Alternative splicing of BRAF transcripts and characterization of C-terminally truncated B-Raf isoforms in colorectal cancer
Alternative splicing of BRAF transcripts and characterization of C-terminally truncated B-Raf isoforms in colorectal cancer
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DOI：
10.1002/ijc.28061
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发表时间：
2013-08-01
影响因子：
6.4
通讯作者：
Kolligs, Frank T.
中科院分区：
文献类型：
--
作者：
Hirschi, Benjamin;Kolligs, Frank T.
BRAF原癌基因在人类肿瘤的一个亚群中发生突变，包括结直肠癌。最近发现了一种缺乏外显子14和15的剪接变体（BRAF del E14/15）。然而，变异的频率、蛋白异构体的激酶活性、它的生物学功能以及它来自哪个等位基因仍然未知。来自结直肠癌细胞系和结肠上皮的BRAF mRNA被逆转录、亚克隆并筛选选择性剪接。通过DNA测序分析了互剪接转录物的新转录物变异和等位基因来源。转染不同BRAF变体的表达构建体后，通过Western blotting检测B-Raf亚型的激酶活性。还发现了另外四个BRAF转录物变异，导致基因产物的c端截断。在正常和肿瘤结肠细胞中，选择性剪接的频率为4.7 - 16.7%。结果表明，野生型和V600E等位基因均可产生替代转录本。所有不一致的B-Raf蛋白变体都被发现是激酶死亡的，不能共激活全长B-Raf。总之，我们提出了一种检测异常剪接转录本的高灵敏度方法。外显子14,15,15b， 16b和16c的选择性剪接发生在正常结肠癌和结直肠癌细胞的相当一部分BRAF mRNA中，并且与亲本等位基因的V600E突变状态无关。非功能性转录物的剪接影响细胞B-Raf的整体活性，可能是降低对生长信号敏感性的一种机制。
The BRAF proto-oncogene is mutated in a subset of human tumors, including colorectal cancer. A splicing variant lacking exons 14 and 15 (BRAF del E14/15) has been described recently. However, the frequency of the variant, the kinase activity of the protein isoform, its biological function, and which allele it is derived from remains unknown. BRAF mRNA from colorectal cancer cell lines and colonic epithelium was reversely transcribed, subcloned, and screened for alternative splicing. New transcript variants and allelic origin of alternatively spliced transcripts were analyzed by DNA sequencing. Kinase activity of the B-Raf isoforms was determined by Western blotting after transfections with expression constructs of the different BRAF variants. Four additional BRAF transcript variants resulting in C-terminal truncation of the gene product were found. Alternative splicing was found at frequencies from 4.7 to 16.7% in normal and neoplastic colorectal cells. Alternative transcripts were shown to be derived from both wild-type and V600E alleles. All nonconsensus B-Raf protein variants were found to be kinase-dead and failed to coactivate full-length B-Raf. In conclusion, we present a highly sensitive method for the detection of aberrantly spliced transcripts. Alternative splicing of exons 14, 15, 15b, 16b and 16c occurs in a considerable fraction of BRAF mRNA in normal colon and colorectal cancer cells and is independent of the V600E mutational status of the parental allele. Splicing of nonfunctional transcripts affects overall cellular B-Raf activity and might represent a mechanism to decrease sensitivity to growth signals.