Fig 1.
PSMD11 regulates cellular circadian clocks, but PSMD4 and PSMD12 do not.
(A), (B), (C) PSMD11, PSMD4 and PSMD12 knockdown cellular circadian profiles. Representative bioluminescence records of circadian rhythms in U2 OS Per2:dLuc reporter cells. n = 3 independent experiments (D) Baseline, amplitude and phase results of A-C that are calculated with WaveClock algorithm. (E) mRNA levels detected with QPCR to check the RNAi knockdown effects. mRNA levels are normalized to GAPDH. Statistical analyses were performed with unpaired t tests. Error bars represent SEM. Representative Western blot images to confirm the protein level effect is shown below for each gene. Stain-free loading controls are shown in S1B Fig. Uncropped controls and images for each Western blot image are represented in ‘S1 Raw images’.
Fig 2.
Proteasomal role of PSMD11 on circadian clock.
(A) Absence of PSMD11 increases PER2 and CRY2 protein levels. PER1, PER2, CRY1 and CRY2 levels are detected with Western blot when PSMD11 is knocked down with an siRNA cocktail and control (siNEG) samples. n = 3, significancy is analyzed with unpaired t tests. (B) PSMD11 mediates regulation of PER2 and CRY2 abundances possibly through FBXL3/FBXL21. PER1, PER2, CRY1, and CRY2 levels are detected with Western blot when FBXL3, FBXL21, and/or PSMD11 is knocked down with an siRNA cocktail. PER2 and CRY2 levels in siPSMD11 (lane 5) are marked with blue squares and their levels when both FBXLs are knocked down along with PSMD11 are marked with green squares (lane 8) for easier comparison. FBXL21 mRNA levels are represented on S2C Fig. Quantification of CRY2 and PER2 on the represented blot is shown on the right. This experiment has one biological replicate. Experiments on this figure are performed in U2 OS cells.
Fig 3.
Temporal regulation of NRON complex at cytoplasmic and nuclear fractions.
(A) PER2, CRY1 and PSMD11 protein levels in whole cell lysates from CT30, CT36, CT42 and CT48. Multiple bands in PER2 blot are caused by hyperphosphorylated PER2 proteins. Stain-free loading control is represented at S3D Fig. Quantifications of three independent experiments are represented on the right. Baseline, amplitude and phase are calculated with cosinor method. P-values for PER2, CRY1 and PSMD11 respectively; 0.06, 0.08, and 0.11 (B) Spatiotemporal analysis of NRON complex components in U2 OS cells. NRON complex component proteins -IQGAP1, KPNB1, CSE1L, CRY2, CRY1, GSK3β, CSNK1ε, and PSMD11- were detected in the fractions 22 to 25 in cytoplasmic and nuclear enriched samples at CT30, CT36, CT42 and CT48 by Western blot. Arrow indicates a non-specific band. Cytoplasmic and nuclear fractions for each protein are run on the same gel. Peak time point of the complex components at each cellular fraction is marked with red asterisks. (C) Quantification of all proteins detected relative to CT30 cytoplasmic fraction from two independent experiments is shown. Baseline, amplitude and phase are calculated with cosinor method. P-values for cytoplasmic fraction and nuclear fraction respectively; 0.019 and 0.0191.
Fig 4.
PSMD11 regulates circadian clock by regulating nuclear transport of the NRON complex.
(A) Indicated proteins are detected in control (siNEG) and siPSMD11 samples in SEC enriched cytoplasmic and nuclear fractions of unsynchronized U2 OS cells. Cytoplasmic and nuclear fractions for each protein are run on the same gel, then cut and pasted together for representation. Arrow indicates a non-specific band. Significantly affected blots by siPSMD11 are marked with blue asterisks on Western blot images. (B) Graphs represent Western blot quantifications of three independent experiments shown in (A). Significancy is analyzed with paired t test. P-values of the significant analyses are displayed on the graph. (C) Pulldown assay using whole cell lysates of U2 OS cells overexpressing PSMD-V5 plasmids or GFP. Pull down experiment repeated two times.
Fig 5.
NRON complex regulates circadian clock through PSMD11 and has a potential to regulate other pathways.
A model to explain the role of the NRON complex at the perinuclear area to transfer proteins between cytoplasm and nucleus. The complex is used by circadian clock and NFAT pathways and potentially is used by different pathways as well. PSMD11 controls the regulation of circadian clock through the complex, other PSMD proteins are suspected to control other pathways as a part of the complex. Created with BioRender.com.