CRY1 Lysine 151 Regulates Circadian Rhythms Through Ubiquitination-Independent Protein Interactions

Post-translational modifications (PTMs) of CRY1 are critical for mammalian circadian clock function. However, the roles of specific ubiquitinated or acetylated lysine residues—and their acute effects on circadian rhythms—remain poorly characterized. To investigate the functional significance of CRY1 lysine 151 (K151), we leveraged a circadian rescue assay in Cry1-/-Cry2-/- (DKO) cells, wherein transfection of the native-promoter-driven CRY1 construct [P(Cry1)-CRY1] restores rhythmicity, as previously described [16]. We hypothesized that K151 undergoes regulatory ubiquitination. To test this, we generated deubiquitination-mimetic mutants (K151R and K151Q) via site-directed mutagenesis, substituting lysine (K) with arginine (R) or glutamine (Q). These mutants (or wild-type [WT] CRY1) were co-transfected with a Per2-promoter-driven luciferase reporter (containing E-box elements) into DKO cells (Figure 1A,B). The results showed that wild-type (WT) CRY1 rescued circadian rhythms within a period of ~24 h (Figure 1C), which was consistent with our previous study [16]. Surprisingly, both K151 mutants markedly shortened the circadian period: K151Q: −2.25 h (n = 3, *** p < 0.001 vs. WT, Student’s t-test); K151R: −1.4 h (n = 3,** p < 0.01 vs. WT, Student’s t-test) (Figure 1C). These results establish K151 as a key residue governing circadian periodicity in mammals.

Given the established role of ubiquitination in regulating core clock protein stability, we examined whether deubiquitination-mimetic mutations at CRY1 lysine 151 (K151R/Q) alter the CRY1 half-life (t_1/2), as described in Ref. [30]. We monitored the degradation kinetics of C-terminal luciferase-tagged CRY1 variants (CRY1::LUC) in HEK293T cells following protein synthesis inhibition with cycloheximide (CHX). The luciferase activity was monitored in living cells within 6 h to determine the t_1/2 of the proteins. The results revealed that while all CRY1::LUC fusions degraded faster than luciferase alone (Figure 2A), the K151Q and K151R mutants exhibited prolonged half-lives compared to WT CRY1 (** p < 0.01 and * p < 0.05, respectively, n = 3, Student’s t-test, Figure 2B). We further confirmed this stability phenotype using Flag-tagged constructs. After the transfection of HEK293T cells with Flag-tagged CRY1 (WT or mutant) plasmids, cells were treated with CHX and harvested at the 3rd, 6th, and 9th h. Western blot analysis of CHX-treated samples demonstrated the enhanced stability of both mutants at the 6 h time point relative to WT (n = 3, p < 0.01 by two-way ANOVA), though protein levels converged to comparable amounts by 9 h (Figure 2C,D).

To determine whether degradation involved proteasomal pathways, we treated transfected cells with the proteasome inhibitor MG132. This intervention completely blocked the degradation of both WT and mutant CRY1 proteins (n = 3, ** p < 0.01, *** p < 0.001 by Student’s t-test, Figure 2E,F). Crucially, the degradation kinetics remained indistinguishable between genotypes in the absence of MG132 (Figure 2F), demonstrating that proteasome-dependent turnover occurs independently of K151 modification status. Collectively, these data indicate that while K151 mutations transiently stabilize CRY1, they do not alter its fundamental dependence on proteasomal degradation.

Building upon the distinct regulatory roles of SCF ubiquitin ligase complexes—where SCF-FBXL3 mediates CRY degradation while its paralog FBXL21 stabilizes nuclear CRY—we investigated whether K151 mutations alter CRY1’s engagement with these E3 ligases. To achieve this, WT or CRY1 mutants and FBXL3 (or FBXL21) were co-expressed as fusion proteins with N- and C-terminal luciferase fragments in HEK293T cells. We observed that K151Q and K151R mutants maintained wild-type binding affinity for FBXL3 but exhibited a stronger interaction with FBXL21 (n = 9, ** p < 0.01 by Student’s t-test, Figure 3A), which was confirmed with the co-immunoprecipitation of CRY1-WT and K151Q/R with FBXL21 proteins (Figure 3B). Given FBXL21’s compartment-specific functions, we examined whether K151 mutations affect CRY1 localization. Fluorescence imaging of GFP-tagged CRY1 variants in U2OS cells revealed comparable nuclear enrichment across all genotypes (Figure 3C), indicating that the subcellular distribution remains unaltered by K151 substitutions. This conserved nuclear retention, coupled with enhanced FBXL21 binding (Figure 2B), provides a mechanistic rationale for the observed stabilization of K151 mutants.

To functionally validate these interactions, we tracked the degradation kinetics of GFP-CRY1 variants co-expressed with FBXL3 or FBXL21 in CHX-treated cells. Time-lapse imaging demonstrated three key outcomes: First, FBXL21 exerted its expected stabilizing effect on all CRY1 forms (n = 3, * p < 0.05, *** p < 0.001 by two-way ANOVA, Figure 3D). Second, FBXL3 comparably accelerated the degradation of wild-type and mutant CRY1 (n = 3, *** p < 0.001 by Two-way ANOVA, Figure 3E). Third, K151 mutants displayed substantially prolonged half-lives relative to wild-type, specifically when co-expressed with FBXL21 (n = 3, * p < 0.05 by Student’s t-test, Figure 3F). Crucially, the degradation kinetics mediated by FBXL3 remained unaltered by K151 mutations. These data collectively establish that K151 modulates CRY1 stability through preferential engagement with the stabilizing factor FBXL21 rather than through altered ubiquitination by the canonical SCF-FBXL3 degradation machinery.

To assess the functional consequences of K151 mutations on molecular clock regulation, we first measured CRY1’s transcriptional repression capacity using E-box-driven luciferase assays in HEK293T cells. The co-expression of CLOCK/BMAL1 activated luciferase expression, while wild-type (WT) CRY1 under CMV promoter control [P(CMV)] suppressed this activity to ~20% of the baseline (Figure 4A). Paradoxically, despite their enhanced protein stability (Figure 2B), both K151R and K151Q mutants exhibited substantially attenuated repression (n = 3; ** p < 0.01 by Student’s t-test), approximately 78% and 64% of that of the wild type (Figure 4A,B).

Given CRY1’s established role in inhibiting CLOCK/BMAL1-DNA binding through direct interactions with BMAL1 [31], we hypothesized that K151 mutations compromise these core clock protein engagements. Luciferase complementation assays confirmed substantially weakened binding affinity between K151Q/R mutants and BMAL1, and the binding rate was approximately 50–60% of the wild type (n = 3; ** p < 0.01, *** p < 0.001 by Student’s t-test, Figure 4C). In addition, the binding affinity between K151Q/R mutants and PER2 was approximately 87% of the wild type, while there was no statistically significant reduction in PER2 associated with K151R (Figure 4D). We further detected the interaction between CRY1 (WT or mutant) and BMAL1 through the co-immunoprecipitation assay (Figure 4E), the results of which displayed a similar binding affinity, as shown in Figure 4D. Crucially, when testing the effects on the CLOCK-BMAL1 heterodimer interface, both WT and mutant CRY1 comparably reduced CLOCK-BMAL1 binding, indicating the preserved disruption of activator complex assembly (n = 3; ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Student’s t-test, Figure 4F). These collective findings demonstrate that residue K151 governs circadian periodicity primarily by modulating CRY1’s interactions with transcriptional regulators BMAL1, rather than through altered heterodimer dissociation.

CRY1/2 double-knockout (DKO) cells were generously provided by the laboratory of Dr. Erquan Zhang at the Beijing Institute of Life Sciences (Beijing, China). The HEK293T (Cat# CL-0005) and U2OS (Cat# CL-0236) cell lines were commercially obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China). All cell lines were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Beijing, China; Cat# C11995500BT) supplemented with 10% fetal bovine serum (FBS; ExCell Bio, Suzhou, China; Cat# FSP500) and 100 U/mL of penicillin–streptomycin (BIOEXPLORER, Guangzhou, China; Cat# B1351-101) under standard culture conditions (37 °C, 5% CO₂) [37].

Real-time circadian reporter assays was performed as previously described [16]. CRY1/2 double-knockout (DKO) cells were plated in 35 mm dishes (NEST, Wuxi, China; Cat# 706001) (3–5 × 10⁴ cells/dish) and cultured overnight. Cells were co-transfected with 1 μg of pGL3-P(Per2)-luc reporter, 50 ng of CRY1 plasmid, and 950 ng of pcDNA3.1 (normalization control) using X-tremeGENE HP DNA Transfection Reagent (Roche, Mannheim, Germany; Cat# 0636623600). Three days after transfection, the cells were treated with 0.1 mM of dexamethasone (DEX; Sigma, Saint Louis, MO, USA, Cat# D4902) for 2 h for synchronization and then switched to XM medium [38] for bioluminescence recording in a LumiCycle (36 °C), as previously described [16].

U2OS cells were seeded at a density of 2–4 × 10⁵ cells per well in a 12-well plate and cultured at 37 °C in a 5% CO₂ incubator for 24 h. For transfection, 2 μg of GFP-tagged CRY1 plasmid (wild-type or mutant) was mixed with Highgene Transfection Reagent (ABclonal, Wuhan, China; Cat# RM09014) and then added dropwise to the cells. Then, 24 h after transfection, the cells were fixed with 4% paraformaldehyde (PFA, Biosharp, Hefei, China; Cat# BL539A) at room temperature for 15 min. The fixative was then removed and the cells were washed three times with PBS (BIOEXPLORER, Guangzhou, China; Cat# B1139-066). For nuclear staining, 100 μL of Hoechst 33,258 (Biosharp, China; Cat# BL803A) solution was added to each well and incubated at room temperature for 10 min in the dark. The staining solution was discarded, and the cells were washed three times with PBS. Finally, the samples were observed using a Nikon ECLIPSE Ti2 microscope (Nikon, Tokyo, Japan) with 20×/0.45 objectives, and images were processed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to analyze the subcellular localization of mCRY1.

To interrogate direct protein–protein interactions, we implemented a split-luciferase reporter system based on functional complementation [16,29,39]. The N-terminal luciferase fragment (NLuc) was fused to CRY1 (wild-type or mutant), while the C-terminal fragment (CLuc) was fused to the interactional paternal, including FBXL3, FBXL21, BMAL1, and PER2. Cells were co-transfected with NLuc-mCRY1 and CLuc-fusion constructs using HighGene Transfection Reagent. Then, 24 h post-transfection, the cells were changed to DMEM medium containing 100 μM of D-luciferin Potassium Salt (GoldBio, St. Louis, MO, USA; Cat# LUCK), and then bioluminescence signals were recorded using a SpectraMax i3x microplate reader (Molecular Devices, Shanghai, China) to quantify the protein interactions.

HEK293 cells were seeded in 96-well white plates at 1–3 × 10⁴ cells/well in DMEM supplemented with 10% FBS and transfected using HighGene Transfection Reagent with 6 ng of luciferase reporter, as described previously [16], plus core clock components (5 ng of CRY1 expression plasmid, 10 ng of BMAL1, and 15 ng of CLOCK), adjusting to 200 ng of total DNA/well with pcDNA3.1. Bioluminescence signals were recorded 24 h post-transfection, as described in Section 4.4.

To evaluate the protein stability of CRY1, we employed a GFP-based degradation assay. HEK293T cells were transiently transfected with plasmids encoding GFP-tagged CRY1 (wild-type or mutant variants). Then, 24 h post-transfection, the cells were treated with 100 μg/mL of cycloheximide (CHX; MedChem Express, Shanghai, China; Cat# HY-12320) to block new protein synthesis. The fluorescence intensity was monitored at 2 h intervals over an 8 h period using a Nikon ECLIPSE Ti2 inverted microscope equipped with a 10×/0.45 objective lens. All images were acquired under consistent exposure conditions and subsequently quantified using ImageJ software to determine the relative protein degradation rates.

We used RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China; Cat# P0013B) supplemented with a complete protease inhibitor cocktail (Roche, Shanghai, China; Cat# 4693116001). Protein concentrations were determined by the BCA assay (Thermo Fisher Scientific, Waltham, MA, USA; Cat# 23225), with equal amounts (20 μg/lane) resolved by 10% SDS-PAGE and electro-transferred to PVDF membranes (Millipore, Burlington, MA, USA; Cat# IPVH00010). After blocking with 5% non-fat milk in TBST, membranes were incubated with primary antibodies overnight at 4 °C, followed by appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized by a Vilber FUSION FX7 chemiluminescence imager (Vilber, Eberhardzell, Germany) and quantified using ImageJ software. The following antibodies were used for protein detection: anti-GAPDH (Proteintech Group, Wuhan, China; Cat#60004-1-Ig), anti-FLAG (MBL, Beijing, China; Cat# M185-3L), and anti-HA (Huaxingbio, Beijing, China; Cat# HX1820). The secondary antibodies used include goat anti-mouse IgG-HRP (Proteintech Group, Wuhan, China; Cat# SA00001-1) and goat anti-rabbit IgG-HRP (Huaxingbio, Beijing, China; Cat# HX2031). The Flag-tagged CRY1 plasmids (wild-type or mutant) were transfected into HEK293T cells. Then, 24 h post-transfection, the cells were treated with 100 μg/mL of CHX and 20 μM of MG132 (MedChem Express, Shanghai, China; Cat# HY-13259) to block protein synthesis and degradation, respectively. Cells were harvested at 0, 3, 6, and 9 h after the CHX treatment to assess protein stability over time.

HEK293T cells were transiently transfected with luciferase-tagged CRY1 (LUC-CRY1) plasmid (wild-type or mutant variants). Then, 24 h post-transfection, protein synthesis was inhibited by treatment with CHX in fresh medium containing 100 µM of D-luciferin. Bioluminescence was monitored at 30 min at 36 °C intervals for 8 h using a SpectraMax i3x microplate reader (Molecular Devices). t_1/2 of protein was calculated via the one-phase exponential decay fitting function in GraphPad PRISM (version 9.00, GraphPad Software, Inc., San Diego, CA, USA) [30].

HEK293T cells were co-transfected with FlAG-CRY1, HA-tagged FBXL21, or BMAL1 expression plasmids. Then, 48 h post-transfection, the cells were treated with 20 μM of MG132 (proteasome inhibitor) for 6 h prior to harvest. The cells were lysed in ice-cold buffer containing 25 mM of Tris-HCl (pH 7.6), 150 mM of NaCl, 1% NP-40, and 0.5 mM of EDTA, supplemented with a protease inhibitor cocktail. For immunoprecipitation, cell lysates were incubated with anti-FlAG antibody at 4 °C overnight, followed by the addition of 40 μL of protein A/G magnetic beads (Beyotime Biotechnology, Shanghai, China; Cat# P2108) for 3 h at 4 °C. The immunocomplexes were washed five times with lysis buffer before elution in 1× SDS loading buffer. Precipitated proteins were resolved by SDS-PAGE and analyzed via immunoblotting using anti-HA and anti-FlAG antibodies.

In all experiments, unless noted, error bars represent SEM. The numbers of repeats and statistical tests used are indicated in the figure legends. Statistical analyses were performed using GraphPad PRISM (version 9.00, GraphPad Software, Inc., San Diego, CA, USA). Comparisons between two groups were made using Student’s unpaired t-test. Half-lives were calculated using a one-phase exponential decay function and then analyzed with Student’s t-test. Comparisons between WT and mutants for CHX-chase assay in Section 4.6, Section 4.7 and Section 4.8 were performed using two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.