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

• Applicant: Professor Dr. Reinhard Kunze
• Subject Area: Plant Genetics and Genomics
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420731442

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.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Thomas Schmülling