Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay pathways

In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs. Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs in A. thaliana. While PROMPTs are overall rare in A. thaliana, about 100 clear cases of exosome-sensitive PROMPTs were identified. We also found that PROMPTs become sources of 21-22 nt small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPT production. In addition, we found ~200 transcription start sites within 3'-UTR-encoding regions that produce long unspliced, exosome-sensitive antisense RNAs. The previously characterized non-coding (nc) RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of ncRNAs. Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations. Overall design: small RNA-seq sequencing of leaf and young flower bud tissues in wt, hen2-5 and rrp4-2 mutants

在动物体内，RNA聚合酶II（RNA polymerase II）介导的转录从基因启动子双向起始，在正向链产生前体mRNA（pre-mRNAs），在反向链产生启动子上游转录本（promoter upstream transcripts，PROMPTs）。启动子上游转录本会被核外切体（nuclear exosome）快速降解。此前基于新生RNA（nascent RNA）技术的研究认为，拟南芥（Arabidopsis thaliana）不会产生启动子上游转录本。本研究使用稳态RNA（steady-state RNA）测序技术，对核RNA降解（包括外切体介导的降解）缺陷的突变体进行分析，重新评估拟南芥中启动子上游转录本的存在情况。尽管拟南芥中的启动子上游转录本整体较为稀少，但本研究共鉴定出约100个对外切体敏感的典型启动子上游转录本案例。本研究还发现，在外切体缺陷突变体中，启动子上游转录本可作为21-22核苷酸小干扰RNA（small interfering RNAs）的产生来源，这或许可以解释植物为何进化出抑制启动子上游转录本生成的机制。此外，本研究在3'非翻译区（3'-UTR）编码区域内发现了约200个转录起始位点，这些位点可产生长链未剪接、对外切体敏感的反义RNA（antisense RNAs）。此前已被鉴定的、调控关键种子休眠调控因子延迟发芽1（DELAY OF GERMINATION1，DOG1）表达的非编码RNA（non-coding RNA，ncRNA），便是这类反义RNA的典型代表。带有对外切体敏感的反义RNA的基因座中，转录因子基因（transcription factor genes）的占比显著偏高，这表明这类非编码RNA可能广泛参与基因表达的调控。最后，本研究对拟南芥中的可变启动子（alternative promoters）使用情况进行了评估，并对比了现有转录起始位点（TSS）注释的准确性。实验整体设计：对野生型（wild type，wt）、hen2-5及rrp4-2突变体的叶片和幼花蕾组织进行小RNA测序（small RNA-seq）