Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0061-7) contains supplementary material, which is available to authorized users.
Highlights
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]
In this two-step biochemical reaction, an OH group of the branch point (BP) adenosine within the branch point sequence (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
To understand what might contribute to the observed alternative exon 3^9 splicing of vemurafenibresistant BRAF RNA, we analyzed sequence structures of the 5′ and 3′ splice sites in BRAF introns 3 and 8 and the 5′ splice site of BRAF intron 9 considering that exon definition may play an important role in defining an upstream 3′ splice site [13, 15, 16]
Summary
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]. The vemurafenib-resistant melanoma cell line C3 SKMEL239 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. 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 [12,13,14]. These recognition steps are followed by two transesterification reactions during spliceosome assembly. 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
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