Causes and consequences of Pol II read-through in Arabidopsis.

In plants, the molecular function(s) of the nuclear localised 5’-3’ EXORIBONUCLEASES (XRNs) are unclear, however their activity is reported to have a significant effect on gene expression and SAL1-mediated retrograde signaling. Using Parallel Analysis of RNA Ends (PARE) we document a dramatic increase in uncapped RNA substrates of the XRNs in both sal1 and xrn2xrn3 mutants. We then demonstrate that a major consequence of reducing SAL1 or XRN activity is RNA Polymerase II (Pol II) 3’ read-through. This occurs at 72% of expressed genes, demonstrating a major genome-wide role for the XRNs and the torpedo model of transcription termination in Arabidopsis. Read-through is often speculated to have a negative effect on transcript abundance, however we do not observe this. Rather, we identify a strong association between read-though and increased mRNA expression of tandemly orientated downstream genes, strongly correlated with the proximity and expression of the upstream gene. We observe read-though in the proximity of the promoters of 903 genes upregulated in the sal1-8 retrograde signaling mutant; thus, read-though may directly account for up to 23% of genes upregulated in sal1-8. Using APX2 and AT5G43770 as exemplars, we genetically uncouple read-through loci from downstream genes to validate the principle of read-through mediated mRNA regulation. Thus, this read-through phenomenon has potential to be a mechanism for regulating gene expression and retrograde signaling.

在植物中，定位于细胞核的5'-3'核糖核酸外切酶（EXORIBONUCLEASES, XRNs）的分子功能尚未明确，但已有研究显示其活性可对基因表达及SAL1介导的逆行信号转导产生显著影响。本研究采用RNA末端平行分析（Parallel Analysis of RNA Ends, PARE）技术，证实sal1与xrn2xrn3突变体中XRNs的未加帽RNA底物水平显著升高。随后我们证明，降低SAL1或XRN活性的主要效应是引发RNA聚合酶II（RNA Polymerase II, Pol II）的3'端通读现象，该现象覆盖72%的表达基因，这表明XRNs及拟南芥中转录终止的鱼雷模型在全基因组层面发挥关键调控作用。此前学界普遍推测3'端通读现象会降低转录本丰度，但本研究未观察到此现象。与之相反，我们发现通读现象与串联排布的下游基因的mRNA表达上调存在显著关联，且该关联强度与上游基因的表达水平及其与下游基因的间距密切相关。我们在sal1-8逆行信号转导突变体的903个上调基因的启动子区域附近观测到通读现象，因此通读现象可直接解释sal1-8突变体中23%的上调基因事件。我们以APX2和AT5G43770为范例，通过遗传学手段将通读位点与下游基因解偶联，验证了通读介导的mRNA调控机制。综上，该3'端通读现象有望成为调控基因表达与逆行信号转导的潜在分子机制。