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

  • ️Sun Jan 01 2023

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

Tzu-Wei Chuang et al. Nucleic Acids Res. 2023.

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.

© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

Identification of Y14-associated RNAs from chromatin-enriched fractions. (A) The diagram illustrates the procedure for identifying Y14-bound RNAs in the chromatin-enriched fraction of HEK293 cells that transiently expressed FLAG-Y14 through immunoprecipitation using anti-FLAG. (B) Reactome pathway analysis of proteins encoded by Y14-associated mRNAs. The bar graph shows the top 10 enriched pathways ranked by the enrichment ratio, i.e. the number of observed genes divided by the number of expected genes from each reactome pathway. (C) Graph showing the distribution of the number of exons per gene (Y14-bound RNAs versus non-Y14-bound RNAs). The average number of exons of Y14-associated mRNAs (middle panel; ***P<0.001) and lncRNAs (right panel; n.s., not significant) is shown. (D) Pie charts show the percentage of different classes of Y14-associated RNAs (mRNA, lncRNA, other types of ncRNA) (left) and lncRNAs (right). (E) Bar graph shows 26 annotated and high-abundance lncRNAs having an average FPKM value >15 in Y14-IP. Orange and blue represent fold enrichment and FPKM value, respectively. A dot indicates lncRNAs selected for experimental verification. (F) HEK293 cells were transiently transfected with empty (vec) or FLAG-Y14-expressing vector. Cell lysates were subjected to immunoprecipitation (IP) using anti-FLAG, followed by RT–PCR using primers specific for the indicated lncRNAs or β-actin (ACTB, control). Immunoblotting was performed using anti-FLAG. (G) HEK293 cells were transiently transfected with empty (vec), or FLAG-Y14- or FLAG-eIF4A3-expressing vector. IP, RT–PCR and immunoblotting were as in (F).

Figure 2.
Figure 2.

HOTAIRM1 is associated with the NHEJ complex. (A) Domains of Y14 (RRM, RNA recognition motif) and its mutants (WV and SA) and C-terminal truncated version. (B) HEK293 cells were transfected with empty (–) or FLAG-Y14-expressing vector (WT, SA or WV mutant; WT represents the wild type), followed by immunoprecipitation (IP), and RT–PCR or immunoblotting. Bottom: bar graphs show the relative co-precipitation efficiency of HOTAIRM1, Ku80 and Ku70 with each Y14 version (mean ± SD). n (the number of experimental repeats) = 4. (C) HEK293 cells were transiently transfected with empty (vec), or FLAG-Y14- or GFP–FLAG-Ku70-expressing vector, followed by immunoprecipitation along with RT–PCR or immunoblotting using anti-FLAG. (D) Recombinant His-tagged Y14, Y14ΔC or Ku70/80 was incubated with total HeLa cell RNAs, followed by pull-down using nickel resin. Bound RNAs were detected by RT–PCR. Bottom panel shows SDS–PAGE of recombinant proteins. (E) Oligonucleotides (AS, antisense; bHM1, biotinylated; gHM1, GapmeR) and short hairpin RNA (shHM1) complementary to HOTAIRM1. E indicates exon. (F) HEK293 cells were transfected with empty vector (vec) or FLAG-Y14 vector. Anti-FLAG immunoprecipitates were mock treated (lane 2) or incubated with antisense oligonucleotides in the presence of RNase H (lanes 3–6). Immunoblotting was performed using antibodies against the indicated proteins. NSO and ACTB represent non-specific and β-actin-targeting antisense oligonucleotides, respectively. Bottom: the numbers indicate the relative level of Ku70 and Ku80 in Y14 co-precipitates after destruction of HOTAIRM1. n = 3; n.d., not detectable. (G) HEK293 cell lysates were mock incubated (lane 2) or incubated with biotinylated oligonucleotides (lanes 3 and 4), followed by pull-down using strepatavidin agarose and immunoblotting or RT–PCR. bNSO: biotinylated non-specific oligonucleotide. (H) HeLa cells were transfected with empty vector (vec) or FLAG-Y14 vector or together with the indicated GapmeR. Anti-FLAG immunoprecipitates were subjected to immunoblotting and RT–PCR. gNSO: non-specific GapmeR. gHM1 disrupted the interaction of Y14 with NBS1 and NHEJ factors by >90%.

Figure 3.
Figure 3.

HOTAIRM1 accumulates at DNA damage sites and contributes to genome integrity. (A) HeLa cells were transfected with the indicated siRNA or GapmeR. RT–PCR and immunoblotting were performed 48 h post-transfection. Bottom: the bar graph shows fold increase of γH2AX after knockdown of Y14 or HOTAIRM1 (mean ± SD; n = 3). (B) HeLa cells were transfected as in (A), followed by the comet assay. Bar graph shows the percentage of DNA in the comet tail (mean ± SD; ***P<0.001 for a two-tailed test); for each sample, 46 or 57 cells were quantified. (C) U2OS cells were untransfected but were mock or Ku-55933 treated as indicated (upper three rows) or transfected with GapmeRs (lower two rows). Except for a set of untransfected cells (–LMI, the top row), laser microirradiation (405 nm; +LMI) was performed. Cells were subsequently immediately fixed for in situ hybridization using bHM1-3 as the probe and subsequently subjected to immunofluorescence microscopy using anti-γH2AX. Arrowhead, LMI track. Scale bar, 10 μm. (D) Diagram shows HOTAIRM1–MS2 chimeric RNA and truncations. U2OS cells were transfected with the control vector (MS2 only) or a HOTAIRM1–MS2-expressing vector and the GFP–MCP-expressing vector (GFP signals represent HOTAIRM1), followed by indirect immunofluorescence microscopy using anti-γH2AX. Bar graph shows the relative efficiency (GFP/γH2AX intensities) of truncated HOTAIRM1 fragments in localization to the DSB sites; the full-length HOTAIRM1 was set to 100% (mean ± SD; ***P<0.001 for a two-tailed test); for each sample, six or 11 cells were quantified. Scale bar, 20 μm. RT–PCR shows the expression of MS2 and full-length and truncated MS2–HOTAIRM1.

Figure 4.
Figure 4.

HOTAIRM1 is required for the recruitment of DNA repair factors to DSB sites and regulates Ku foci dynamics. (A) U2OS cells that stably expressed the GFP fusion with MDC1, Ku70 or Ku80 were mock transfected or transfected with the shHORAITM1 (shHM1)–mCherry-expressing vector. Cells were subjected to laser microirradiation followed by live-cell imaging using confocal microscopy. Representative confocal images show accumulation of GFP fusion proteins at sites (white-outlined rectangles or circles) of laser microirradiation at the indicated time points. NT (non-treated) indicates samples before microirradiation. mCherry represents shHOTAIRM1-expressing cells. Graphs to the right show fluorescence intensities of GFP fusion proteins at the irradiated region. Intensity was quantified periodically up to 10 min, normalized, and is presented as the mean and SD (P-values as indicated) for 12 or 15 cells in each experiment. The RT–PCR data indicate the efficiency of HOTAIRM1 knockdown. Scale bar, 10 μm. (B) U2OS cells were transfected with gNSO or gHM1. Cells were not irradiated (–IR) or exposed to 10 Gy of IR and harvested at the indicated time points post-IR. Cells were treated with Triton X-100 and RNase A according to Britton et al. (43). Indirect immunofluorescence microscopy was performed using anti-γH2AX and anti-Ku70. Dot graph shows relative fluorescence intensity of Ku70; for each sample, 21–29 cells were measured (–IR was set to 1; mean ± SD; P-values for a two-tailed test, *<0.05, ***<0.001, n.s. not significant). Scale bar, 10 μm. RT–PCR in both panels shows HOTAIRM1 knockdown efficiency.

Figure 5.
Figure 5.

HOTAIRM1 participates in DSB repair. (A) Experimental design for quantitative measurement of NHEJ-mediated repair of DSBs. Transfection of HeLa cells with the Cas9/sgRNA (sgHPRT) vector induces DSBs. Incorporation of the double-stranded oligonucleotide ‘Ins’ into the DSB sites evaluated by PCR and qPCR represented DNA repair efficiency. (B) HeLa cells were mock transfected (lane 1) or transfected for 48 h with one or more of the following reagents: the Cas9/sgHPRT plasmid, Ins and GapmeR (lanes 4 and 5) or siRNA (lanes 6–8), as indicated. Genomic DNA was recovered for PCR using the primer set I/R or F/R. Bar graph shows qPCR of lanes 3–8 (sg + Ins was set to 1; mean ± SD; n = 6; ***P<0.001 for a two-tailed test). Immunoblotting and RT–PCR indicate the knockdown efficiency of HOTAIRM1, Y14 and LIG4 and transfection efficiency of Cas9/sgHPRT; lanes are numbered to correspond with the PCR analysis (upper panel). (C) HeLa NHEJ reporter cells in which the chromosomally integrated GFP gene is disrupted by an intron and flanked by I-SceI sites. I-SceI-induced cleavage mimics a DSB. GFP expression was restored after the cleavage repaired through NHEJ. (D) HeLa NHEJ reporter cells were mock transfected or transfected with pSCE (the I-SceI expression vector) and GapmeR or siRNA as indicated. The number of GFP-positive cells was counted 48 h post-transfection. Bar graph represents relative repair efficiency; samples without GapmeR/siRNA transfection were set to 1 (mean ± SD; n = 5; ***P<0.001 for a two-tailed test). Immunoblotting and RT–PCR show knockdown efficiency.

Figure 6.
Figure 6.

HOTAIRM1 is associated with the mRNA surveillance factors. (A) Procedure for identification of HOTAIRM1-interacting proteins. HEK293 cell lysates were incubated with three bHM1 oligonucleotides followed by affinity selection using streptavidin. Selected proteins were analyzed by MS. (B) KEGG pathway enrichment analysis of the 688 HOTAIRM1 partners that were identified. Bar graph shows the top enriched Gene Ontology terms (P-value <0.05) from the KEGG Pathway Database. (C) STRING analysis of the 166 HOTAIRM1-interacting proteins (Supplementary Table S6) that function in gene expression, DNA replication or DNA repair. The diagram shows protein–protein interaction (PPI) networks for these proteins (PPI enrichment P-value <1.0e-16). NHEJ and mRNA surveillance are highlighted in red. RNA polymerase II (RNA pol II) subunits and the PRP19–CDCL5 complex are enclosed by a dotted yellow line and represent hub proteins. BER, base excision repair; MR, mismatch repair; NER, nucleotide excision repair. (D) Affinity selection of HOTAIRM1 was as in (A). RT–PCR and immunoblotting were performed to detect HOTAIRM1 and its interacting partners.

Figure 7.
Figure 7.

mRNA surveillance factors are involved in DSB repair. (A) U2OS cells were mock irradiated (–IR) or irradiated with 10 Gy (+IR) followed by immunofluorescence microscopy using antibodies against the indicated proteins. Scale bar, 20 μm. (B) U2OS cells were transfected with gNSO or gHM1. Laser microirradiation was performed, followed by immunofluorescence microscopy using antibodies against the indicated proteins. Arrowheads indicate laser-irradiated cells. Fluorescence intensities along a white line across a nucleus were measured in arbitrary units. Line-scan profiles of fluorescence intensity are shown to the right. RT–PCR shows HOTAIRM1 knockdown efficiency. Scale bar, 20 μm. (C) The DSB repair assay was performed as in Figure 5B. HeLa cells were transfected with the Cas9/sgHPRT vector, Ins and siRNA as indicated. Genomic DNA was collected at 48 h post-transfection and subjected to qPCR. Bar graph is shown as in Figure 5B (mean ± SD; n = 4; ***P<0.001 for a two-tailed test). (D) Schematic drawing of Cas9/sgHPRT-generated DSBs and production of dilncRNAs in the DSB-flanking regions. HeLa cells were transfected with the Cas9/sgHPRT vector, Ins, siRNA or GapmeR as indicated. RT–qPCR was performed using the primers indicated in the diagram. Bar graphs show relative levels of dilncRNAs (mean ± SD; n = 4; ***P<0.001 for a two-tailed test). (E) HeLa cells were transfected with the Cas9/sgHPRT vector, Ins, siC or siSMG6 together with the empty vector (vec) or the siRNA-resistant wild-type or mutant SMG6 expression vector (WT-res or mtPIN-res). qPCR and immunoblotting were performed as in (C) (mean ± SD; n = 3; P-values for a two-tailed test, **<0.01, ***<0.001). (F) HeLa cells were transfected as in (E). RT–qPCR was performed as in (D) (mean ± SD; n = 4; ***P<0.001 for a two-tailed test).

Figure 8.
Figure 8.

HOTAIRM1 participates in DSB repair via its assocition with DNA repair and NMD factors. Left: a DSB repair model is depicted without HOTAIRM1. Upon DSB induction, the Ku heterodimer forms a complex with DNA-PK, binding to the DNA ends. DNA ligase IV and its cofactor XRCC4 and regulator XLF participate in DNA ligation. This model does not exclude the involvement of RNA. Right: a model shows HOTAIRM1-mediated DSB repair. HOTAIRM1 accumulates at DSBs and is essential for efficient recruitment of NHEJ and NMD factors (Upf1/SMG6) to DSBs and subsequent DSB repair. SMG6 regulates dilncRNA turnover. Depletion of HOTAIRM1 causes dilncRNA accumulation and may inhibit Ku dissociation from DSB sites. The ATM activity is essential for the integrity of the HOTAIRM1 ribonucleoprotein complex and its localization at DSB sites.

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