- Letter to the Editor
- Open Access
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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- Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–54.PubMedView ArticleGoogle Scholar
- Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Brocker EB, LeBoit PE, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353:2135–47.PubMedView ArticleGoogle Scholar
- McArthur GA, Chapman PB, Robert C, Larkin J, Haanen JB, Dummer R, Ribas A, Hogg D, Hamid O, Ascierto PA, et al. Safety and efficacy of vemurafenib in BRAF(V600E) and BRAF(V600K) mutation-positive melanoma (BRIM-3): extended follow-up of a phase 3, randomised, open-label study. Lancet Oncol. 2014;15:323–32.PubMedPubMed CentralView ArticleGoogle Scholar
- Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507–16.PubMedPubMed CentralView ArticleGoogle Scholar
- Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O’Dwyer PJ, Lee RJ, Grippo JF, Nolop K, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363:809–19.PubMedPubMed CentralView ArticleGoogle Scholar
- Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA, Emery CM, Stransky N, Cogdill AP, Barretina J, et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature. 2010;468:968–72.PubMedPubMed CentralView ArticleGoogle Scholar
- Montagut C, Sharma SV, Shioda T, McDermott U, Ulman M, Ulkus LE, Dias-Santagata D, Stubbs H, Lee DY, Singh A, et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 2008;68:4853–61.PubMedPubMed CentralView ArticleGoogle Scholar
- Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, Wubbenhorst B, Xu X, Gimotty PA, Kee D, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell. 2010;18:683–95.PubMedPubMed CentralView ArticleGoogle Scholar
- Sun C, Wang L, Huang S, Heynen GJ, Prahallad A, Robert C, Haanen J, Blank C, Wesseling J, Willems SM, et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature. 2014;508:118–22.PubMedView ArticleGoogle Scholar
- Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G, Shi H, Atefi M, Titz B, Gabay MT, et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature. 2011;480:387–90.PubMedPubMed CentralView ArticleGoogle Scholar
- Salton M, Kasprzak WK, Voss T, Shapiro BA, Poulikakos PI, Misteli T. Inhibition of vemurafenib-resistant melanoma by interference with pre-mRNA splicing. Nat Commun. 2015;6:7103.PubMedPubMed CentralView ArticleGoogle Scholar
- Graveley BR. Sorting out the complexity of SR protein functions. RNA. 2000;6:1197–211.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng ZM. Regulation of alternative RNA splicing by exon definition and exon sequences in viral and Mammalian gene expression. J Biomed Sci. 2004;11:278–94.PubMedPubMed CentralView ArticleGoogle Scholar
- Fu XD, Ares M Jr. Context-dependent control of alternative splicing by RNA-binding proteins. Nat Rev Genet. 2014;15:689–701.PubMedPubMed CentralView ArticleGoogle Scholar
- Berget SM. Exon recognition in vertebrate splicing. J Biol Chem. 1995;270:2411–4.PubMedView ArticleGoogle Scholar
- Sterner DA, Carlo T, Berget SM. Architectural limits on split genes. Proc Natl Acad Sci USA. 1996;93:15081–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Hamm J, Mattaj IW. Monomethylated cap structures facilitate RNA export from the nucleus. Cell. 1990;63:109–18.PubMedView ArticleGoogle Scholar
- Sun H, Chasin LA. Multiple splicing defects in an intronic false exon. Mol Cell Biol. 2000;20:6414–25.PubMedPubMed CentralView ArticleGoogle Scholar
- Dou Y, Fox-Walsh KL, Baldi PF, Hertel KJ. Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site. RNA. 2006;12:2047–56.PubMedPubMed CentralView ArticleGoogle Scholar
- Kondo Y, Oubridge C, van Roon AM, Nagai K. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. Elife. 2015;4:e04986.View ArticleGoogle Scholar
- Rossi F, Forne T, Antoine E, Tazi J, Brunel C, Cathala G. Involvement of U1 small nuclear ribonucleoproteins (snRNP) in 5′ splice site-U1 snRNP interaction. J Biol Chem. 1996;271:23985–91.PubMedView ArticleGoogle Scholar
- Malca H, Shomron N, Ast G. The U1 snRNP base pairs with the 5′ splice site within a penta-snRNP complex. Mol Cell Biol. 2003;23:3442–55.PubMedPubMed CentralView ArticleGoogle Scholar
- Green MR. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708.PubMedView ArticleGoogle Scholar
- Zhuang YA, Goldstein AM, Weiner AM. UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc Natl Acad Sci USA. 1989;86:2752–6.PubMedPubMed CentralView ArticleGoogle Scholar
- Gao K, Masuda A, Matsuura T, Ohno K. Human branch point consensus sequence is yUnAy. Nucleic Acids Res. 2008;36:2257–67.PubMedPubMed CentralView ArticleGoogle Scholar
- Corvelo A, Hallegger M, Smith CW, Eyras E. Genome-wide association between branch point properties and alternative splicing. PLoS Comput Biol. 2010;6:e1001016.PubMedPubMed CentralView ArticleGoogle Scholar
- Mercer TR, Clark MB, Andersen SB, Brunck ME, Haerty W, Crawford J, Taft RJ, Nielsen LK, Dinger ME, Mattick JS. Genome-wide discovery of human splicing branchpoints. Genome Res. 2015;25:290–303.PubMedPubMed CentralView ArticleGoogle Scholar
- Desmet FO, Hamroun D, Lalande M, Collod-Beroud G, Claustres M, Beroud C. Human splicing finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng ZM, Tao M, Yamanegi K, Bodaghi S, Xiao W. Splicing of a Cap-proximal human papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5′ Cap. J Mol Biol. 2004;337:1091–108.PubMedView ArticleGoogle Scholar
- Zheng ZM, He P, Baker CC. Selection of the bovine papillomavirus type 1 nucleotide 3225 3′ splice site is regulated through an exonic splicing enhancer and its juxtaposed exonic splicing suppressor. J Virol. 1996;70:4691–9.PubMedPubMed CentralGoogle Scholar
- Zheng ZM, Reid ES, Baker CC. Utilization of the bovine papillomavirus type 1 late-stage-specific nucleotide 3605 3′ splice site is modulated by a novel exonic bipartite regulator but not by an intronic purine-rich element. J Virol. 2000;74:10612–22.PubMedPubMed CentralView ArticleGoogle Scholar
- Vogel J, Hess WR, Borner T. Precise branch point mapping and quantification of splicing intermediates. Nucleic Acids Res. 1997;25:2030–1.PubMedPubMed CentralView ArticleGoogle Scholar
- Ajiro M, Zheng ZM. E6^E7, a novel splice isoform protein of human papillomavirus 16, stabilizes viral E6 and E7 oncoproteins via HSP90 and GRP78. MBio. 2015;6:e02068.PubMedPubMed CentralView ArticleGoogle Scholar
- Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: a web resource to identify exonic splicing enhancers. Nucleic Acids Res. 2003;31:3568–71.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng ZM, Quintero J, Reid ES, Gocke C, Baker CC. Optimization of a weak 3′ splice site counteracts the function of a bovine papillomavirus type 1 exonic splicing suppressor in vitro and in vivo. J Virol. 2000;74:5902–10.PubMedPubMed CentralView ArticleGoogle Scholar
- Ajiro M, Jia R, Zhang L, Liu X, Zheng ZM. Intron definition and a branch site adenosine at nt 385 control RNA splicing of HPV16 E6*I and E7 expression. PLoS One. 2012;7:e46412.PubMedPubMed CentralView ArticleGoogle Scholar
- Majerciak V, Yamanegi K, Zheng ZM. Gene structure and expression of Kaposi’s sarcoma-associated herpesvirus ORF56, ORF57, ORF58, and ORF59. J Virol. 2006;80:11968–81.PubMedPubMed CentralView ArticleGoogle Scholar
- Taggart AJ, DeSimone AM, Shih JS, Filloux ME, Fairbrother WG. Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo. Nat Struct Mol Biol. 2012;19:719–21.PubMedPubMed CentralView ArticleGoogle Scholar
- Tran Q, Roesser JR. SRp55 is a regulator of calcitonin/CGRP alternative RNA splicing. Biochemistry. 2003;42:951–7.PubMedView ArticleGoogle Scholar
- Mercado PA, Ayala YM, Romano M, Buratti E, Baralle FE. Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene. Nucleic Acids Res. 2005;33:6000–10.PubMedPubMed CentralView ArticleGoogle Scholar
- Gozani O, Potashkin J, Reed R. A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol Cell Biol. 1998;18:4752–60.PubMedPubMed CentralView ArticleGoogle Scholar
- Lardelli RM, Thompson JX, Yates JR III, Stevens SW. Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing. RNA. 2010;16:516–28.PubMedPubMed CentralView ArticleGoogle Scholar
- Kfir N, Lev-Maor G, Glaich O, Alajem A, Datta A, Sze SK, Meshorer E, Ast G. SF3B1 association with chromatin determines splicing outcomes. Cell Rep. 2015;11:618–29.PubMedView ArticleGoogle Scholar
- Brosi R, Hauri HP, Kramer A. Separation of splicing factor SF3 into two components and purification of SF3a activity. J Biol Chem. 1993;268:17640–6.PubMedGoogle Scholar
- Corrionero A, Minana B, Valcarcel J. Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A. Genes Dev. 2011;25:445–59.PubMedPubMed CentralView ArticleGoogle Scholar
- Kramer A. Purification of splicing factor SF1, a heat-stable protein that functions in the assembly of a presplicing complex. Mol Cell Biol. 1992;12:4545–52.PubMedPubMed CentralView ArticleGoogle Scholar
- Berglund JA, Abovich N, Rosbash M. A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev. 1998;12:858–67.PubMedPubMed CentralView ArticleGoogle Scholar
- Crisci A, Raleff F, Bagdiul I, Raabe M, Urlaub H, Rain JC, Kramer A. Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins. Nucleic Acids Res. 2015. doi:10.1093/nar/gkv952.PubMedPubMed CentralGoogle Scholar
- Reed R, Maniatis T. The role of the mammalian branchpoint sequence in pre-mRNA splicing. Genes Dev. 1988;2:1268–76.PubMedView ArticleGoogle Scholar
- Ibrahim EC, Schaal TD, Hertel KJ, Reed R, Maniatis T. Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers. Proc Natl Acad Sci USA. 2005;102:5002–7.PubMedPubMed CentralView ArticleGoogle Scholar
- Zheng ZM, Huynen M, Baker CC. A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly. Proc Natl Acad Sci USA. 1998;95:14088–93.PubMedPubMed CentralView ArticleGoogle Scholar
- Noble JC, Pan ZQ, Prives C, Manley JL. Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites. Cell. 1987;50:227–36.PubMedView ArticleGoogle Scholar
- Zheng ZM, Baker CC. Parameters that affect in vitro splicing of bovine papillomavirus type 1 late pre-mRNAs. J Virol Methods. 2000;85:203–14.PubMedView ArticleGoogle Scholar