Background: Loss of the p53-inducibleLINC01021in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze howLINC01021affects the p53-induced transcriptional program. Methods: Using a CRISPR/Cas9-approach, we deleted the p53 binding site in theLINC01021promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated withLINC01021. Results: Loss of the p53-inducibility ofLINC01021resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared toLINC01021wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such asNOXAandFAS,displayed significantly stronger induction when the p53-inducibility ofLINC01021was abrogated. Loss of the p53-inducibility ofLINC01021resulted in alternative splicing of a small number of mRNAs, such asARHGAP12,HSF2, andLYN. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners ofLINC01021by mass spectrometry. Conclusions: Our results suggest thatLINC01021may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.
背景：在p53功能完整的结直肠癌细胞系中，p53诱导型LINC01021的缺失会导致对DNA损伤性化疗药物的敏感性增加。本研究系统分析了LINC01021如何影响p53诱导的转录调控网络。方法：我们采用CRISPR/Cas9技术敲除SW480结直肠癌细胞中LINC01021启动子区的p53结合位点，并在激活外源性p53后进行RNA测序分析。通过RNA亲和纯化结合质谱分析技术鉴定与LINC01021相互作用的蛋白质。结果：在外源性p53激活后，与LINC01021野生型细胞相比，LINC01021的p53诱导性缺失导致显著调控的mRNA数量增加约1.8倍。当LINC01021的p53诱导性被消除时，部分直接p53靶基因（如NOXA和FAS）的诱导表达显著增强。LINC01021的p53诱导性缺失还导致少量mRNA（如ARHGAP12、HSF2和LYN）发生选择性剪接。质谱分析鉴定出多个参与前体mRNA剪接的RNA结合蛋白与LINC01021存在相互作用。结论：我们的研究结果表明，LINC01021可能通过环境依赖性地调控部分p53靶基因的表达和剪接，从而限制p53介导的转录变化的范围和强度。
LINC01021Attenuates Expression and Affects Alternative Splicing of a Subset of p53-Regulated Genes
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