Alternative splicing in Ras transformed cells

背景通过RNAi合成致死筛选，我们已经确定了一些对RAS突变型癌细胞的生存所需的关键的mRNA剪接因子。在这个项目中，我们旨在解决以下问题：1)RNA剪接因子亚集的缺失导致RAS突变的合成致死性的机制；2)在RAS突变细胞中哪些细胞蛋白被差异剪接；3)与RAS野生型细胞相比，RAS突变细胞中mRNA剪接模式的变化如何影响其生存能力。2)用于定量mRNA剪接模式变化的RNA-SEQ协议。2012财年ACCOMPLISHMENT我们已经验证了针对一些mRNA剪接因子的shRNA，这些剪接因子被证实是KRAS合成致死的。我们已经建立了稳定表达针对这些因素的救援性cDNA的细胞系。我们已经进行了RNA-SEQ实验，以表征KRAS野生型和突变细胞中有或没有剪接因子敲除的mRNA剪接模式。我们已经确定了候选基因，其适当的剪接和表达对KRAS突变细胞的生存至关重要，目前我们正在研究这种依赖的机制。

BACKGROUNDThrough a RNAi synthetic lethal screen we have identified a number of key mRNA splicing factors to be required for the viability of Ras mutant cancer cells.PURPOSEIn this project we aim to address the following questions: 1) the mechanism by which depletion of a subset of mRNA splicing factors causes synthetic lethality with Ras mutation; 2) which cellular proteins are differentially spliced in Ras mutant cells; 3) how the changes in mRNA splicing pattern in Ras mutant cells affect their viability compared to Ras wild type cells.SIGNIFICANT MATERIALS AND METHODS1) shRNAs that target splicing factors. 2) RNA-seq protocol for quantifying mRNA splicing pattern changes.FY2012 ACCOMPLISHMENTWe have validated shRNAs that target a number mRNA splicing factors that are identified to be synthetically lethal with KRAS. We have generated cell lines stably expressing rescue cDNAs to these factors. We have carried out RNA-seq experiments to characterize mRNA splicing patters in KRAS wild type and mutant cells with or without splicing factor knockdown. We have identified candidate genes whose proper splicing and expression are critical for the viability of KRAS mutant cells and we are currently investigate the mechanisms underlying such dependency.