The long non-coding RNA LINDA restrains cellular collapse following DNA damage in Arabidopsis thaliana

The genomic integrity of every organism is endangered by various intrinsic and extrinsic stresses. To maintain the genomic integrity, a sophisticated DNA damage response (DDR) network is activated rapidly after DNA damage. Notably, the fundamental DDR mechanisms are conserved in eukaryotes. However, knowledge about many regulatory aspects of the plant DDR is still limited. Important, yet little understood, regulatory factors of the DDR are the long non-coding RNAs (lncRNAs). In humans, 13 lncRNAs functioning in DDR have been characterized to date, whereas no such lncRNAs have been characterized in plants yet. By meta-analysis, we identified the long intergenic non-coding RNA induced by DNA damage (LINDA) that responds strongly to various DNA double-strand break-inducing treatments, but not to replication stress induced by mitomycin C. After DNA damage, LINDA is rapidly induced in an ATM- and SOG1-dependent manner. Intriguingly, the transcriptional response of LINDA to DNA damage is similar to that of its flanking hypothetical protein-encoding gene. Phylogenetic analysis of putative Brassicales and Malvales LINDA homologs indicates that LINDA lncRNAs originate from duplication of a flanking small protein-encoding gene followed by pseudogenization. We demonstrate that LINDA is not only needed for the regulation of this flanking gene, but also for fine-tuning of the DDR after the occurrence of DNA double-strand breaks. Moreover, ?linda mutant root stem cells are unable to recover from DNA damage, most likely due to hyper-induced cell death. Overall design: We performed RNA-Seq experiments on wildtype (Col-0) and ?linda mutants after induction of DNA damage by treatment of 14-day-old seedlings with either 80 µg/ml Zeocin or 2 kJ UV-C. Mock treated seedlings or seedlings grown under normal light conditions were used as controls for Zeocin or UV-C treatment, respectively. Samples were harvested 3 hours after induction of DNA damage. We harvested four samples per genotype per treatment.

所有生物体的基因组完整性均会受到多种内源性与外源性胁迫的威胁。为维持基因组完整性，DNA发生损伤后会快速激活一套精密的DNA损伤应答（DNA damage response, DDR）网络。值得注意的是，真核生物的核心DDR机制具有高度保守性。然而，目前学界对植物DDR诸多调控层面的认知仍较为有限。DDR中一类重要却鲜有深入研究的调控因子是长链非编码RNA（long non-coding RNA, lncRNA）。迄今为止，人类中已有13种参与DDR的lncRNA被鉴定，但植物中尚未有相关lncRNA被表征。通过荟萃分析，我们鉴定出了DNA损伤诱导的基因间长链非编码RNA（long intergenic non-coding RNA induced by DNA damage, LINDA），其可对多种DNA双链断裂诱导处理产生强烈响应，但对丝裂霉素C诱导的复制胁迫无应答。DNA损伤发生后，LINDA会以ATM和SOG1依赖的方式被快速诱导。有趣的是，LINDA对DNA损伤的转录应答模式与其侧翼的假定蛋白编码基因高度相似。对十字花目（Brassicales）和锦葵目（Malvales）的推定LINDA同源序列进行系统发育分析，结果显示LINDA lncRNA起源于侧翼的小型蛋白编码基因的复制事件，随后发生假基因化。我们证实，LINDA不仅参与调控该侧翼基因的表达，还可对DNA双链断裂发生后的DDR进行精细调控。此外，Δlinda突变体的根干细胞无法从DNA损伤中恢复，这极有可能源于细胞死亡的过度诱导。总体实验设计：我们对野生型（Col-0）及Δlinda突变体幼苗开展RNA测序（RNA-seq）实验：将14日龄幼苗分别用80 μg/ml博莱霉素（Zeocin）或2 kJ UV-C处理以诱导DNA损伤，以Mock处理幼苗或正常光照条件下生长的幼苗分别作为博莱霉素或UV-C处理的对照。于DNA损伤诱导3小时后收集样本。每个基因型的每个处理组均设置4个生物学重复。