Luciferase Reporters for NMD Regulation In Vivo

In order to express luciferase NMD reporters in vivo, HeLa cells (grown to 60–80% confluency) were transfected with the reporter constructs in pcDNA 3.1 (+) vectors (described above). Short hairpin RNA (shRNA)-mediated RNA interference (RNAi) degradation⁵⁵ was applied to knockdown UPF1 as described previously^{35 (link),53 (link),55} . The transfection mix consisted of 20 ng/µl reporter plasmid, 20 ng/µl pSUPuro plasmid (1:1 mixture of pSUPuro UPF1 against two target sequences^{53 (link)}) in Opti-MEM containing 3% (v/v) Dogtor (OZ Biosciences) transfection reagent. As a negative control, a pSUPuro plasmid containing a randomized target sequence was used (pSUPuro Scr). After 12 h the medium was replaced by DMEM +/+ containing 1.5 µg/ml Puromycin (Santa Cruz Biotechnology). The antibiotic selection was carried out for 48 h until the medium was replaced with DMEM +/+ to let the cells recover for approx. 24 h. To obtain a list of NMD-sensitive RNAs 3 × 10⁵ HeLa cells per well were seeded in 6-well plates. Twenty-four hours later, the cells were transfected with 52 pmol of siRNA using Lullaby reagent (OZ Biosciences). After 48 h, the cells were re-transfected as before. Protein and total RNA were isolated after one additional day. The siRNA sequence 5′-GAUGCAGUUCCGCUCCAUU-3′ was used for targeting UPF1.