Fig 1: ZEB1-AS1 directly interacts with LRPPRC in ECs. (A). Nuclear fraction experiment and qRT-PCR detected the abundance of ZEB1-AS1 in the nucleus and cytoplasm. GAPDH was set as the positive control for cytoplasm, and U1 served as the positive controls for the nucleus. (B) The subcellular distribution of ZEB1-AS1 was visualized by RNA Fluorescent in situ hybridization (FISH) in HUVECs. ZEB1-AS1 was mainly distributed in cytoplasm of HUVECs. (C). Prediction of 421–630 nt ZEB1-AS1 structure was based on minimum free energy (MFE) and partition function (http://rna.tbi.univie.ac.at/). (D) RNA pulldown assay was performed using ZEB1-AS1 incubated with cytoplasm extracts of HUVECs, followed by silver staining. An arrow indicates LRPPRC. (E) Verification of LRPPRC protein by using mass spectrometry. (F) The subcellular distribution of LRPPRC (cytoplasm) was visualized by immunofluorescence in HUVECs. (G) The interaction between ZEB1-AS1 and LRPPRC was confirmed by RNA pulldown and western blotting. GAPDH served as negative controls. (H) RIP was performed using anti-LRPPRC and control IgG antibodies, followed by qRT-PCR to examine the enrichment of ZEB1-AS1 and U6. U6 served as negative controls. **P < 0.01 compared respective IgG group (Mann–Whitney U-test).
Fig 2: ZEB1-AS1 regulates mRNA stability of NOD2 via recruiting LRPPRC. (A) LRPPRC was silenced by specific siRNAs at both transcript (down panel) and protein (upper panel) levels, ∗∗P < 0.01 (Mann–Whitney U-test). (B) NOD2 expression was detected in HUVECs overexpressed with ZEB1-AS1 and/or silenced with LRPPRC, ∗P < 0.05 (Mann–Whitney U-test). (C) HUVECs lysates were incubated with in vitro synthesized, biotin-labeled control LacZ DNA probes or DNA probes against ZEB1-AS1 for the biotinylated oligonucleotide pulldown assay. The precipitates from the pulldown were analyzed by qRT-PCR to detect the interacting mRNAs. ∗∗P < 0.01 (Mann–Whitney U-test) compared to respective LacZ probes. (D) RIP was performed using anti-LRPPRC and control IgG antibodies, followed by qRT-PCR to examine the enrichment of NOD2 and U6. U6 served as negative controls. ∗∗P < 0.01 (Mann–Whitney U-test) compared to respective IgG controls. (E) HUVECs expressing si-ZEB1-AS1#2 or si-LRPPRC were treated with actinomycin D (5 μg/mL) for the indicated periods of time. ∗P < 0.05 (Mann–Whitney U-test) compared to si-NC group (F). HUVECs expressing si-NC, or p-ZEB1-AS1, or p-ZEB1-AS1 + si-LRPPRC were treated with actinomycin D (5 μg/mL) for the indicated periods of time. Total RNA was purified and then analyzed using qRT-PCR to examine the mRNA half-life of NOD2. ∗P < 0.05 (Mann–Whitney U-test) compared to si-NC group, and #P < 0.05 (Mann–Whitney U-test) compared to p-ZEB1-AS1 group.
Fig 3: ZEB1-AS1 modulates ox-LDL-induced ECs injury by stabilization of NOD2 mRNA. (A,B) HUVECs (treated with ox-LDL or not) were transfected with si-NC or si-LRPPRC. Cell viability (A) and LDH release (B) were detected were measured by MTT assay and flow cytometry, respectively, *P < 0.05 (Mann–Whitney U-test). (C,D) Cell viability (C) and apoptosis (D) were evaluated by MTT assay and flow cytometry assay, respectively, *P < 0.05 (Mann–Whitney U-test). (E) A scheme of the proposed mechanisms. ox-LDL treatment increases ZEB1-AS1 expression by sequestering p53 from binding to the promoter region of ZEB1-AS1 gene, which activates the transcription and upregulates ZEB1-AS1 level in HUVECs. Overexpression of ZEB1-AS1 upregulates NOD2 level by guiding LRPPRC to stabilize its mRNA, resulting in decreased viability and increased apoptosis of ECs.
Fig 4: SINEs inhibit the nuclear export of Cas9 mRNA in an XPO1-dependent manner.a KPT330 reduces cytoplasmic Cas9 mRNA, as determined by the ratio of cytoplasmic Cas9 mRNA to total Cas9 mRNA using RT-qPCR. Significant difference is determined using Student’s t-test. b SINEs reduce cytoplasmic Cas9 mRNA in a dose-dependent manner, as determined by RT-qPCR. mRNA expression is normalized to drug-free group. a, b ß-actin is used as an internal control. c Screening siRNA screen for XPO1 knockdown. d Evaluation of the efficiencies of siRNA knockdown of XPO1 mRNA expression in the absence and presence of 0.5 µM KPT330, as determined by RT-qPCR. e The effects of XPO1 knockdown on Cas9 mRNA transport in the absence and presence of 0.5 µM KPT330. d, e Significant difference between sham and XPO1 knockdown is determined using Student’s t-test. f RIP experiment showing HuR and LRPPRC binding with Cas9 mRNA. g The effects of KPT330 treatment on the expression of XPO1, HuR and LRPPRC proteins. h Cartoon illustrating SINE-mediated modulation of Cas9 mRNA transport. The above data are shown as mean ± SD (n = 2 or 3 biologically independent samples).
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