ABSTRACT

RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5^V569E and MAP4K5^V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.

摘要

来自编辑和可变剪接的 RNA 变体形成了蛋白质信号传导的重要调控阶段。在本报告中，我们应用了一个集成的 DNA 和 RNA 变异检测工作台来定义在人类黑色素瘤细胞模型中偏离参考基因组的 RNA 变异的范围。RNA 变体可分为 (i) 经典 ADAR 样或 APOBEC 样 RNA 编辑事件和 (ii) 多核苷酸变体 (MNV)，包括三个和六个碱基对框内非规范未映射外显子。我们专注于验证这些类别的代表性基因。首先，通过 Sanger 测序验证CDK13基因中的聚集非同义 RNA 编辑 (AI)，以确认 RNA 变异检测工作台的完整性。其次， MAP4K5中高度保守的 RNA 变体检测到的基因很可能是由非规范的三碱基外显子剪接引起的。从MAP4K5基因座产生的两种 RNA 变体偏离基因组参考序列并产生 V569E 或 V569del 同种型变体。低剂量的剪接抑制剂表明 MAP4K5-V569E 变体来自 SF3B1 依赖性剪接事件。重组 SBP 标记的 MAP4K5 ^V569E和 MAP4K5 ^{V569del的质谱分析}转染细胞系统中的蛋白质下拉用于识别这两种 MAP4K5 同种型的蛋白质-蛋白质相互作用并提出可能的功能。这些数据共同突出了这种集成的 DNA 和 RNA 变异检测平台在检测癌细胞中的 RNA 变异并支持未来对癌症组织中 RNA 变异检测的分析的实用性。