)) tract and branch point, frequently follow the illustrated consensus motifs, whereas the sequences of enhancer or silencer elements depend upon the specific RNA-binding protein that recognizes them. Consensus motifs are illustrated as sequence logos, where the height of each nucleotide corresponds to its approximate genome-wide frequency in bits. Sequence motifs are illustrated as genomic DNA sequence rather than pre-mRNA sequence (“T” instead of “U”). Note that splicing factors such as SR proteins and hnRNPs frequently play context-dependent regulatory roles. (b) Simplified schematic of intron excision and ligation of two adjacent exons. The steps shown are: recognition of the 5′ and 3′ splice sites by the U1 and U2 small nuclear ribonucleoprotein complexes (snRNPs), assembly of the snRNPs into the active spliceosome, the excision of the intron lariat and the ligation of the two exons. (c) Schematic of constitutive and alternative splicing events. Light blue: constitutive sequence that always forms part of the mature mRNA; dark blue: alternative sequence that can be either included or excluded in the mature mRNA.

Figure 2. Commonly mutated spliceosomal proteins and their associations with specific cancer types

(a) The incidence and cancer distribution of the most frequently mutated spliceosomal genes are shown. (b) The spectrum of mutations that have been identified in U2AF1 (U2 small nuclear RNA auxiliary factor 1), SRSF2 (serine/arginine-rich splicing factor 2), ZRSR2 (zinc finger, RNA-binding motif and serine/arginine-rich 2) and SF3B1 (splicing factor 3B, subunit 1). Mutations shown in bold text occur at hotspots; other illustrated mutations are recurrent but rare. As ZRSR2 mutations do not occur at hotspots, very rare or private mutations are shown as examples. (c) Schematic illustrating the potential reasons that spliceosomal gene mutations are mutually exclusive with one another: they either converge on a common downstream target or result in synthetic lethality. (d) The frequency of specific mutations in SF3B1 across various histological subtypes of cancer. AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; CMML, chronic myelomonocytic leukemia; HD, HEAT domain; PPP1R8, binding site for protein phosphatase 1 regulatory subunit 8; RARS, refractory anemia with ringed sideroblasts; RCMD-RS, refractory cytopenia with multilineage dysplasia and ringed sideroblasts; RRM, RNA recognition motif; RS, arginine/serine-rich domain; sAML, secondary AML; UHM, U2AF homology motif; ZN, zinc finger domain.

Figure 3. Current understanding of the mechanistic consequences of spliceosomal gene mutations for RNA splicing

(a) Splicing factor 3B, subunit 1 (SF3B1) mutations (mutant form shown in red) alter 3′ splice site (ss) selection by permitting or enhancing recognition of cryptic upstream 3′ splice sites. It is not yet known how mutations affecting SF3B1 alter its target protein:RNA and/or protein:protein interactions to drive this cryptic splice site recognition. Sequence motifs are illustrated as genomic DNA sequence rather than pre-mRNA sequence (“T” instead of “U”). (b) U2 small nuclear RNA auxiliary factor 1 (U2AF1) mutations alter 3′ splice site consensus sequences. Wild-type U2AF1 recognizes the consensus motif yAG|r at the intron|exon boundary (y = pyrimidine, r = purine, ‘|’ = intron-exon boundary). S34F or S34Y mutations (shown in red) promote recognition of cAG|r over tAG|r, whereas Q157P or Q157R mutations (shown in red) promote recognition of yAG|g over yAG|a. (c) Zinc finger, RNA-binding motif and serine/arginine-rich 2 (ZRSR2) mutations (shown in red) cause loss of ZRSR2 function to induce splicing defects, primarily involving the aberrant retention of U12-type introns. (d) Serine/arginine-rich splicing factor 2 (SRSF2) mutations (shown in red) alter exonic splicing enhancer (ESE) preferences. Wild-type SRSF2 recognizes the consensus motif SSNG (S = C or G), whereas mutant SRSF2 preferentially recognizes the CCNG motif over the GGNG motif.

Figure 4. Links between splicing factors and diverse biological processes and potential methods for therapeutic manipulation of splicing

(a) Changes in the abundance, post-translational modifications and/or subcellular localization of splicing factors such as serine/arginine-rich splicing factor 1 (SRSF1) can cause DNA damage or influence the DNA damage response (DDR). Splicing factors have been linked to DNA damage and the DDR both directly (for example, insufficient levels of splicing factors can cause R loop formation and genomic instability) and indirectly (for example, through downstream changes in splicing). (b) The steps involved in nonsense-mediated decay (NMD) are shown. The exon junction complex (EJC; grey) is deposited upstream of exon-exon junctions on the processed mRNA, and is displaced by the ribosome (blue) during the pioneer (first) round of translation. Ribosome stalling at a premature termination codon (PTC) >50 nt upstream of an EJC promotes interactions between release factors (purple) and UPF1 (green), recruitment of other NMD components (orange), and RNA degradation by endo- and exonucleases (beige). In contrast, if only a normal termination codon (TC) is present, then all EJCs are displaced by the ribosome during the pioneer round of translation and NMD is not triggered. Red stop signs indicate stop codons. (c) Compounds and oligonucleotides that can disrupt or modulate normal splicing catalysis or alter splice site recognition through distinct pathways include (i) drugs affecting U2 small nuclear ribonucleoprotein (snRNP) function, formation, and/or interaction with pre-mRNA, (ii) drugs affecting post-translational modifications of serine/arginine-rich (SR) proteins and potentially other splicing factors, and (iii) oligonucleotides to manipulate specific mRNA isoforms that may be important in tumor maintenance. CLK, CDC2-like kinase; SF3B1, splicing factor 3B, subunit 1; SRPK, SR protein kinase.