Figure 7. Translational repression promotes the decay of mRNAs by UPF1_LL in the NMD pathway and down‐regulates normally protected transcripts.

1. mRNA decay measurement using Roadblock‐qPCR (Watson et al, 2021). RNA was isolated from HEK‐293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/ml puromycin. mRNA half‐lives were estimated by fitting the data to a single‐phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1_LL was compared to the siNT in the absence and presence of puromycin treatment using the extra‐sum‐of‐squares F test. Error bars indicate mean ± SD (n = 4 biological replicates). See also Dataset EV3 for P values associated with each statistical comparison.
2. RT–qPCR analysis of indicated transcripts following transfection of HEK‐293 cells with indicated siRNAs and treatment with 50 µg/ml puromycin for 4 h. Relative fold changes are in reference to vehicle‐treated, NT siRNA. 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 transcript isoforms with validated poison exons (Lareau et al, 2007; Ni et al, 2007). See also Dataset EV3 for P values associated with each statistical comparison.
3. Density plot of changes in relative mRNA abundance as determined from RNA‐seq following treatment of HEK‐293 cells with 50 µg/ml of puromycin for 4 h. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K–W test, with Dunn's correction for multiple comparisons.
4. Density plot as in (C), following UPF1_LL‐specific knockdown.

Source data are available online for this figure.