Novel exon was identified between canine BRCA2 exon 1 and exon 2
We identified a novel exon between canine BRCA2 exon 1 and exon 2 via RT-PCR using samples from two ovaries and one testis (Fig. 1 A). These PCR products were sequenced to determine their splicing patterns (Accession No. LC547963, LC547964, LC547965, LC547966, LC547967 and LC547968). We identified six splicing variants in addition to the registered sequence (Fig. 1 B). These variants were identified between originally reported exon 1 and exon 2 in 5′ UTR of canine BRCA2 gene and complied with the GU-AG rule. These splice variants resulted in mRNA transcripts that varied in size from 248 bp to 988 bp. We referred to the splicing variants as I-VI, I being the variant that encodes the longest transcript to VI being the shortest. Novel splicing variant I contained 8 start codons, which potentially translate short and truncated proteins, and splicing variant VI, the shortest variant, contained 5 start codons, whereas the registered sequence does not have a start codon in 5′ UTR. It is not easy to determine the precise expression level by RT-PCR, but splicing variants I, II, V, VI and registered sequence were main transcripts (Fig. 1 A). We also tested the expression of these splicing variants in cultured cells derived from dogs. Three out of four cell lines showed the presence of these spliced variants similar to testis and ovary tissues (Fig. 1 C) and the splicing variants I, II, V, VI and registered sequence showed higher mRNA levels.
Expression pattern and levels of splicing variants were affected by serum starvation
Expression level of BRCA2 was regulated by cell cycle and the condition of the cells. Thus, we tested the effect of serum starvation, which introduced G1 phase arrest, on cultured cell lines. The expression pattern of splicing variants in CHM-p cell lines changed due to serum starvation. However, MDCK, CIP-p and CNM-p showed maintained the same expression pattern even after serum starvation (Fig. 2 A). CHM-p cells expressed only splicing variants I and III after serum starvation (Fig. 2 A). In all the four cell lines mentioned above, the expression level of BRCA2 was reduced by serum starvation (Fig. 2 B). The relative ratio of expression levels of BRCA2 with the shortest registered sequence to total BRCA2 was also changed (Fig. 2 C). MDCK and CIP-p cells showed an increased relative expression ratio of registered sequence, but it was decreased in case of CNM-p and CHM-p (Fig. 2 C).
Human cultured cell lines also expressed similar splicing variant
Canine tissues and cultured cells expressed splicing variants in canine BRCA2 5′ UTR, and expression ratio of splicing variants was regulated by the condition of the cells. We hypothesized that regulation of BRCA2 gene expression via splicing variants could also be present in humans. We tested this using two human derived cell lines, HeLa and 293T. Splicing variants including whole intron 1 sequence was expressed in these cell lines (Fig. 3 A and B, Accession No. LC547969). The novel splicing variant contained 8 start codons, which potentially translate short and truncated proteins, whereas the registered sequence did not have a start codon in 5′ UTR. Although expression pattern of splicing variants was not changed by serum starvation, the relative ratio of registered sequence and shorter splicing variant, was reduced or increased with a slight increase in BRCA2 expression level in HeLa or 293T cells (Fig. 3 C). We also tested the effect of X-ray irradiation as some genes have been reported to change the ratio of splicing variants to regulate its expression level in response to DNA damage [23,24]. However, relative ratio of the registered sequence did not show any drastic change until 60 min after 10 Gy X-ray treatment (Fig. 3 E).
Novel splicing variants of canine and human BRCA2 suppressed translational efficiency
The novel splicing variants of BRCA2 were expressed in canine tissues and both canine and human derived cell lines and these ratios were changed by the conditions in which the cells were maintained. We speculated that these splicing variants regulate the translational efficiency of BRCA2. To evaluate the translational efficiency of BRCA2, luciferase assay with quantitative PCR were employed (Fig. 4 A). To evaluate the translational efficiency, we designed plasmids containing CMV promoter followed by each 5′ UTR and luciferase. These constructs might also contribute to the suppression of the transcriptional efficiency. Thus, to avoid the effect on transcriptional efficiency, we quantified the expression level of firefly luciferase and the transfection efficiency of the plasmid DNA containing firefly luciferase gene and genomic DNA of human BRCA2 exon 27 was determined via quantitative PCR. In canine BRCA2, the splicing variants, I, II, V, VI, and registered sequence were major variants in tissues and cell lines. Thus, we evaluated these five variants. Figure 4 B shows the relative expression level of firefly luciferase expression levels. Unexpectedly, splicing variant I and II of canine BRCA2 and splicing variant I of human BRCA2 suppressed the CMV promoter activity (Fig. 4 B). In parallel, luciferase assay was performed to evaluate the translational activity. The values of the transcriptional activity were used to normalize the relative translational activity. The splicing variants of canine BRCA2 excluding splicing variant I and splicing variant I of human BRCA2 were found to be associated with decreased translational activity.
Intron 1 of canine and human BRCA2 also suppressed BRCA2 promoter activity
Novel splicing variants of canine and human BRCA2 suppressed CMV promoter activity in addition to the translational suppression. Thus, we next tested the effect of the DNA sequence of various splicing variants on BRCA2 promoter activity. Prior to this, canine and human BRCA2 intron 1 region were compared to find conserved DNA sequences, because novel exon region identified in intron 1 region were different from registered sequence and conserved sequence was the most potential silencer element (Fig 5). Three conserved regions were identified in canine and human BRCA2. The 5′-terninal 15 bp region was located on BRCA2 promoter region. The other two regions, human BRCA2 +460-+617 bp and +732-+942 bp, within canine BRCA2 intron 1 were also found to be conserved. Thus, we expected these two conserved DNA sequences to have a silencer element. Because canine BRCA2 splicing variant II, which did not have 3′-terminal 95 bp of intron 1, still suppressed the effect of CMV promoter activity, thus around the sequence +460-+841 bp in human BRCA2 corresponding +413-+787 bp in canine BRCA2 should also be associated with silencer elements. To avoid the translational effect, the human BRCA2 intron 1 region without promoter sequence (-187-+310 bp) was inserted upstream of human BRCA2 promoter (Fig 6 A). As expected, +443-843 bp region in human BRCA2 decreased the promoter activity similar to human BRCA2 intron 1 region (+311-942 bp). Next, we attempted to identify where the silencer element was located within the 500 bp sequence. Sequential deletion mutants in 5′ and 3′ ends were compared with the promoter activity (Fig 6 B and C). The region +743-+842 bp contained cis-regulatory silencer element (Fig 6 C). The region of +443-+542 bp also contained a silencer, as the presence of this region showed significantly reduced promoter activity than that of +543-+842 bp region (Fig 6 C) and +443-+542 bp region tended to reduce the promoter activity but it was not significant (Fig 6 B; p=0.0295, by holm’s method if p value was less than 0.0125, it would be significant.). Unexpectedly, the region around +643-+742 bp harbored enhancer element, and +543-+642 bp region contained silencer element from Figure 6 C, but not Figure 6 B. Thus, we speculated that cis-regulatory elements could be separated in some constructs. To study this region in-detail, three distinct constructs were tested (Fig 6 D). As we expected, +593-+692 bp region contained silencer element. However, surprisingly, +543-+642 bp region acted as enhancer element. To identify the enhancer elements around +742 bp in intron 1, we also analyzed +693-+792 bp region (Fig 6 E). This region showed slightly decreased BRCA2 promoter activity. Thus, we concluded that the silencer and enhancer element were close to be separated, and the effect of silencer element in this region was stronger than enhancer element in this region.