Fig. 1 ∣. NMD targets encode neuronal cell proteins that function in diverse neurogenic programs.

a, Venn diagram defining SH-SY5Y NMD targets based on upregulation by UPF1 siRNA (UPF1 KD) relative to control siRNA (log₂[fold change] > 0 and control siRNA reads per kilobase of transcript, per million mapped reads >1) and increased abundance in p-UPF1 immunoprecipitations (IPs) relative to both the input sample and rabbit IgG immunoprecipitations (log₂[fold change] > 1 and reads in p-UPF1 immunoprecipitation > 10). n represents the number of mRNAs. See also Supplementary Table 1. b, Gene Ontology term enrichment analysis using defined NMD targets. Only the top ten ranked functional terms are shown. P values were calculated using Fisher’s exact test. c, Western blots of lysates of SH-SY5Y cells transiently transfected with control siRNA or two different UPF1 siRNAs. Here and elsewhere, the left-most lanes under the wedge analyse serial threefold dilutions of the lysate. The results represent three independent biological replicates. Ctl, control. d, Histogram of the RT-qPCR quantitations of the specified mRNAs, each normalized to the level of its pre-mRNA, using lysates from siRNA-transfected SH-SY5Y cells. The results are presented as means with s.d. (n = 3 independent biological replicates). P values pertain to comparisons with control siRNA-transfected samples (*P < 0.05; **P < 0.01; ***P < 0.001) and were determined by two-sided t-test. e, RT-qPCR quantitations of the specified NMD targets, normalized to quantitations of GAPDH mRNA, in SY-SY5Y cells transiently transfected with the specified siRNA and cultured 3 d later in the presence (5 μg ml⁻¹) of the transcriptional inhibitor actinomycin D. Note the biphasic nature of the NMD target decay curves, where NMD is largely confined to newly synthesized mRNA. All of the results are shown as means and s.d. values (n = 3 independent biological replicates at each time point). Statistical source data and unprocessed blots are provided online.