Deregulation of splicing factors and breast cancer development

388 | Journal of Molecular Cell Biology (2015), 7(5), 388 – 401 doi:10.1093/jmcb/mjv027 Published online May 5, 2015 Review Deregulation of splicing factors and breast cancer development Marco Silipo, Hannah Gautrey, and Alison Tyson-Capper* Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK * Correspondence to: Alison Tyson-Capper, E-mail: It is well known that many genes implicated in the development and progression of breast cancer undergo aberrant alternative splicing events to produce proteins with pro-cancer properties. These changes in alternative splicing can arise from mutations or single-nucleotide polymorphisms (SNPs) within the DNA sequences of cancer-related genes, which can strongly affect the activity of splicing factors and influence the splice site choice. However, it is important to note that absence of mutations is not sufficient to prevent misleading choices in splice site selection. There is now increasing evidence to demonstrate that the expression profile of ten splicing factors (including SRs and hnRNPs) and eight RNA-binding proteins changes in breast cancer cells compared with normal cells. These modifications strongly influence the alternative splicing pattern of many cancer-related genes despite the absence of any detrimental mutations within their DNA sequences. Thus, a comprehensive assessment of the splicing factor status in breast cancer is important to provide insights into the mechanisms that lead to breast cancer development and metastasis. Whilst most studies focus on mutations that affect alternative splicing in cancer-related genes, this review focuses on splicing factors and RNA-binding proteins that are themselves deregulated in breast cancer and implicated in cancer-related alternative splicing events. Keywords: alternative splicing, splicing factors, RNA-binding proteins, breast cancer Introduction The majority of human protein-coding genes produce multiple mRNA transcripts by alternative splicing. This RNA processing event is mediated by a complex interplay of splicing factors (SFs) and defined RNA sequences (splice sites) within pre-mRNA transcripts. Splice sites are short conserved sequences located within the pre-mRNA, which define the start/end of the exons and introns (Srebrow and Kornblihtt, 2006) (Table 1 and Figure 1). Splice sites act as binding sites for the spliceosome—a multi component complex comprising of five small nuclear ribonucleoproteins (snRNPs) working with 100–200 different non-snRNPs (McManus and Graveley, 2011). Selection of different splice sites is under the control of the splicing factors to either promote or prevent the recruitment of the spliceosome to splice sites (Cartegni et al., 2002) (Figure 1). It is now established that aberrant alternative splicing events are associated with the onset and aggression of breast cancer (Dutertre et al., 2010). One such example is that of the BRCA1 gene. BRCA1 is a tumour suppressor and mutations within its genetic sequence are associated with breast cancer (Orban and Olah, 2003). Alternative splicing of BRCA1 can generate variants that lack functional domains of the protein, thus compromising its tumour suppressor activity (Orban and Olah, 2003). The use of splicing assays reveals that mutations within specific sequences can affect the binding of splicing factors and this in turn strongly influences the splicing pattern of BRCA1 (Raponi et al., 2014). It is not uncommon that point mutations that affect the binding of splicing factors to the pre-mRNA can lead to erroneous control over the spliceosome, resulting in inaccurate alternative splicing of cancer-related genes (Cartegni et al., 2002). As such, the production of proto-oncogene splice variants might be favoured over tumour suppressor splice variants. In addition to mutations at binding sites, changes in the nuclear concentration of splicing factors can also have repercussions on splice site selection (Figure 2). Deregulation of splicing factors, and also other RNA-binding proteins, disrupt alternative splicing patterns of many genes associated with cancer (Grosso et al., 2008). This review focuses on splicing factors that have critical roles in regulating cancer-related genes associated with development and progression of breast cancer. Received December 15, 2014. Revised February 12, 2015. Accepted February 24, 2015. # The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. Splicing factors Splicing factors are RNA-binding proteins that interact with specific RNA sequences, or motifs, known as exonic splicing enhancers or silencers (ESEs, ISEs) and intronic splicing enhancer and Splicing factors and breast cancer | 389 Table 1 Splice sites and spliceosome members. Splice site Location Motif Spliceosome member 5′ splice site (5′ SS or donor splice site) Exon/intron junction Small nuclear ribonucleic particle (snRNP) U1 Branch point Intron Polypyrimidine tract 3′ splice site (3′ ss or acceptor splice site) Intron Intron/exon junction AG/GURAGU / ¼ exon/intron boundary R ¼ purine (either G or A) YNYURAY N ¼ any nucleotide Y ¼ pyrimidine (either C or U) A ¼ pivotal adenine for the splicing process (Srebrow and Kornblihtt, 2006) 15 –20 uridine/cytosine repetition YAG/ Y ¼ pyrimidine (either C or U) / ¼ intron/exon boundary (Collins and Guthrie, 2001) Small nuclear ribonucleic particle (snRNP) U2 U2AF large subunit (U2AF65) U2AF small subunit (U2AF35) This table shows consensus splice sites within pre-mRNA sequences, together with spliceosome members that specifically bind to the splices to trigger the splicing process. Nucleotide sequences and location of each splice site are also indicated. Figure 1 Regulation of splicing. Pre-mRNA contains four conserved sequences required for the splicing process; snRNP U1 binds the 5′ ss therefore defining the end of the Exon 1; the small subunit of the dimeric protein U2AF (U2AF35) binds the 3′ ss therefore defining the end of the intron and the beginning of Exon 2. Both U2AF35 and the larger subunit U2AF65, when bound to the polypyrimidine tract (defined by uridine repetition), support the recruitment of snRNPU2 to the branch point (marked by an adenine). The figure highlights that the binding of U2AF to both 3′ splice site and polypyrimidine tract is inhibited by a nearby exonic splicing silencer (ESS) and an intronic splicing silencer (ISS). This prevents the recruitment of snRNP U2 to the branch point. Exon/intron splicing enhancers (ESEs, ISEs) and exon/intron splicing silencers (ESSs, ISSs) are defined recondition RNA for splicing factors that, in turn, either promote or prevent the splicing process. Splicing factors either guide or block the interactions between the spliceosome and splice sites. Different splicing factors can also compete with each other for binding the same motif, t (...truncated)