DEK binds to pre-mRNAs and regulated alternative splicing of Hippo signaling genes in HeLa cell [RIP-seq]

Purpose: Explore how DEK genome widely bind RNA and affect the alternative splicing in cancer Methods: DEK was overexpressed by transfecting HeLa cells with a DEK-overexpressing plasmid and RNA_seq was used to analyze gene expression profile and splicing pattern of DEK overexpression and control cells. Then, an improved RIP-seq was conducted to explore genome-widely binding characteristic of DEK in HeLa cell. Bioinformatics analysis and qPCR/RIP-PCR were performed to identify and validate the bound and regulated targets of DEK, respectively Results:The results showed DEK overexpression did not affect transcript level expression of those high expressed genes (with FPKM > 1), but splicing pattern of 411 genes (RASGs) was regulated by DEK in HeLa cells, which were enriched in Hippo signaling pathway. Moreover, DEK broadly bind the RNA of a total of 11, 112 genes, with a biased binding the GGUAA motifs at the CDS and intronic regions Conclusions: our results indicated DEK could broadly bind and regulate the pre-mRNA splicing process, which provide new insights of mechanisms that DEK functions in various biological processes including cancer RIP-seq was conducted to explore genome-widely binding characteristics of DEK in HeLa cell

研究目的：探究DEK蛋白如何广泛结合RNA，并影响癌症中的可变剪接过程。研究方法：通过向HeLa细胞转染DEK过表达质粒以实现DEK过表达，随后利用RNA测序（RNA-seq）分析DEK过表达组与对照组细胞的基因表达谱及剪接模式；继而开展改良型RNA免疫沉淀测序（RIP-seq），以探究DEK在HeLa细胞中的全基因组结合特征；最后通过生物信息学分析与qPCR/RIP-PCR实验，分别鉴定并验证DEK的结合靶标与调控靶标。研究结果：DEK过表达不会影响FPKM>1的高表达基因的转录水平，但可调控HeLa细胞中411个基因（RASGs）的剪接模式，这些基因富集于Hippo信号通路。此外，DEK可广泛结合总计11112个基因的RNA，且偏好结合编码区（CDS）与内含子区域内的GGUAA基序。研究结论：本研究结果表明，DEK能够广泛结合并调控前体mRNA剪接过程，为DEK在包括癌症在内的多种生物学过程中发挥功能的分子机制提供了全新的研究视角。