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

近年来发现在真核细胞中表达数千个长的非编码RNA（lncRNA），其中约三分之一由转座因子序列组成或含有转座因子序列。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，在哺乳动物细胞中占主导地位，功能特征。在筛选通过DNA双链断裂（DSB）诱导的电离辐射（X射线）转录再激活的拟南芥转座子时，我们发现了约80种新的上调或下调的lncRNA。几乎所有这些lncRNA的调节都依赖于蛋白激酶ATM，ATM是激活DNA损伤修复机制的信号级联的关键调节剂。我们已经开始对lncRNA XlincR进行功能分析，该基因通过遗传毒性治疗特异性上调。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（LIG 4）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)我们将通过测试它们对不同基因毒素的反应来研究X射线诱导的lncRNA的特异性。对于14个最强调控的lncRNA，其中一半含有转座子，T-DNA插入线是可用的，将进行DNA损伤敏感性测定和表型分析。(2)为了阐明XlincR在DDR中的作用，我们将通过不同的基因毒素及其发育和组织特异性来确定其调节。在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体中的转录DNA损伤反应来确定。使用CRISPR/Cas，我们将通过用随机序列替换XlincR来产生真正的功能丧失突变体，并且XlincR内部的两个短开放阅读框架被破坏。为了直接可视化不同基因型中DNA损伤和修复的水平和动力学，将应用彗星测定。将通过野生型xlincr突变体的RNA测序鉴定Xlincr靶基因。此外，我们将研究是否可以启动拟南芥增加DNA损伤抗性，如果XlincR参与。(3)我们将研究组蛋白去甲基化酶REF 6与XlincR、FLC和FT之间的调控网络。总的来说，我们预计，该项目将导致识别lncRNA作为新的调节因子，在DDR的A。thaliana.

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.