CRISPR-Cas9-mediated knockout and knockin: - For 3BΔ2BD, two pX330 plasmids carrying two guide RNA sequences were co-transfected into HCT116 cells using JetOptimus (PolyPlus) as described above. After 2–3 weeks, single clones were isolated and screened for genomic deletion. - For resistance marker-based knockouts, donor plasmids carrying antibiotic resistance genes and homology arms along with pX330 plasmid expressing guide RNAs that targeted Cas9 close to the insert site were co-transfected using JetOptimus (PolyPlus). - For MYC-UPF2, we were unable to achieve efficient knock-in without selection. We used a resistance marker-based knock-in approach where a donor plasmid carrying hygromycin resistance marker-P2A-MYC tag in frame with UPF2 ORF was co-transfected with pX330 expressing guide RNA targeting a site close to the UPF2 start codon. Hygromycin resistant clones were isolated and screened for correct insert. All DNA sequences edited via CRISPR-Cas9 were confirmed by Sanger sequencing. For pulse-chase assays, 75,000 HeLa Tet-off cells were plated in each well of a 12-well plate. After 24 h, reporter mRNA and protein expression plasmids were transfected using JetPrime, following manufacturer’s protocol (using 3:1 ratio of reagent to µg DNA). Cells were transfected with 200 ng pTet2 β39 plasmid, 20 ng β-GAP internal control, 10 ng pcDNA3ez-YFP, and 20 ng pcDNA3ez-FLAG-UPF3A/B. 100 ng/ml Tetracycline was used to suppress β39 expression. After 24 h, tetracycline was removed to induce β39 expression overnight (~16 h). Tetracycline (1 µg/ml) was added, and cells were harvested in 0.5 ml TRIzol at indicated time points. For steady state assays, cells were transfected and induced in the same way as the reporter decay assay. At the day of harvesting, 1 µg/ml of tetracycline was added to all cells and cells are harvested 4–6 h post transcription shut-off. RNA was extracted as above and Northern blotting was performed as described previously.