LncRNA HOTAIRM1 functions in DNA double-strand break repair via its association with DNA repair and mRNA surveillance factors

Abstract

The eukaryotic exon junction complex component Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. Using immunoprecipitation-RNA-seq, we identified a set of Y14-associated long non-coding RNAs (lncRNAs). The lncRNA HOTAIRM1 serves as a strong candidate that mediates the interaction between Y14 and the NHEJ complex. HOTAIRM1 localized to near ultraviolet laser-induced DNA damage sites. Depletion of HOTAIRM1 delayed the recruitment of DNA damage response and repair factors to DNA lesions and compromised the efficiency of NHEJ-mediated DSB repair. Identification of the HOTAIRM1 interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in a HOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude that HOTAIRM1 serves as an assembly scaffold for both DNA repair and mRNA surveillance factors that act in concert to repair DSBs.

Co-phase separation of Y14 and RNA in vitro and its implication for DNA repair

“…Wild-type Y14, but not W73V, could co-condensate with RNA. Moreover, HOTAIRM1 promoted LLPS of Y14, supporting their coordinated function in DNA damage repair (Chuang et al, 2023). As has been observed for several RBPs such as Fus and hnRNP A1 (Maharana et al, 2018), low RNA:protein ratios could promote LLPS of Y14, whereas higher concentrations of RNA inhibited LLPS.…”

“…Y14 interacts with DNA repair factors via the lncRNA HOTAIRM1, of 1044 nucleotides in length (Chuang et al, 2023). Therefore, we tested whether HOTAIRM1 could promote Y14 LLPS under certain circumstances.…”

“…HeLa cells were maintained as above for U2OS cells. To assess the NHEJ activity, 1 µg of the Cas9/sgHPRT expressing vector (sgHPRT targeting sequences: AAAGGGTGTTTATTCCTCA), 25 pmol of dsDNA oligonucleotides Insertion (Ins dsDNA-1: 5'-TTAATTGAGTTGTCATATGTTAATAACCGG-3'; Ins dsDNA-2: 5'-ACCGTTATTAACATATGACAACTCAATTA-3', Du et al, 2018), 40 pmol Y14 targeting siRNA (5′-ggguauacucuaguugaaa-3′, Thermo Fisher Scientific) and 4 µg of siRNA-resistant Y14-expressing vectors were transfected into 2×10 5 HeLa cells (Chuang et al, 2023). Genomic DNA was collected 48 hours post-transfection and extracted by PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific).…”

“…Chromatin fractionation of transfected U2OS cells was essentially performed as describe in Chuang et al (2023). In brief, cells were incubated with the cytoskeleton (CSK) buffer containing 10 mM PIPES (pH 7.0), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.7% Triton X-100 at 4˚C for 3 min.…”

Deciphering shared attributes of plant long non-coding RNAs through a comparative computational approach

“…For example, in humans, a lncRNA named DNA damage-sensitive RNA1 (DDSR1) plays a critical role in modulating DNA repair by homologous recombination 71 . The lncRNA, HOTAIRM1 serves as an assembly scaffold for non-homologous end joining (NHEJ) factors (Upf1/SMG6) to DNA double-strand breaks and subsequently helps in DSB repair 72 . Another lncRNA LINP1 , was reported to direct the NHEJ-mediated DNA repair by interacting with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes 73 .…”