Fig. 1

NMD is hyperactivated during mouse cortex development. a Venn diagram defining mouse N2A-cell NMD targets based on their upregulation by Upf1 siRNA (UPF1-KD) relative to control siRNA (log₂ fold-change > 0, adjusted P value < 0.05) as determined by RNA-sequencing (RNA-seq), denoted in blue, and their increased immunoprecipitation (IP) using antibody (α) to phosphorylated UPF1 (p-UPF1) and RNA IP (RIP)-seq footprinting relative to size-matched input Neuro2A(N2A) RNA (log₂ fold-change > 1, adjusted P value < 0.05), shown in red. n, number of transcripts. b Western blots of lysates of N2A cells cultured with either control (Ctl) siRNA or Upf1 siRNA used in a. Results represent three independently performed experiments. Here and elsewhere, leftmost lanes under the wedge represent 3-fold serial dilutions to show that results are within the linear range of analysis. c As in b, but before (−) or after IP using α-p-UPF1 or, as a control, rabbit IgG (rIgG). Results represent three independent experiments. d Western blot of cortex from wild-type (WT) mice at the denoted day of embryonic (E) or postnatal (P) development using the specified antibody. Results represent three independent experiments. e As in d, but using Fmr1-knockout (KO) mouse cortex. Results represent three independent experiments. f Line plot of the ratio of UPF1 normalized to GAPDH as quantitated from d and e. Means ± S.D., where n = 3 (WT) and 3 (Fmr1-KO). g As in f, but for the ratio of p-UPF1 normalized to GAPDH. Means ± S.D., where n = 3 (WT) and 3 (Fmr1-KO). (*)P < 0.05 or (**)P < 0.01 compares Fmr1-KO cells relative to WT cells (two-sided unpaired t-test). h Volcano plot of RNA-seq data deriving from 2-day cultures of neurons from WT or Fmr1-KO mouse P1 cortex. All results derive from independently derived samples in triplicate. i Using data from h, cumulative fraction analysis, where the category “Others” consists of cellular transcripts not defined in a as NMD targets. n, number of transcripts. P values were calculated by the two-sided Wilcoxon rank-sum test. j Histogram representation of RT-qPCR demonstrating that NMD is hyperactivated in cultured neurons from the P1 cortex of Fmr1-KO mice. The level of each NMD target was normalized to the level of the pre-mRNA from which it derives, and the normalized level in WT mice is defined as 1. Means with S.D., where n = 3 (WT) and 3 (Fmr1-KO). (*)P < 0.05, (**)P < 0.01 or (***)P < 0.001 compares Fmr1-KO cells relative to WT cells (two-sided unpaired t test)