Long non-coding RNAs in the DNA damage response of Arabidopsis thaliana

长非编码RNA在拟南芥DNA损伤反应中的作用

• 批准号: 420731442
• 负责人: Professor Dr. Reinhard Kunze
• 金额: --
• 依托单位: Arbeitsgruppe Molekulare Pflanzengenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/420731442?language=en
• 关键词: Long; non; coding; RNAs; DNA

In recent years it was discovered that in eukaryotic cells thousands of long non-coding RNAs (lncRNAs) are expressed and that approximately one third of them consist of or contain transposable element sequences. It is assumed that most lncRNAs are involved in gene regulation and in transposon silencing, but currently only few lncRNAs, predominanty in mammalian cells, are functionally characterized. In a screen for Arabidopsis thaliana transposons that are transcriptionally re-activated by DNA double strand break (DSB)-inducing ionizing radiation (X-rays), we discovered ~80 novel up- or downregulated lncRNAs. The regulation of almost all of these lncRNAs is dependent on the protein kinase ATM, which is the key regulator of the signaling cascade that activates the DNA damage repair machinery. We have started to functionally analyse the lncRNA XlincR, which is specifically upregulated by genotoxic treatments. We found that in a XlincR-T-DNA mutant the transcription of the DNA damage repair-associated DNA Ligase IV (LIG4) gene is elevated. We therefore hypothesize that XlincR, and presumably also other DNA damage-associated lncRNAs, are involved in the DNA damage response (DDR). (1) We will investigate the specificity of the X-ray induced lncRNAs by testing their response to different genotoxins. For 14 of the most strongly regulated lncRNAs, half of them containing transposons, T-DNA insertion lines are available that will be subjected to DNA damage sensitivity assays and phenotypical analyses. (2) To unravel the role of XlincR in the DDR, we will determine its regulation by different genotoxins and its developmental and tissue specificity. The position in the DDR regulatory network will be determined by analysing its transcriptional DNA damage response in DNA repair, replication and cell cycle mutants. Using CRISPR/Cas we will generate a true loss-of-function mutant by replacing XlincR with a random sequence, and a line where the XlincR-internal two short open reading frames are destroyed. For directly visualizing the level and kinetics of DNA damage and repair in different genotypes the Comet assay will be applied. XlincR target genes will be identified by RNA sequencing of wild type xlincr-mutants. Furthermore we will investigate whether Arabidopsis can be primed for increased DNA damage resistance and if XlincR is involved. (3) We will investigate the regulatory network between the histone demethylase REF6 and XlincR, FLC and FT. Overall, we anticipate that this project will lead to the identification of lncRNAs as novel regulatory factors in the DDR of A. thaliana.

近年来，人们发现真核细胞中有数千种长链非编码rna （lncrna）表达，其中约三分之一由转座元件序列组成或含有转座元件序列。据推测，大多数lncrna参与基因调控和转座子沉默，但目前只有少数lncrna被功能表征，主要存在于哺乳动物细胞中。在筛选被DNA双链断裂（DSB）诱导的电离辐射（x射线）转录重新激活的拟南芥转座子时，我们发现了约80个新的上调或下调lncrna。几乎所有这些lncrna的调控都依赖于蛋白激酶ATM，它是激活DNA损伤修复机制的信号级联的关键调节因子。我们已经开始对lncRNA XlincR进行功能分析，它在基因毒性治疗中被特异性上调。我们发现，在XlincR-T-DNA突变体中，DNA损伤修复相关的DNA连接酶IV （LIG4）基因的转录升高。因此，我们假设XlincR以及其他DNA损伤相关的lncrna参与了DNA损伤反应（DDR）。(1)我们将通过检测x射线诱导的lncrna对不同基因毒素的反应来研究其特异性。对于14种最强烈调控的lncrna，其中一半含有转座子，T-DNA插入系可用于DNA损伤敏感性分析和表型分析。(2)为了揭示XlincR在DDR中的作用，我们将确定不同基因毒素对XlincR的调控及其发育和组织特异性。DDR调控网络中的位置将通过分析其在DNA修复、复制和细胞周期突变中的转录DNA损伤反应来确定。使用CRISPR/Cas，我们将生成一个真正的功能缺失突变体，方法是用一个随机序列替换XlincR，并在其中一行XlincR内部的两个短开放阅读帧被破坏。为了直接可视化不同基因型DNA损伤和修复的水平和动力学，将应用Comet测定。XlincR靶基因将通过野生型XlincR突变体的RNA测序进行鉴定。此外，我们将研究拟南芥是否可以增加DNA损伤抗性，以及XlincR是否参与其中。(3)我们将研究组蛋白去甲基化酶REF6与XlincR、FLC和FT之间的调控网络。总的来说，我们预计该项目将导致鉴定lncrna作为拟南芥DDR的新调控因子。