Fig 2: UPF1 phosphorylation is necessary for the regulation of lncRNA-HEIH via SMG5.a The indicated NMD factors were depleted by siRNA in Huh7 cells. b Huh7 cells treated with 100 nM okadaic acid were employed for RT‒qPCR. c The UPF1 variants (MYC-UPF1-12ST > A and MYC-UPF1-8ST > A), which contained twelve or eight serines/threonine to alanine mutations, increased the level of unphosphorylated UPF1. The level of target mRNA detected by RT‒qPCR was normalized to that of GAPDH mRNA (a–c). PC positive control, NC negative control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant.

Fig 3: ZIKV infection disrupts the NMD pathway. (a) Transcript levels of NMD substrates and a housekeeping gene control from Huh7 cells or NPCs mock infected or infected with ZIKV strain P6-740 or the contemporary clinical isolate PRVABC59. Cells were infected at a multiplicity of infection (MOI) of 0.1 or 1 and harvested at 48 hours postinfection (hpi). Data are represented as means plus standard errors of the means (SEM) (error bars). Statistical significance (and P values) were calculated by unpaired Student’s t test and indicated as follows: *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant. Three independent experiments were performed. (b) Venn diagram showing overlap of significantly upregulated genes associated with ZIKV infection of NPCs and UPF1 knockdown (KD) in HeLa cells. RNA-Seq analyses of mock-infected or ZIKV-infected NPCs harvested at 56 hpi and control siRNA-treated or UPF1 siRNA-treated HeLa TO cells harvested at 72 h posttransfection (hpt). The GeneProf hypergeometric probability calculator was then used to generate a hypergeometric P value. ****, P ≤ 0.0001. (c) Transcript levels of housekeeping genes and select genes involved in cell cycle arrest and apoptosis that were identified in panel b. Huh7 cells were mock infected or infected with ZIKV PRVABC59 at an MOI of 0.1 or 1 and harvested at 48 hpi. Data are represented as means plus SEM. The P values were calculated by unpaired Student’s t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant. n = 3 independent experiments.

Fig 4: UPF1-host transcript interactions are decreased during ZIKV infection of NPCs.A) irCLIP schematic describing the workflow to obtain sequencing data. After 48 hrs of ZIKV infection, UPF1 and RNA are crosslinked using UV light, followed by UPF1 pulldown and protease degradation to expose the UPF1-bound RNA for sequencing. B) Metagene profile of all transcripts sequenced from the irCLIP experiment. The graphs show Reads Per Kilobase of transcript, per Million mapped reads (RPKM) values for positions in the Coding Domain Sequence (CDS) and the 3’UTR. Experiment was produced from 2 biological replicates. Representative metagene plots (FREM2, NELL2, POU3F4, MDFIC) of loci found to have a loss of UPF1 interaction. C) A metagene plot of GAPDH and ACTB are shown as controls. D) Metagene plot of reads mapping to the ZIKV genome, with the 3’UTR marked. E) Tabular breakdown of read map percentages from the UPF1-CLIP experiment.

Fig 5: LncRNA-HEIH regulates HCC tumorigenesis.a Volcano plot showing the log2-fold change in expression between (i) UPF1-depleted Huh7 cells and control Huh7 cells performed by Lee et al.17 and (ii) UPF1-depleted HEK-293T cells and control HEK-293T cells performed by Ge et al.23. b Integrative Genomics Viewer (IGV) display of the increased level of lncRNA-HEIH upon UPF1 depletion from RNA sequencing in (a). The levels of lncRNA-HEIH were measured by RT‒qPCR from UPF1-depleted (c) or MYC-UPF1-overexpressing (d) Huh7 cells. WB was performed to evaluate the level of endogenous or exogenous UPF1. The indicated cell lines transfected with lncRNA-HEIH (e) or silncRNA-HEIH (f) were used to measure cell growth over a 96 h period. The relative level of mRNA was normalized to that of GAPDH mRNA in (c, d). g Similar to (f), however, UPF1 was depleted in the presence or absence of lncRNA-HEIH. Cell migration assays (h) and invasion assays (i) were performed using the indicated HCC cell lines transfected with lncRNA-HEIH. Relative migration and invasion rates were quantified. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant.