Vemurafenib-resistant BRAF selects alternative branch points different from its wild-type BRAF in intron 8 for RNA splicing
© Ajiro and Zheng. 2015
Received: 4 November 2015
Accepted: 14 December 2015
Published: 21 December 2015
One mechanism of resistance of the melanoma-associated BRAF kinase to its small molecule inhibitor vemurafenib is by point mutations in its intron 8 resulting in exons 4–8 skipping. In this report, we carried out in vitro BRAF RNA splicing assays and lariat RT-PCR to map the intron 8 branch points in wild-type and BRAF mutants. We identify multiple branch points (BP) in intron 8 of both wild-type (wt) and vemurafenib-resistant BRAF RNA. In wt BRAF, BPs are located at -29A, -28A and -26A, whereas in a vemurafenib-resistant BRAF splicing mutant, BPs map to -22A, -18A and -15A, proximal to the intron 8 3′ splice site. This finding of a distal-to-proximal shift of the branch point sequence in BRAF splicing in response to point-mutations in intron 8 provides insight into the regulation of BRAF alternative splicing upon vemurafenib resistance.
BRAF proto-oncogene encodes a serine/threonine kinase regulator of the MAP kinase pathway, and activating BRAF mutations are found in 40–60 % of melanoma, with 90 % of them containing the V600E mutation [1, 2]. Vemurafenib, a potent inhibitor of (V600E) BRAF in melanoma cells, is currently in clinical use [3–5]. However, patients treated with vemurafenib develop resistance by activation of alternative signaling pathways [6–9] or by inducing alternative splicing of BRAF to exclude the RAS-binding domain encoded by exons 3–5 . The vemurafenib-resistant melanoma cell line C3 SKMEL-239 produces BRAF exon 3^9 splicing and contains two intronic point-mutations at positions -435 (C-to-A) and -51 (C-to-G) from the BRAF intron 8 3′ splice site. In a minigene system the -51 mutation, located in the computationally predicted branch point (BP), was found to be sufficient to recapitulate BRAF exon 3^9 splicing [10, 11].
Each intron of eukaryotic primary RNA transcripts (pre-mRNAs) has a 5′ splice site with a GU dinucleotide and a 3′ splice site with an AG dinucleotide. The 3′ splice site also contains a BP in a 7-nt or 5-nt branch point sequence (BPS) and a run of 15–40 pyrimidines (usually Us), called polypyrimidine tract (PPT), between the BPS and the 3′ end AG dinucleotide. Defining the exon–intron boundary in pre-mRNA splicing is the first step in the accurate recognition of an intron 5′ splice site by U1 snRNA, of BPS by U2 snRNA, and of a 3′ splice site by U2AF (U2 auxiliary factors) modulated by many cellular splicing factors [12–14]. These recognition steps are followed by two transesterification reactions during spliceosome assembly. In this two-step biochemical reaction, an OH group of the BP adenosine within the BPS performs a nucleophilic attack on a phosphodiester bond of the intron-5′ exon junction, resulting in the first step in the 5′ exon being cleaved off and forming a lariat intermediate by a branching reaction of the intron 5′ end G to the BP adenosine via a 5′-to-2′ phosphodiester link. The second step is to cleave the intron from the lariat intermediate by another nucleophilic attack of the OH group from the cleaved 5′ exon on a phosphodiester bond of the intron-3′ exon junction and join the cleaved 5′ exon to the cleaved 3′ exon. Thus, if an intron 5′ and 3′ splice sites are of consensus sequence, they sequentially bind three different splicing factors in order to assemble the spliceosome. However, the splice sites in higher eukaryotes are usually not well conserved and binding of splicing factors to pre-mRNAs with non-consensus sequence is often inefficient. In addition, pre-mRNA splicing is subject to regulation by other intronic or exonic cis-elements, intronic splicing enhancers (ISE) or silencers (ISS) and exonic splicing enhancer (ESE) or silencer (ESS), often located at a distance. The combination of the strength of the various cis-regulatory elements and the local availability of splicing factors determines alternative splicing outcome [13, 14].
In this report, we experimentally mapped the BPS in BRAF intron 8 that controls the constitutive RNA splicing of wild-type (wt) BRAF exon 8^9 and discovered an alternative BPS in the intron 8 of a vemurafenb-resistant mutant (mt) BRAF pre-mRNA.
Result and discussion
BRAF intron 3 and intron 8 are suboptimal
In general, a consensus 3′ splice site is composed of three critical elements: BPS, PPT (usually with a stretch of U residues), and an AG dinucleotide at the 3′ end of the intron. Mammalian consensus BPSs are YNYURAC [23, 24] or YUNAN, [25–27] with 90 % of BPSs occurring within 19–37 (median 25) nucleotides upstream of the 3′ AG dinucleotides and 78 % of the BP nucleotides within a BPS being an adenosine . Analysis of the intron 3 and intron 8 3′ splice sites using Human Splice Finder (http://www.umd.be/HSF/)  revealed that both introns bear a non-consensus 7-nt BPS within the distance range in intron 3, but further upstream (46 nts) in intron 8. The intron 8 3′ splice site is also predicted to have multiple non-consensus 5-nt BPSs within the distance range to its 3′ AG dinucleotide (Fig. 1b). Moreover, both introns have a weak PPT interspersed by purines with runs of uridines no longer than three. Altogether, the weak nature of these 3′ splice sites would subject them to regulation by RNA cis-elements or trans-acting factors.
Reconstitution of wt exon 8^9 and mt exon 3^9 splicing of BRAF in vitro
Identification of distinct sets of alternative BPs for wt and mt BRAF splicing by lariat RT-PCR
In summary, our data demonstrate that wt and mt BRAF RNA select a distinct set of alternative BPs in the intron 8 for splicing, with the wt BRAF using distal BPs (-29A, -28A and -26A) to the intron 3′ splice site for the exon 8^9 splicing and the mt BRAF using proximal BPs (-22A, -18A and -15A) to the intron 3′ splice site for the exon 3^9 splicing (Fig. 4e).
Flexibility or redundancy in BP selection has a role in alternative splicing and was described in both viral [33, 35–37] and human gene expression [27, 38]. Recent genome-wide BP mapping studies indicate that a large proportion of introns have more than one BP, generally clustered in close proximity in relation to the 3′ splice site [27, 38], although a BP could be found in rare case further upstream of a 3′ splice site [27, 38]. Since the predicted -51C  from the 3′ splice site of intron 8 identified by ESEfinder  or by Human Splice Finder  was not mapped as an authentic BP in this study, our data imply that the observed mutations (-435 C-to-A and -51 C-to-G) in the mt BRAF pre-mRNA might disrupt the binding of trans-acting factors, such as SRSF6 (SRp55) [11, 39, 40] and SF3b/3a [41–45], to the -51 region and thereby prevent the recruitment of SF1 and U2 snRNA [46–48] to select an authentic distal BP for splicing of BRAF RNA. Consequently, loss of splicing factor binding to the -51 region and activation of a proximal BP usage might lead to skipping of exons 4–8 in splicing of mt BRAF. The minigene system in this report constructed in a classical way [49–52] has some advantage over the minigene in other study . The latter had an extremely large (>1 kb) middle exon (an exon 4/8 fusion exon inserted with a strawberry reporter) and a BRAF exon 9 as a terminal exon fused with a GFP reporter . An oversized internal exon larger than 500 nts has been shown to affect exon definition and thereby RNA splicing . In summary, our observation provides further insight into the molecular mechanisms toward understanding the regulation of alternative splicing of BRAF upon vemurafenib resistance in melanoma.
RT-PCR, in vitro splicing assay and lariat RT-PCR
RT-PCR is performed as described  for wt SKMEL-239 cells and C3 SKMEL-239 melanoma cells. Two primer sets were used separately with the primer pair of 3F and 9R for detection of both the constitutive and alternative BRAF RNA splicing and 8F and 9R only for the constitutive exon 8^9 splicing (Additional file 1: Table S1; Fig. 2a). GAPDH RNA was detected with a primer pair described  as a loading control.
BRAF pre-mRNAs were prepared by in vitro transcription with T7 RNA polymerase from two-exon, one-intron DNA templates prepared by overlapping PCR [31, 36]. The wt BRAF template has a truncated intron 8 originally from SKMEL-239 cells and the mt BRAF template from C3 SKMEL-239 cells has a chimeric intron 3 and intron 8 of which the intron 3 5′ splice site (64 nts) was fused with the intron 8 3′ splice site (440 nts) including the point mutations in the intron (Fig. 3a). See primer details for template preparation in Additional file 1: Table S1.
In vitro splicing assay was performed as described [29, 36, 53]. Briefly, 4 ng of 32P-labeled pre-mRNAs were incubated with HeLa cell nuclear extract at 30 °C for a 2 h in vitro splicing reaction and followed by extraction of splicing products. The splicing products were resolved by electrophoresis on a 6 % denaturing PAGE gel. Autoradiograph was captured by PhosphorImager Storm 860 (GE Healthcare Life Sciences, Pittsburgh, PA).
For lariat RT-PCR [31–33], in vitro splicing products from 100 ng of cold pre-mRNAs were reverse transcribed by Superscript II (Life technologies, Thermo Fisher Scientific) using a primer R and amplified by PCR with a primer pair of R and F1 first followed by a nested primer pair of R and F2 (Fig. 4a; Additional file 1: Table S1). The lariat RT-PCR products were subcloned into the pCR2.1 TOPO vector (Life Technologies) and sequenced.
MA participated in the design of the study, performed all experiments and analysis of the data. ZMZ designed the study and analyzed the data. MA and ZMZ drafted. Both authors read and approved the final manuscript.
We thank Tom Misteli and Maayan Salton for providing us the cellular materials of wt SKMEL-239 cells and C3 SKMEL-239 melanoma cell lines and critical reading of the manuscript.
Both authors declare that they have no competing interests.
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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