Skip to main content
. 2022 Apr 11;41(10):e108898. doi: 10.15252/embj.2021108898

Figure EV5. Reduced translation efficiency promotes UPF1LL activity.

Figure EV5

  1. Schematic of the RNA‐seq experimental workflow and conditions for UPF1LL knockdown and puromycin treatment.
  2. Density plot of relative mRNA abundance as determined by RNA‐seq following treatment of HEK‐293 cells with 50 µg/ml puromycin. mRNAs were binned according to destabilization in CLIP‐UPF1LL or CLIP‐UPF1SL overexpression experiments, as determined by REMBRANDTS analysis (Alkallas et al, 2017). Statistical significance was determined by K–S test.
  3. Quantification of UPF1LL isoform expression in control and puromycin‐treated HEK‐293 cells from rMATS analyses (n = 3 biological replicates) (Shen et al, 2014).
  4. RT–qPCR analysis of indicated transcripts following treatment of HEK‐293 cells with indicated concentrations of puromycin for 4 h. Relative fold changes are in reference to vehicle‐treated control. Significance of puromycin treatment on relative transcript abundance was compared to the vehicle‐treated control. Asterisk (*) indicates P < 0.05, as determined by two‐way ANOVA. Black dots represent individual data points, and error bars indicate mean ± SD (n = 3 biological replicates). Dashed lines indicate log2 (fold change) of ± 0.5. PTC+ indicates the use of primers specific to the transcript isoform with a validated poison exon (Lareau et al, 2007; Ni et al, 2007). See also Dataset EV3 for P‐values associated with each statistical comparison.
  5. Density plot of relative mRNA abundance as determined by RNA‐seq following treatment of HEK‐293 cells with 25 µg/ml or 100 µg/ml puromycin. mRNAs were binned according to sensitivity to 50 µg/ml puromycin and UPF1LL knockdown. Statistical significance was determined by K–S test.
  6. Density plot of relative mRNA stability as determined by REMBRANDTS analysis of RNA‐seq following treatment of HEK‐293 cells with 50 µg/ml puromycin for 4 h (Alkallas et al, 2017). mRNAs were binned by changes in relative mRNA abundance in puromycin. Statistical significance was determined by K–S test.
  7. Density plot of relative mRNA stability as determined by REMBRANDTS analysis of RNA‐seq following UPF1LL knockdown in HEK‐293 cells and treatment with 50 µg/ml puromycin for 4 h (Alkallas et al, 2017). mRNAs were binned by changes in relative mRNA abundance in puromycin with UPF1LL knockdown. Statistical significance was determined by K–W test, with Dunn's correction for multiple comparisons.

Source data are available online for this figure.