BRCA1 (breast cancer early-onset 1) alternative splicing levels are regulated in a cell-cycle- and cell-type-specific manner, with splice variants being present in different proportions in tumour cell lines as well as in normal mammary epithelial cells. The importance of this difference in the pathogenesis of breast cancer has yet to be determined. Developing an understanding of the impact of BRCA1 isoform ratio changes on cell phenotype will be of value in breast cancer and may offer therapeutic options. In the present paper, we describe the splicing isoforms of BRCA1 exon 11, their possible role in cancer biology and the importance of maintaining a balanced ratio.
- alternative splicing
- breast cancer early-onset 1 (BRCA1)
Splicing is an important mechanism which contributes to pre-mRNA maturation. Control of this process determines which pre-mRNA sequences are removed (introns) and which are spliced to form the mature RNA (exons). The machinery driving the splicing process is the spliceosome, a complex of many interacting proteins and snRNAs (small nuclear RNAs) .
In order to perform accurate splicing, the spliceosome must recognize the donor site at the exon–intron junction and the acceptor site at the intron–exon junction. These elements are necessary, but not always sufficient. Splice site strength often depends on nearby sequence elements known as splicing enhancers and silencers .
Different combinations of splice site selections allow a large number of mRNA and protein isoforms to be generated from a lower number of genes; this process, known as alternative splicing, is a critical mechanism and generates transcriptome and proteome complexity. Alternative splicing has been found to be associated with diseases, including cancer, and can influence cell proliferation, motility and response to drugs .
Mutations in splicing regulatory sequences as well as alteration in the levels of splicing regulatory proteins may affect alternative splicing. BRCA1 (breast cancer early-onset 1) mutations predicted to affect BRCA1 function have been found in 40–45% cases of hereditary breast cancer. In addition, a large number of BRCA1 sequence variants have been found in patients but their clinical significance is unknown. These unclassified variants could include potential splicing mutations .
Several mRNA splicing isoforms have been identified for the BRCA1 gene in normal tissues. Among those that result from exon-skipping events and retain the translational reading frame are variants that skip exon 5, exon 11 (all of it or most of it), exons 2–10, exons 9–11, exons 14–17 and exons 14–18 . Some of these splicing isoforms have been associated with breast and ovarian cancer . In particular, the relative levels of BRCA1 isoforms associated with exon 11 alternative splicing appears to be different between normal and cancer tissues/cell lines as well as between the phases of the cell cycle . These isoforms include BRCA1 full-length (inclusion of all exons), Δ11 (skipping of exon 11), Δ11q (partial skipping of exon 11, throughout the use of a donor site within exon 11), Δ9,10,11q (skipping of exons 9, 10 and partial skipping of exon 11) and IRIS isoforms (skipping of exons 12–24, but retaining a short segment from intron 11) (Figures 1, 2 and 3). With the exception of IRIS, it is unclear whether altered levels of exon 11 splicing isoforms play a pathogenic role in cancer.
BRCA1 protein domains and multiple roles
The breast cancer susceptibility gene BRCA1 is a tumour-suppressor gene that was first identified on the basis of its linkage to early-onset breast and ovarian cancer in women . The BRCA1 gene has 24 exons, including two untranslated exons, and encodes a protein of 1863 amino acids with three distinct regions of protein interaction: the RING domain, the RAD51-interaction domain and the BRCT (BRCA1 C-terminus) domain .
The N-terminal RING finger domain has been found to bind to several proteins, including formation of heterodimers with BARD1 (BRCA1-associated RING domain protein) . Both BRCA1 and BARD1 contain the RING finger domain that seems to be important for several tumour-suppression functions .
The RAD51-interaction domain is enclosed by exon 11 and is involved in repair of double-strand DNA breaks . It also contains multiple protein-binding sites, in addition to those for the RAD51 and RAD50 complex . Loss of RAD51 binding may increase the risk of cancer as there could be an increment in the amount of damaged DNA.
The C-terminus of BRCA1 contains an acidic domain that can function as a transcriptional activation domain [8,13]. This domain contains a tandem repeat of approximately 95 amino acids called BRCT . This domain is also found in proteins such as BARD1 which is involved in cell-cycle control and DNA repair .
BRCA1 has been implicated in diverse cancer-related activities, including roles in cell-cycle progression, DNA repair, DNA damage-responsive cell-cycle checkpoints, transcription regulation, ubiquitination, chromosome remodelling and apoptosis .
The presence of different BRCA1 splicing isoforms that are naturally expressed in various cellular settings may be the reason that BRCA1 is involved in such different activities.
The Δ11 isoform
The Δ11 isoform is composed of 21 coding exons, arising from skipping of exon 11 (Figure 1). Exon 11 is a large exon of 3.4 kb. The acceptor site (at the intron–exon junction) and the donor site (at the exon–intron junction) of exon 11 are predicted to have weak splice site consensus sequences and therefore may be also surrounded by additional regulatory sequences in order to be recognized. We have shown recently that such regulatory sequences exist at the beginning of exon 11 and they are important for regulating BRCA1 alternative splicing . The identification of which factors bind to these sequences would provide insight into the mechanism of BRCA1 alternative splicing regulation and especially the maintenance of BRCA1 isoform ratios.
Although studies have attempted to determine the role of the Δ11 isoform, its importance in cancer has yet to be determined and has been implicated in both cell death and cell proliferation (Table 1).
Both BRCA1-null mice and mice that exclusively express BRCA1 Δ11 undergo embryo lethality [18,19]. However, embryos with exclusive expression of BRCA1 Δ11 die later, suggesting that, at least in part, the Δ11 isoform can compensate for the lack of the other BRCA1 isoforms during early embryogenesis.
Embryos exclusively expressing BRCA1 Δ11 (Δ11/Δ11 embryos) and also heterozygous for a p53 allele are able to survive, probably due to a reduction of the apoptotic barrier. These mice are susceptible to premature aging and also tumour formation [19,20]. In addition to these mouse models, in vitro studies have utilized cells that exclusively express BRCA1 Δ11.
Studies of mouse embryonic fibroblasts homozygous for the allele BRCA1 Δ11 (Δ11/Δ11) showed poor proliferation, suggesting a role of the BRCA1 Δ11 isoform in preventing tumour formation. However, these cells (BRCA1 Δ11/Δ11 fibroblasts) when immortalized proliferated faster than control immortalized fibroblasts . A consideration is that the Δ11 isoform is inefficient at binding the protein RAD51 . This can alter the capacity of Δ11/Δ11 cells to repair DNA double-strand breaks and cause an accumulation of defects in the cell cycle. This genetic instability and accumulation of defects may well explain the inconsistent behaviour of Δ11/Δ11 cells and mice that have been shown to potentially induce both apoptosis and a tendency to malignant transformation.
Besides studying the exclusive expression of the Δ11 isoform, Kim et al.  investigated mice lacking the Δ11 isoform. Female mice showed hyperplasia and spontaneous tumour in the gynaecological system, suggesting that the BRCA1 Δ11 isoform is involved in repressing tumour formation . However, the cDNA knockin approach employed in this study, to specifically block BRCA1 alternative splicing from exon 10 to exon 12, could have generated mutant mice lacking not only the Δ11 isoform, but also other BRCA1 isoforms. For instance, as suggested by Kim et al. , the possibility cannot be excluded that depletion of BRCA1 IRIS, rather than depletion of the Δ11 isoform, is responsible for the hyperplasia and spontaneous tumour observed. As both BRCA1 IRIS and the Δ11 isoform seem to be involved in tumour formation neither hypothesis appeals.
A further possibility is that loss of balance between each isoform (e.g. the selective expression of the remaining BRCA1 isoforms or a combination of this with depletion of the Δ11 and BRCA1 IRIS isoforms) is causative of the phenotypes observed. In addition, overexpression (using retroviral transduction) of the Δ11 isoform in mammary epithelial cells showed hyperplasia in mice injected with these cells .
The Δ11q isoform
The Δ11q isoform derives from the alternative choice of a donor site within human BRCA1 exon 11 (Figure 2). It should be noted that the Δ11q isoform has not been described in mouse. Importantly, the splice site used within human BRCA1 exon 11 to produce the Δ11q isoform is not predicted in the murine sequence.
In the Δ11q isoform, part of exon 11 (nucleotides 905–4215) is excluded. As a consequence of this exclusion, the isoform (like the Δ11 isoform) lacks the NLS (nuclear localization signal). Isoforms lacking the NLS can be transported in the nucleus via an alternative mechanism that requires Ubc9 . However, when Ubc9 is depleted, the Δ11q isoform becomes exclusively cytoplasmic and promotes growth and survival of breast cancer cells .
Although cytoplasmic Δ11q seems to play a role in tumour formation, the overexpression of BRCA1 Δ11q is able to inhibit growth of breast cancer cells  (Table 2). This ambiguous capacity of BRCA1 Δ11q to induce or repress cell proliferation in different contexts seems strictly related to its localization; overexpression may cause an increase in nuclear Δ11q that promotes apoptosis, whereas nuclear depletion (cytoplasmic retention) of Δ11q has the opposite effect.
To highlight further the different functional roles of BRCA1 isoforms, a study of human mammary epithelial cells has shown that the expression levels of many genes are altered in a BRCA1 isoform-specific manner . Specifically, overexpression of Δ11q induced activation of 22 genes, whereas overexpression of the full-length isoform (that included all BRCA1-coding exons) did not. Moreover, whereas overexpression of the full length also repressed several genes, transfection of Δ11q did not result in repression.
This BRCA1 isoform-specific gene regulation is intriguing and suggests a hypothesis that a balanced ratio of BRCA1 isoforms is required to maintain normal cell physiology.
BRCA1 also encodes a 1399 residue polypeptide, termed BRCA1 IRIS. It consists of an open reading frame from codon 1 in exon 2 up to the first 34 codons in intron 11 (Figure 3). It is not completely clear whether this recently described BRCA1 IRIS isoform is the product of alternative splicing at the intron 11 donor site or simply the result of an alternative promoter usage as described previously . A possible explanation is that the use of a different promoter may also affect the rate of transcription which in turn may affect splicing at the intron 11 donor site with the production of BRCA1 IRIS mRNA.
BRCA1 IRIS plays an important positive role in DNA replication . Unlike full-length BRCA1, BRCA1 IRIS does not interact with BARD1; it is exclusively chromatin-associated, present in G0 cells and overexpression promotes cell proliferation, breast cancer and cisplatin resistance in ovarian cancer cells [26–30]. The effect on cells of BRCA1 IRIS depletion or overexpression is shown in Table 3.
Knockdown experiments of BRCA1 isoforms targeting exon 12, have claimed that down-regulation of the full-length isoform can cause overexpression of BRCA1 IRIS because of mRNA stabilization . Whether this effect is related only to depletion of the full-length isoform or is also due to depletion of other isoforms that include exon 12 (e.g. Δ11 and Δ11q) needs to be determined.
The recent discovery of this oncogene-like BRCA1 IRIS isoform is of potential significance as it could represent a therapeutic target in breast cancer. In addition, discovery of BRCA1 IRIS should challenge the interpretation of various data. For instance, as suggested by ElShamy and Livingston , the embryonic lethality observed in BRCA Δ11/Δ11 mice was probably attributable to a loss of BRCA1 IRIS isoform rather than to the exclusive expression of the Δ11 isoform.
Moreover, mRNA levels of the full-length, Δ11 and Δ11q BRCA1 isoforms could have been misinterpreted considering that detection methods using oligonucleotides or probes upstream of intron 11 do not discriminate the IRIS isoform.
The fact that the physiological and pathogenic role of most BRCA1 isoforms tends to be opposite strongly suggests that cell fate can be switched in a particular direction that is dependent on changes in the overall splicing ratio rather than changes in a specific isoform. Understanding the significance of this ratio in cancer/apoptosis may be crucial in determining cancer predisposition and would also offer therapeutic options.
Currently, unclassified variants found to affect the ratio of natural BRCA1 splicing isoforms in patients with breast cancer are not classified as pathogenic mutations. Establishing that an unbalanced ratio can predispose an individual to cancer is fundamental in order to change the way that these variants are classified. Central to this, it will be critical a full understanding of the potential role of BRCA1 splicing.
D.B., M.R. and C.T. are supported by EURASNET, Cancer Research UK and the University of Southampton.
We thank Professor Diana Eccles.
RNA UK 2012: An Independent Meeting held at The Burnside Hotel, Bowness-on-Windermere, Cumbria, U.K., 20–22 January 2012. Organized and Edited by Raymond O'Keefe and Mark Ashe (Manchester, U.K.).
Abbreviations: BRCA1, breast cancer early-onset 1; BARD1, BRCA1-associated RING domain protein; BRCT, BRCA1 C-terminus; NLS, nuclear localization signal
- © The Authors Journal compilation © 2012 Biochemical Society