Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada.
PLoS One. 2013;8(1):e53469. doi: 10.1371/journal.pone.0053469. Epub 2013 Jan 3.
Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.
在癌症患者的恶性细胞中已经鉴定出透明质酸合酶 1 (HAS1) 的异常前体 mRNA 剪接变体。生物信息学分析表明，内含子序列的变化可能是异常剪接的基础。在 HAS1 迷你基因构建体中引入缺失和突变，以确定可影响异常内含子剪接的区域，比较转染子与多发性骨髓瘤 (MM) 患者的剪接模式。如这里所示，HAS1 内含子 3 和 4 中的引入遗传变异可以促进在 MM 患者的恶性细胞中检测到的异常剪接。HAS1Vd 是这里首次鉴定的新型内含子剪接变体。HAS1Vb 是以前在患者中鉴定的内含子剪接变体，跳过外显子 4 并利用与 HAS1Vd 相同的内含子 4 替代 3'剪接位点。对于未改变内含子 3 和 4 的转染构建体，HAS1Vd 转录物很容易检测到，通常排除 HAS1Vb。相比之下，在 MM 患者中，HAS1Vb 比 HAS1Vd 更常见。在 HAS1 迷你基因中，内含子 4 的缺失与内含子 3 的突变相结合导致从 HAS1Vd 表达转变为 HAS1Vb 表达。异常剪接的上调，这里以 HAS1Vb 的表达为例，表明受内含子序列中多个遗传变化的影响。对于 HAS1Vb，这包括增强的外显子 4 跳跃和增加使用替代 3'剪接位点。因此，引入的 HAS1 内含子 3 突变与引入的 HAS1 内含子 4 缺失的组合促进了先前显示具有临床意义的异常剪接模式的转变。大多数 MM 患者在内含子 4 中存在遗传变异，并且如这里所示，近一半患者在 HAS1 内含子 3 中存在反复突变。我们的工作表明，MM 患者中异常内含子 HAS1 剪接可能依赖于 MM 患者中频繁出现但在健康供体中不存在的内含子 HAS1 缺失和突变。
