Global characterization of direct substrates of nonsense-mediated mRNA decay in Caenorhabditis elegans

Purpose: We present a genome-wide investigation to distinguish mRNA substrates directly regulated by nonsense-mediated mRNA decay (NMD) from indirect secondary effects using true NMD null alleles Methods: mRNA profiles of wild type (N2), smg-1(r910), and smg-1(r910) smg-2(r915) animals were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. We further performed deep sequencing in triplicate on RNAs that co-immunoprecipitate with the central effector of NMD, SMG-2, in both smg-1(r910) and smg-1(r910) smg-2(r915) mutant animals. The sequence reads that passed quality filters were analyzed at both the gene level and the intron level using Tophat2 and edgeR. qRT-PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: We found that a significant portion of genes with increased expression in an NMD-deficient background are direct substrates of SMG-2. Among the list of direct targets, we find many mRNAs from pseudogenes, alternative splice isoforms harboring PTCs, mRNAs encoding RNA processing factors, and two recently expanded gene families are directly regulated by the NMD pathway. mRNA profiles of wild type (N2), smg-1(r910), and smg-1(r910) smg-2(r915) C. elegans were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. RNAs that co-immunoprecipitate with SMG-2 in smg-1(r910) and smg-1(r910) smg-2(r915) nematodes were also sequenced, in triplicate, using Illumina HiSeq2000.

【研究目的】本研究开展全基因组范围的探究，利用真实的无义介导的mRNA降解（nonsense-mediated mRNA decay, NMD）功能缺失等位基因，区分直接受NMD调控的mRNA底物与间接次级效应产物。
【实验方法】本研究采用Illumina HiSeq2000测序平台，对野生型（N2）、smg-1(r910)以及smg-1(r910) smg-2(r915)三种秀丽隐杆线虫（C. elegans）样本进行三次生物学重复的深度测序，以获取mRNA表达谱。此外，我们分别在smg-1(r910)与smg-1(r910) smg-2(r915)突变体线虫中，对与NMD核心效应蛋白SMG-2发生免疫共沉淀的RNA进行三次生物学重复的深度测序。对通过质量过滤的测序读段，分别在基因水平与内含子水平采用Tophat2与edgeR软件进行数据分析。同时采用SYBR Green检测法完成实时定量反转录聚合酶链反应（qRT-PCR）验证。
【实验结果】本研究采用优化后的数据分析流程，将每个样本中约3000万条测序读段比对至小鼠基因组（版本mm9）；通过BWA分析流程在野生型与Nrl纯合缺失（Nrl−/−）小鼠视网膜中鉴定出16014条转录本，采用TopHat流程则鉴定出34115条转录本。RNA测序（RNA-seq）数据证实了25个已知持家基因的稳定表达，其中12个经qRT-PCR验证。RNA-seq数据与qRT-PCR结果在超过四个数量级的区间内呈线性相关，拟合优度（R²）为0.8798。约10%的转录本在野生型与Nrl−/−小鼠视网膜中呈现差异表达（折叠变化≥1.5，p值<0.05）。另有25个基因的表达变化经qRT-PCR验证，证实了RNA-seq方法的高灵敏度。对差异表达基因进行层级聚类分析后，发现多个尚未被注释的基因可能参与视网膜功能调控。采用BWA与TopHat流程进行的数据分析结果存在显著重叠，但二者在转录组分析中可提供互补的研究视角。
【研究结论】本研究发现，在NMD缺陷背景下表达上调的基因中，有相当一部分是SMG-2的直接底物。在直接靶标列表中，我们鉴定到大量来自假基因的mRNA、携带提前终止密码子（Premature Termination Codons, PTCs）的可变剪接异构体、编码RNA加工因子的mRNA，以及两个近期发生扩增的基因家族，它们均受NMD通路的直接调控。本研究的数据集包含采用Illumina HiSeq2000测序平台完成三次生物学重复的野生型（N2）、smg-1(r910)以及smg-1(r910) smg-2(r915)秀丽隐杆线虫的mRNA表达谱；同时还包含在smg-1(r910)与smg-1(r910) smg-2(r915)线虫中与SMG-2免疫共沉淀的RNA的三次生物学重复测序数据。