Fig. 3: Coronin 2B knockdown impairs the formation of the transcription initiation complex.
From: Coronin 2B deficiency induces nucleolar stress and neuronal apoptosis
A Coronin 2B is associated with UBF in vivo. UBF was immunoprecipitated from the nuclear extracts of cortical neurons cultured for 14 days. Western blot analysis was performed to determine the associations of coronin 2B and UBF with their respective antibodies and mouse IgG as a negative control. B, C Coronin 2B interacts with UBF in vitro. Cell lysates from HEK293T cells expressing coronin 2B-HA or pcDNA3.0 vector control were subjected to immunoprecipitation with antibody against HA (B) and UBF (C), respectively. Western blot analysis revealed the reciprocal interaction between UBF and coronin 2B. D–G The formation of the transcription initiation complex is impaired in coronin 2B-deficient cortical neurons. Cortical neurons were cultured for 9 days and then infected with shCoro2B lentivirus or corresponding scramble control. After 5 days, Pol I was immunoprecipitated from the nuclear extracts with an antibody against RPA194. The co-immunoprecipitation of UBF and TAFIp110 was analyzed on immunoblots with respective antibodies. The quantification is shown in (E) and (G) (from three independent experiments, unpaired Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant). H Schematic of rDNA repeats and primers used for chromatin immunoprecipitation (ChIP) assay. H42 is at the promoter; H4, H8, and H13 span the transcription region; and H15, H18, and H34 are at the intergenic spacer regions. I, J The rDNA occupancy of UBF is compromised in coronin 2B-deficient neurons. ChIP assay was performed on cortical neurons transduced with shCoro2B lentivirus or corresponding scramble control. Antibodies against UBF and mouse IgG as a control were used for rDNA immunoprecipitation. Precipitated DNA was assayed by qPCR using the primers shown in (H). The results are presented as the percentage of input values normalized to the control. Data were mean ± SEM (from three independent experiments; Mann–Whitney U-test in (I), unpaired Student’s t-test in (J); *p < 0.05, **p < 0.01, ***p < 0.001).
