Upf2 in NMD pathway

Purpose: Probe the transcriptome-wide changes in the expression pattern between WT and Sertoli-specific Upf2 KO testes Methods: Total RNA were extracted from WT and Sertoli-specific Upf2 KO testes in triplicates and subject to deep-sequencing in Ion Torrent seq platform. 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: Our study represents the first detailed analysis of Upf2-mediated NMD pathway in Sertoli cell development Testis mRNA profiling was generated from postnatal day 4 WT and Amh-cKO (Sertoli specific Upf2 KO) testes, in triplicates.研究目的：探究野生型（Wild Type, WT）与支持细胞特异性Upf2基因敲除（Knock Out, KO）小鼠睾丸之间的全转录组表达模式变化。 实验方法：分别从野生型与支持细胞特异性Upf2基因敲除小鼠睾丸中提取总RNA，设置三次生物学重复，并在Ion Torrent测序平台上进行高通量测序。 实验结果：通过优化后的数据分析流程，我们将每个样本约3000万条序列读段比对至小鼠基因组（mm9组装版本），并分别采用BWA流程与TopHat流程，在野生型与Nrl基因敲除（Nrl-/-）小鼠的视网膜中分别鉴定出16014条转录本与34115条转录本。RNA测序（RNA-sequencing, RNA-seq）数据证实了25个已知持家基因的稳定表达，其中12个通过实时定量逆转录聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）得到验证。RNA-seq数据与qRT-PCR结果在超过四个数量级的表达范围内呈现线性相关，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（倍数变化≥1.5且P值<0.05）。通过qRT-PCR验证了25个基因的表达变化，证实了RNA-seq方法的高灵敏度。对差异表达基因进行层次聚类分析，发现了多个尚未被注释的基因，这些基因可能与视网膜功能相关。采用BWA与TopHat流程进行的数据分析显示二者存在显著的结果重叠，但同时也为转录组分析提供了互补的视角。 研究结论：本研究首次详细分析了支持细胞发育过程中Upf2介导的无义介导的mRNA降解（Nonsense-mediated mRNA Decay, NMD）通路。本研究的睾丸mRNA转录组分析数据来自出生后第4天的野生型小鼠与抗缪勒管激素条件性基因敲除（Amh-cKO，即支持细胞特异性Upf2基因敲除）小鼠的睾丸，设置三次生物学重复。