Genome-wide analysis of cellular NMD substrates that regulated by Upf1, PNRC2 or CTIF in HeLa cell

The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein (CBP) 80 and 20. During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A) mRNAs increases in the cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is one of NMD-inducing features. Using chimeric reporter constructs and confocal microscopy, we find that the uORF of CDKN1A mRNA is actively translated and modulates a translational efficiency of the main downstream ORF. Our findings provide the biological insights into the possible role of NMD in diverse pathways mediated by CDKN1A. The microarray analysis performed to analize the cellular NMD substrates that regulated by Upf1, PNRC2 and/or CTIF in HeLa cell. The hypothesis tested in the present study was that endogenous NMD substrates may co-regulated by Upf1, PNRC2 and CTIF. Results provide important information that vast range of cellular NMD substrates are reqired CTIF. Total RNA obtained from HeLa cells with downregulation of Upf1, PNRC2 or CTIF by siRNA. The up- or down-regulated transcripts were compare to control siRNA treated HeLa cell RNA extract. Significant transcripts were confirmed by replication.

翻译的第一轮发生在被核帽结合复合物（CBC）结合的mRNA上，该复合物由核帽结合蛋白（CBP）80与20组成。在此轮翻译过程中，异常mRNA会被无义介导的mRNA降解（NMD）通路识别并丰度下调，而NMD是mRNA质量控制机制之一。本研究的基因芯片分析显示，在细胞内NMD因子被敲低后，细胞周期蛋白依赖性激酶抑制剂1A（CDKN1A）的mRNA水平会升高。值得注意的是，CDKN1A mRNA含有一个上游开放阅读框（uORF），这是可诱导NMD的特征之一。本研究利用嵌合报告基因构建体与共聚焦显微镜，发现CDKN1A mRNA的上游开放阅读框能够被主动翻译，并调控下游主开放阅读框的翻译效率。本研究结果为NMD在CDKN1A介导的多种通路中可能发挥的作用提供了生物学见解。
本研究通过基因芯片分析，旨在探究海拉细胞（HeLa）中受Upf1、PNRC2及CTIF调控的细胞内NMD底物。本研究验证的假说为：内源性NMD底物可能同时受Upf1、PNRC2与CTIF共同调控。本研究结果提供了重要信息：大量细胞内NMD底物的调控过程依赖于CTIF。本研究利用小干扰RNA（siRNA）敲低海拉细胞中的Upf1、PNRC2或CTIF，随后提取总RNA。将差异表达（上调或下调）的转录本与经对照siRNA处理的海拉细胞RNA提取物进行比较。通过生物学重复验证了显著差异表达的转录本。