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Fig. 5 | Genome Biology

Fig. 5

From: A novel antiviral lncRNA, EDAL, shields a T309 O-GlcNAcylation site to promote EZH2 lysosomal degradation

Fig. 5

The 56-nt portion of EDAL in 5′ end carries the antiviral function. a EDAL secondary structure was predicted by RNAstructure Version 5.8 software (https://rnastructure.software.informer.com/). EDAL was divided into four sections based on sub-structures: EDAL-1 (1–304 nt), EDAL-2 (305–764 nt), EDAL-3 (765–1258 nt), and EDAL-4 (1259–1564 nt). b The full-length EDAL and its truncations were separately transfected into N2a cells for 48 h. The EZH2 and H3K27me3 levels were resolved by Western blotting and the ratio normalized to H3 was calculated. c The full-length EDAL and its truncations were expressed in N2a cells for 12 h and then the cells were infected with RABV at MOI 0.01. At 48 hpi, the virus titers in the cell supernatant were measured. d, e Four sections within EDAL-1 were selected based on the secondary structures (d). The four truncations EDAL-1 deleting 1–43 nt (EDAL-1 ∆1–43), 98–153 nt (EDAL-1 ∆98–153), 160–180 nt (EDAL-1 ∆160–180), and 207–303 nt (EDAL-1 ∆207–303) were cloned into pcDNA3.1, respectively. The different truncations as well as full-length EDAL-1 were overexpressed in N2a cells for 48 h. Then EZH2 and H3K27me3 levels were resolved by Western blotting and normalized to H3 (e). f N2a cells were transfected with pcDNA3.1, pcDNA-EDAL-1, or different truncations of EDAL-1 for 12 h. Then the cells were infected with RABV at MOI 0.01 and the virus titers in supernatant were measured at 48 hpi. g, h The functional domain (FD) of the 56-nt portion of EDAL was cloned into pcDNA3.1 or fused with 3′ end of the other three control lncRNAs (g). Then these lncRNAs were transfected together with pCAGGS-EZH2-flag into N2a cells for 48 h. EZH2 and H3K27me3 levels were analyzed by Western blotting and normalized to H3 (h). i N2a cells were transfected with pcDNA3.1, pcDNA-EDAL, or different recombinant lncRNAs for 12 h. Then the cells were infected with RABV at MOI 0.01 and the virus titers in supernatant were measured at 48 hpi. Statistical analysis of grouped comparisons was carried out by Student’s t test(**P < 0.01; ***P < 0.001). Bar graph represents means ± SD, n = 3. Western blot data are representative of at least two independent experiments

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