Fig. 2: UPF1 posttranscriptionally regulates lncRNA-HEIH expression.

a UPF1 binding sites on lncRNA-HEIH, positive (GADD45B), and negative control (lnc-VPS36-3) using public UPF1 CLIP-seq datasets were visualized. S1, S2, and S3 in lncRNA-HEIH indicate the relative position. b UPF1-bound lncRNA-HEIH in Huh7 cells was analyzed by immunoprecipitation (IP) using an anti-UPF1 antibody. IP eluates were analyzed by WB and RT‒qPCR analysis. c Endogenous lncRNA-HEIH in Huh7 cells was pulled down by antisense oligonucleotide (ASO) or sense oligonucleotide as a negative control. WB was performed to evaluate lncRNA-HEIH-bound proteins. d The relative lncRNA-HEIH levels were measured by RT‒qPCR after transfection of the S2 region of lncRNA-HEIH (lncRNA-HEIH-S2) in Huh7 cells. e Schematic representation of a dual-luciferase construct containing putative WT or Mut UPF1 binding sites from 628 to 746 in lncRNA-HEIH. The two putative wild-type (WT) UPF1-binding sequences (blue bold) in the S2 region, which were mutated to an AT-rich sequence (red bold, Mut), are indicated. f WT or Mut UPF1 binding site-expressing reporter plasmids in (e) were cotransfected with siUPF1 or control siRNA in Huh7 cells. The relative amount of FLuc mRNA normalized to the level of RLuc mRNA was measured by RT‒qPCR. g Schematic representation of lncRNA-HEIH. The full-length lncRNA-HEIH was divided into three regions (S1, S2, and S3). The indicated sequences (WT and Mut) were the same as in (e). h Huh7 cell lysates that were cotransfected with WT or Mut lncRNA-HEIH and MUP plasmid as a transfection control were employed for IP against anti-UPF1 antibody. WB and RT‒qPCR were performed to evaluate the indicated proteins and coimmunoprecipitated lncRNA-HEIH with UPF1. NRS; normal rabbit serum. i Similar to (h), however, samples were from cells transfected with WT or Mut lncRNA-HEIH-S2 plasmid. j Purified GST-UPF1 and in vitro transcribed WT or Mut lncRNA-HEIH-S2 were employed for the in vitro biotinylated RNA binding assay. RNA-bound GST-UPF1 and biotinylated RNA were observed by WB and semi RT‒qPCR (RT-sqPCR), respectively. WT or Mut lncRNA-HEIH plasmids were transfected into UPF1-depleted (k) or UPF1-overexpressing (l) Huh7 cells. The relative level of mRNA was normalized to that of GAPDH mRNA (d, f) or MUP mRNA (h, i). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns not significant.