Nonsense-Mediated RNA Decay Factor UPF1 Is Critical for Posttranscriptional and Translational Gene Regulation in Arabidopsis

9.618Q1       2020 Sep;32(9):2725-2741.

DOI: 10.1105/tpc.20.00244

Vivek K Raxwal 1, Craig G Simpson 2, Jiradet Gloggnitzer 3, Juan Carlos Entinze 4, Wenbin Guo 5, Runxuan Zhang 5, John W S Brown 2 4, Karel Riha 1

一作单位：马萨里克大学中欧理工学院，布尔诺，捷克共和国。

Abstract

Nonsense-mediated RNA decay (NMD) is an RNA control mechanism that has also been implicated in the broader regulation of gene expression. Nevertheless, a role for NMD in genome regulation has not yet been fully assessed, partially because NMD inactivation is lethal in many organisms. Here, we performed an in-depth comparative analysis of Arabidopsis (Arabidopsis thaliana) mutants lacking the NMD-related proteins UPF3, UPF1, and SMG7. We found different impacts of these proteins on NMD and the Arabidopsis transcriptome, with UPF1 having the biggest effect. Transcriptome assembly in UPF1-null plants revealed genome-wide changes in alternative splicing, suggesting that UPF1 functions in splicing. The inactivation of UPF1 led to translational repression, as manifested by a global shift in mRNAs from polysomes to monosomes and the downregulation of genes involved in translation and ribosome biogenesis. Despite these global changes, NMD targets and mRNAs expressed at low levels with short half-lives were enriched in the polysomes of upf1 mutants, indicating that UPF1/NMD suppresses the translation of aberrant RNAs. Particularly striking was an increase in the translation of TIR domain-containing, nucleotide binding, leucine-rich repeat (TNL) immune receptors. The regulation of TNLs via UPF1/NMD-mediated mRNA stability and translational derepression offers a dynamic mechanism for the rapid activation of TNLs in response to pathogen attack.

摘要

无意义介导的RNA衰变（NMD）是一种RNA控制机制，也与基因表达的更广泛调控有关。然而，尚未完全评估NMD在基因组调控中的作用，部分原因是NMD失活在许多生物中都是致死性的。在这里，我们对拟南芥（Arabidopsis thaliana）进行了深入的比较分析。）缺少NMD相关蛋白UPF3，UPF1和SMG7的突变体。我们发现这些蛋白质对NMD和拟南芥转录组的不同影响，其中UPF1的作用最大。UPF1缺体的植物中的转录组装配揭示了全基因组可变剪接的变化，表明UPF1在剪接中起作用。UPF1的失活导致翻译抑制，表现为mRNA从多核糖体向单核糖体的整体转移以及参与翻译和核糖体生物发生的基因的下调。尽管有这些全局变化，但在upf1的多核糖体中富集了NMD靶标​​和低水平半衰期的mRNA表达。突变体，表明UPF1 / NMD抑制异常RNA的翻译。尤其引人注目的是包含TIR结构域，核苷酸结合，富含亮氨酸的重复（TNL）免疫受体的翻译增加。通过UPF1 / NMD介导的mRNA稳定性和翻译抑制抑制TNL的调控为响应病原体侵袭而快速激活TNL提供了一个动态机制。

实验

          To further assess the effects of UPF1 and SMG7 on gene expression, we compared the leaf transcriptomes of upf1 pad4 and smg7 pad4 plants (Gloggnitzer et al., 2014) using RNA-seq. We found that 17,393 and 15,933 genes were expressed in the pad4 vs. upf1 pad4 and pad4 vs. smg7 pad4 contrast pairs, respectively [≥ 1 normalized CPM; (counts per million; scaled to length of transcripts and library size) in at least two samples; Supplemental Data Set 2]. To compare the global impact of UPF1 and SMG7 deletions on the transcriptome, we plotted the kernel density distribution along with a scatter plot of gene expression between the two contrast pairs (Figure 2A).

          Subsequently, differential expression analysis  was performed using the limma-voom pipeline enabled in the 3D RNA-seq tool (Guo et al., 2019). In brief, TPM count obtained from Kallisto were converted to transcript length scaled TPM values for each sample. These transcript length scaled TPM values were further converted to normalized CPM for each sample. Transcripts expressed at low levels were filtered if they did not have ≥1 sample with ≥1 normalized CPM (scaled to length and library size) expression. A gene was determined to be an expressed gene if one of its transcripts was expressed. The resulting expressed transcripts were used to estimate the batch effect among samples, and the batch effect was removed from the samples using RUVr (Risso et al., 2014). 

         随后，使用3D RNA-seq工具中启用的limma-voom管道进行差异表达分析(Guo et al.， 2019)。简而言之，从Kallisto获得的TPM计数被转换为每个样本的转录长度比例TPM值。这些按转录长度比例计算的TPM值进一步转换为每个样本的标准化CPM。低水平表达的转录本如果没有≥1个样本且≥1个标准化CPM(按长度和文库大小缩放)表达，则对其进行过滤。如果一个基因的转录本中有一个被表达了，那么该基因就被确定为表达基因。最终的表达转录本被用来估计样本之间的批效应，并使用RUVr将批效应从样本中移除(Risso等人，2014)。

** Karel Riha **