Nucleolin is required for rRNA transcription and p53 regulation. (A) qPCR for 5?ETS, ITS1, ITS2 and 18S segment of the pre-rRNA in ncl+/+ and ncl?/? zebrafish (n=10 per sample) indicates that rRNA transcripts were significantly lower in ncl?/? embryos compared with their ncl+/+ siblings. canx was used as an internal control. (B) RNA immunoprecipitation (RIP) using a Nucleolin-specific antibody indicates that Nucleolin binds to the 5?ETS and ITS1 region of the 47S rRNA but not to the ITS2 or 18S in wild-type zebrafish (n=100 per replicate and condition). The y-axis indicates fold change of RNA pulldown compared with its absolute expression in the embryos. (C) p53 transcript expression was not significantly altered in ncl?/? mutant zebrafish between 18 and 36?hpf; however, expression of its downstream target p21 was significantly higher between 24 and 30?hpf in the ncl?/? mutants compared with wild-type zebrafish (n=5 per sample). (D) Nucleolin and IgG binding to p53 mRNA was similar in wild-type zebrafish, as observed by RNA immunoprecipitation (n=100 per replicate and condition). The y-axis indicates fold change of RNA pulldown compared with its absolute expression in the embryos. (E) p53 protein levels were higher in ncl?/? mutants at 24?hpf compared with their ncl+/+ siblings and comparable between ncl+/+ and ncl?/? embryos at 36?hpf as observed by western blotting (n=5 per sample). ?-tubulin was used as a loading control. (F) Immunoprecipitation (IP) with a Nucleolin-specific antibody followed by western blotting for p53 and Nucleolin indicates that p53 and Nucleolin bind to each other in wild-type zebrafish (n=25 per replicate and condition). (G) In ncl?/? mutants, Nucleolin expression was significantly reduced compared with controls (n=25 per replicate). ?-tubulin was used as a loading control. (H) At 28?hpf, control zebrafish displayed higher binding of Mdm2 and p53 compared with that seen in mutant zebrafish (n=25 per replicate and condition). (I) Quantification of p53 protein levels in 24?hpf and 36?hpf ncl+/+ and ncl?/? embryos (n=3). (J) Quantification of p53-Mdm2 binding in ncl+/+ and ncl?/? embryos (n=3). (K-L?) ncl?/? mutants have more TUNEL+ cells (red in K,L; white in K?,L?) at 24?hpf compared with their ncl+/+ siblings (n=15 per genotype). (M-N?) By 36?hpf, apoptosis (red in M,N; white in M?,N?) is confined to the midbrain-hindbrain boundary in both ncl+/+ and ncl?/? embryos (n=15 per genotype). (O-P?) On a p53?/? mutant background, the number of TUNEL+ cells (red in O,P; white in O?,P?) in both ncl+/+ and ncl?/? embryos (n=15 per genotype) at 24?hpf is reduced. All experiments were performed three times. Scale bars: 70?µm. Data are represented as meanħs.d. in A-D; circles and squares represent individual data points and horizontal lines represent the mean in I,J. ns, not significant; *P<0.05 (two-tailed, paired Student's t-test).
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