Eg5 UFMylation promotes spindle organization during mitosis

Eg5 UFMylation promotes spindle organization during mitosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Eg5 UFMylation promotes spindle organization during mitosis Ranhui Duan, Guangxu Li, Yuanjiang Huang, Liyi Wei, Hongjing Huang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3754446/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2024 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract UFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays critical roles in the maintenance of endoplasmic reticulum (ER) proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in UFM1 , as well as in its specific E1 enzyme UBA5 and E2 enzyme UFC1 , have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective mutant of Eg5 (K564R) displayed shortened or asymmetrical spindles, and suppressed cell proliferation in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression and cell proliferation. Biological sciences/Cell biology/Post-translational modifications Biological sciences/Cell biology/Cell division Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like protein (UBL) that remains less well-understood compared to other UBLs(1). UFM1 is highly conserved across evolutionary lines, with orthologs found in both Metazoa and plants(2). Even rice shares over 66.27% nucleotide sequence similarity with humans. Although UFM1 shares only 16% amino acid sequence identity with ubiquitin, its tertiary structure closely resembles the ubiquitin fold, featuring specific β-sheets and an α-helix(3). The UFM1 precursor consists of 85 amino acids and is processed to its mature form by specific cysteine proteases, UFSP1 and UFSP2, which cleave the C-terminal dipeptide Ser-Cys, exposing the conserved Gly83 conjugating residue(4). The activation of UFM1 involves the UFM1-specific E1 enzyme, UBA5, through processes of adenylation and thioesterification(5). The UFM1-specific E2 enzyme, UFC1, accepts the activated UFM1 in a trans-esterification reaction(6). The UFM1-specific E3 enzyme, UFL1, catalyzes the covalent binding of UFM1 to substrate lysine residues(7). The DDRGK domain-containing protein 1 (DDRGK1, also known as UFBP1) serves as an E3 ligase adaptor, regulating the UFMylation of substrates(8). UFMylation is a reversible process, as UFM1 can be cleaved from its target proteins by UFSP1 and UFSP2(9). UFMylation regulates various cellular functions, such as hematopoiesis, endoplasmic reticulum (ER) proteostasis, DNA damage response, autophagy, transcriptional regulation, and signaling pathways(2, 10–12). In 2016, we reported for the first time that compound heterozygous variants of UBA5 lead to neurodevelopmental disorders characterized by cerebellar atrophy and developmental delays(13). Subsequently, a series of biallelic variants of UBA5 were discovered in patients, causing severe infantile-onset epileptic encephalopathy primarily characterized by microcephaly(14–19). Furthermore, biallelic mutations in UFM1 or UFC1 have revealed similar microcephalic traits(20, 21). Genes implicated in microcephaly typically play key roles during mitosis. Our recent study in Drosophila demonstrate that knockdown of UFMylation results in reduced brain size, partial embryonic lethality before gastrulation, and mitotic defects in spindle assembly and chromosome separation(22). However, the underlying mechanisms remain to be elucidated, due to the limited identification of relevant substrates. Here, we employed mass spectrometry and cross-referenced other reliable proteomic datasets to identify various substrates, among which Kinesin-5 (Eg5, also referred to as KIF11) is included. Functionally, Eg5 acts as a microtubule-oriented motor protein that moves towards the plus-end, facilitating bipolar spindle assembly during mitosis(23). Inhibition of Eg5 leads to a monopolar spindle, causing cell mitotic arrest and subsequent cell death(24). In this study, we confirmed Eg5 as a novel substrate for UFMylation and pinpointed Lys564 as the crucial UFMylation site among its 90 lysine residues. Eg5 directly interacted with the E3 ligases UFL1 and DDRGK1, and exhibited modification by UFM1 both in vivo and in vitro . During mitosis, Eg5 and UFM1 co-localized at the centrosomes and spindle, and defective UFMylation resulted in reduced spindle localization of Eg5. The UFMylation-defective Eg5 mutant (K564R) displayed shortened or asymmetrical spindles during metaphase, and inhibited cell proliferation in HeLa cells. Our findings suggest that Eg5 UFMylation is significant for understanding the pathogenic mechanisms involving cell cycle dysregulation. MATERIALS AND METHODS Cell culture HEK-293T and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), at 37℃ in a 5% CO2 atmosphere. The cells used in this study were verified to be free from mycoplasma contamination, and the identity of HeLa cells was verified through STR profiling. To generate UFSP2 knockout cell lines, the guide RNA targeting UFSP2 (5′-AGCAGTGACATAAACACC-3′) was cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene, Watertown, MA, USA). HEK293T cells were transfected with PX459 and selected with puromycin (1 µg/ml). After selecting for 5 days, cells were cloned using a limiting dilution and screened for knockout by Sanger sequencing and Western blot analysis. siRNA and plasmid transfection The siRNA duplexes were synthesized by GenePharma (Shanghai, China), and the corresponding sequences were as follows: si-NC: UUCUUCGAACGUGUCACGU; si- UBA5 : GGUUAUACAAGAAGAGGAA; si- UFC1 : GAAAGACAGCAAAGAUGUA; si- UFL1 -1: GGAACUUGUUAAUAGCGGA; si- UFL1 -2: GAGGAGUAAUUUUUACGGA; si- DDRGK1 -1: GAAAAUUGGAGCUAAGAAA; si- DDRGK1 -2: CCAUAAAUCGCAUCCAGGA; si- Eg5 -1: CAGAUUGAUGUUUACCGAA; si- Eg5 -2: CUGGAUAUCCCAACAGGUA; si- Eg5 -3: CGAUGAGUUUAGUGUGUAAAG. UBA5 , UFC1 , UFL1 , DDRGK1 and UFM1 cDNAs were inserted into the pRK5-HA vector. UFM1 cDNA variants with two or three amino acids deleted in the C-terminal (UFM1-ΔC2 or UFM1ΔC3) were cloned into both the pRK5-HA vector and the pcDNA3.1-Flag vector. Eg5 cDNA was cloned into the pRK5-Flag vector. Truncations and point mutations of Eg5 were generated using the MutExpress II Fast Mutagenesis Kit V2 (C214-02, Vazyme, Nanjing, China). Bacteria expressing His-tagged UBA5, UFC1, UFL1, DDRGK1, UFM1-ΔC2 and GST-tagged Eg5 were generated using the pET-28a and pGEX-4T-1 systems, respectively. All plasmids were validated through Sanger sequencing. RNA interference was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and plasmid transfection was performed with Lipofectamine 3000 (Invitrogen), following the manufacturer’s instructions. Mass spectrometry UFSP2 knockout HEK293T cells were transfected with Flag-tagged UFM1-ΔC2 and Flag-tagged UFM1ΔC3, respectively. for 36 h. The cells were then lysed with NP-40 lysis buffer containing 50mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 1×protease inhibitor cocktail (P8340, Sigma, St. Louis, MO, USA). Subsequently, Flag-UFM1-ΔC2 or Flag-UFM1ΔC3 was immunoprecipitated by incubation the lysates with anti-FLAG M2 beads (M8823, Sigma) overnight at 4°C. The protein precipitates were subjected to SDS-PAGE and mass spectrometry. Mass spectrometric analysis of peptide samples was carried out using a Q Exactive Mass Spectrometer (Thermo Fisher, Waltham, MA, USA) at Applied Protein Technology Company (Shanghai, China). Western blot Cells were lysed with RIPA lysis buffer consisting of 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1×protease inhibitor cocktail. Whole cell lysates with equal protein were resolved by SDS-PAGE and subsequently transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 1×TBST (TBS with 0.1% Tween 20), supplemented with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4℃ and with the secondary antibodies for 1 h at room temperature. Primary antibodies were as follows: anti-Flag (F1804, 1:5000, Sigma), anti-HA (C29F4, 1:2000, Cell Signaling, Danvers, MA, USA), anti-UFL1 (PA5-56501, Thermo Fisher, 1:1000), anti-DDRGK1 (21445-1-AP, Proteintech, Hubei, China, 1:2000), anti-Eg5 (23333-1-AP Proteintech, 1:2000), anti-UBA5 (ab177478, Abcam, Cambridge, UK, 1:1000), anti-UFC1 (ab189251, Abcam, 1:1000), anti-UFM1 (15883-1-AP, Proteintech, 1:2000), anti-UFSP2 (ab185965, Abcam, 1:1000), anti-His (ab18184, Abcam, 1:5000), anti-α-tubulin (ab18251, Abcam, 1:5000), and anti-GAPDH (10494-1-AP, Proteintech, 1:500). Secondary antibodies were as follows: HRP-conjugated goat anti-mouse IgG (115-035-166, Jackson ImmunoResearch, West Grove, PA, USA, 1:10000) or HRP-conjugated goat anti-rabbit IgG (111-035-144, Jackson ImmunoResearch; 1:10000). The protein bands were visualized using a ECL-chemiluminescent kit . UFMylation assays For the in vivo UFMylation assays, cells transfected with appropriate plasmids were cultured for 36 h. To prepare cell lysates, the cells were boiled in a buffer containing 150 mM Tris (pH 8.0), 5% SDS, and 30% glycerol for 8 min. Subsequently, the lysates were diluted 20-fold with NP-40 lysis buffer and subjected to immunoprecipitation using anti-FLAG M2 beads overnight at 4°C. The samples were then subjected to SDS-PAGE and Western blot analysis. In vitro UFMylation assays were performed following previous reported method. Recombinant GST-Eg5 were expressed in BL21 cells and purified using the GST-tag Protein Purification Kit (P2262, Beyotime, Jiangsu, China). Additionally, His-UBA5, His-UFC1, His-UFL1, His-DDRGK1 and His-UFM1-ΔC2 were expressed in BL21 cells and purified using the His-tag Protein Purification Kit (P2226, Beyotime). His-UFM1-ΔC2 (5 ng), His-UBA5 (5 ng), His-UFC1 (5 ng), His-UFL1 (5 ng), His-DDRGK1 (5 ng) and GST-Eg5 (10 ng) were mixed in the reaction buffer containing 5 mM ATP and 10 mM MgCl 2 , and incubated at 30°C for 90 min. The mixtures were boiled with the addition of loading buffer containing 5% mercaptoethanol for 5 min. Immunoprecipitation and in vitro binding assays For immunoprecipitation, cells were lysed with NP-40 lysis buffer and the lysates were then incubated with 1 µg of the indicated antibodies and 10 µl of Protein A/G Magnetic Beads (B23201, Biomake, Shanghai, China) overnight at 4°C. For the in vitro binding assays, purified His-UFL1 and His-DDRGK1 were incubated with GST or GST-Eg5 in PBS with 0.2% NP-40 for 2 h at 4°C, followed by pull-down with GSH-resin. The samples were subjected to SDS-PAGE and Western blot analysis. 5% of each supernatant was used as input control. Immunofluorescence staining HeLa cells transfected with the indicated siRNAs or plasmids were cultured for 48 h. The cells were fixed in 4% paraformaldehyde for 30 min, washed in 0.2% PBST (PBS with 0.2% Triton X-100), and blocked in 5% normal goat serum for 1 h at room temperature.Cells were incubated overnight at 4°C with primary antibodies: rabbit anti-UFM1 (ab109305, Abcam, 1:200), mouse anti-Eg5 (ab51976, Abcam, 1:500), rabbit anti-Eg5 (23333-1-AP Proteintech, 1:200), rabbit anti-α-tubulin (ab18251, Abcam, 1:500), or mouse anti-γ-tubulin (ab11316, Abcam, 1:500). Secondary antibodies were as follows: Alexa Fluor 488-conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch, 1:200) or Cy3-conjugated goat anti-rabbit IgG (111-165-003, Jackson ImmunoResearch, 1:200). The cells were then stained with DAPI and mounted with Fluoromount mounting medium (F4680, Sigma). The image was acquired using the LSM 880 (Zeiss, Oberkochen, Germany) or the SP8 (Leica, Wetzlar, Germany) confocal system, and fluorescence intensities were calculated using ImageJ software. Cell viability and colony formation assays For the cell viability assays, 5000 HeLa cells were seeded into 96-well plates and transfected with the indicated siRNAs or plasmids. After culturing for 48h, the cells were treated with a CCK-8 solution (C0037, Beyotime) and incubated for 2 h. The absorbance values at a wavelength of 450 nm (OD450) were measured using an elx800 reader (BioTek, Winooski, VT, USA). For the colony formation assays, HeLa cells were placed in 6-well plates at a density of 500 cells per well. Every three days, the cells were transfected with the indicated siRNAs or plasmids. After 10 days of culturing, during which the clones became visible, the cells were fixed using a 4% paraformaldehyde and stained with 0.1% crystal violet. The colonies were then photographed and quantified. Statistical analysis The statistical software GraphPad Prism 8 was used to carry out unpaired Student's t test to compare two specific datasets and one-way ANOVA for multiple comparisons. The data were presented as mean ± SD of three independent replicates. The values of * P < 0.05, ** P < 0.01, and *** P < 0.001 were considered to be statistically significant. RESULTS Identification of candidate substrates for UFMylation To identify target proteins for UFMylation, we overexpressed Flag-tagged mature UFM1 (UFM1-ΔC2) in UFSP2 knockout HEK293T cells to facilitate UFM1 conjugate formation. In addition, we employed a Flag-tagged conjugation-defective UFM1 lacking Gly83 (UFM1-ΔC3) as a negative control (Fig. 1 A). After verifying the expression levels of Flag-UFM1 (Figure S1 ), we immunoprecipitated the cell lysates with anti-Flag M2 beads, and the precipitated proteins were subjected to SDS-PAGE (Fig. 1 B) and mass spectrometry. We screened for UFMylation substrates by identifying proteins that specifically bound to Flag-UFM1-ΔC2 (Supplementary Table 1). Among these candidates, we selected Eg5 for further investigation due to its pivotal role in the dynamic assembly and function of the mitotic spindle through cross-linking and sliding adjacent microtubules(24). Eg5 interacts with UFL1 and DDRGK1 To determine whether Eg5 is a target protein for UFMylation, we examined its ability to interact with UFL1, the UFM1 E3 ligase, and DDRGK1, a critical regulatory factor for UFMylation. Immunoprecipitation analysis demonstrated that Eg5 is capable of binding to both UFL1 and DDRGK1 (Fig. 1 C-E). Moreover, endogenous Eg5, UFL1 and DDRGK1 exhibited mutual interactions in cells (Fig. 1 F, G). Furthermore, in vitro binding assays showed that Eg5 directly interacted with UFL1 and DDRGK 1(Fig. 1 H), suggesting that Eg5 could be a bona fide substrate of UFMylation. Eg5 is a target substrate for UFMylation To confirm whether Eg5 can undergo UFMylation, we co-expressed Flag-Eg5 with the UFMylation components UBA5, UFC1, UFL1, UFM1 and DDRGK1 in HEK293T cells. In vivo UFMylation assays demonstrated that Eg5 can be UFMylated by both the wild-type UFM1 (UFM1-WT) and mature UFM1 (UFM1-ΔC2), but not by the conjugation-defective UFM1 (UFM1-ΔC3) (Fig. 2 A). Additionally, we observed that individually knocking down UBA5, UFC1, UFL1, or DDRGK1 resulted in a decrease in the UFMylation levels of Eg5 in UFSP2 knockout HEK293T cells (Fig. 2 B), indicating their crucial involvement in the Eg5 UFMylation process. Furthermore, the UFMylation assays validated Eg5 as a target substrate for UFMylation in vitro (Fig. 2 C). These findings unequivocally establish Eg5 as a novel target substrate for UFMylation. Identification of the essential UFMylation site in Eg5 To identify the essential sites for Eg5 UFMylation, we employed three deletion constructs (ΔN, ΔM and ΔC) and transfected them into UFSP2 knockout HEK293T cells, along with the HA-UFM1-ΔC2 plasmid. Among these constructs, UFMylation was specifically observed in the ΔM construct of Eg5 (Fig. 3 A). To further investigate the UFMylation sites, we generated additional deletion constructs targeting specific segments within the ΔM region. Notably, the deletion constructs lacking amino acids 552–641 (ΔD) showed a significant reduction in UFMylation levels (Fig. 3 B). As there are six Lys residues in this region, we individually substituted each Lys residues with Arg in wild-type Eg5. Among these substitutions, the replacement of Lys564 by Arg (referred to as K564R) resulted in a diminished Eg5 UFMylation (Fig. 3 C). In addition, the K564R mutation significantly reduced Eg5 UFMylation in vitro (Fig. 3 D). Collectively, these data demonstrated that K564 is a essential UFMylation site in Eg5. UFMylation deficiency leads to decreased monoubiquitination of Eg5 Previous studies have shown that UFMylation is related to substrate stability and ubiquitination(25–27). To determine whether UFMylation influences the stability of Eg5, we suppressed the expression of UFL1 or DDRGK1 in HeLa cells using siRNAs (Figure S2). We treated the cells with cycloheximide and found that Eg5 protein stability remained unaffected after knockdown of UFL1 or DDRGK1 (Fig. 4 A). Interestingly, the monoubiquitination of Eg5 was significantly decreased by the knockdown of UFL1 or DDRGK1 (Fig. 4 B). However, we did not extensively explore how UFMylation of Eg5 affects its monoubiquitination. UFM1 and Eg5 exhibit co-localization at the centrosome and spindle To gain functional insight into the association between UFMylation and Eg5, we investigated the cellular localization of UFM1 and Eg5. Immunofluorescence staining revealed that during interphase, UFM1 was present in both the nucleus and the cytoplasm, while Eg5 predominantly localized in the cytoplasm. As cells entered prophase, UFM1 and Eg5 start to accumulated at the centrosomes, displaying an overlapping pattern. During metaphase, UFM1 and Eg5 were distributed throughout the spindle apparatus. Subsequently, UFM1 and Eg5 were observed at the spindle poles and midbody in anaphase and telophase (Fig. 5 ). The dynamic co-localization of UFM1 and Eg5 throughout different stages of the cell cycle suggests their potential roles in mitotic processes and spindle organization. UFMylation maintains Eg5 interaction with the mitotic spindle The spatial and temporal dynamics of Eg5 are crucial for its motor function in driving centrosome separation, spindle assembly, and chromosome segregation during mitosis(24). Given the co-localization of UFM1 and Eg5 at the centrosome and spindle, we were intrigued to explore whether UFMylation affects the spatial relationship between Eg5 and the mitotic centrosome or spindle. Indeed, knockdown of UFL1 or DDRGK1 had no influence on the localization of Eg5 at the centrosome during prophase (Fig. 4 A, B). However, the knockdown of UFL1 or DDRGK1 lead to a smaller spindle area and a reduction in both sun and mean EG5 intensity at the spindle during metaphase (Fig. 4 C-F). These results suggested that Eg5 UFMylation is crucial for maintaining its interaction with the mitotic spindle. UFMylation promotes spindle organization and cell proliferation Given the crucial role of Eg5 in mitotic spindle assembly, we proceeded to explore the effects of Eg5 UFMylation on mitotic spindle morphology. We employed siRNAs to suppress Eg5 expression in HeLa cells and observed a significant increase in mitotic arrest, characterized by condensed chromosomes arranged around a monopolar spindle (Figure S3), a phenomenon also observed in other studies(28, 29). Next we co-transfected Flag-Eg5-WT or Flag-Eg5-K564R plasmids to rescue the effects of Eg5 siRNA-3, which targets the 3'-UTR sequence without affecting the expression of exogenous Eg5 (Fig. 5 B). Remarkably, spindle bipolarity was restored when HeLa cells were co-transfected with Eg5 siRNA and Eg5-WT. In contrast, co-transfection of cells with Eg5 siRNA and Eg5-K564R resulted in a significant increase in disorganized spindle defects, such as shorter length and asymmetric morphology (Fig. 5 A, C). Additionally, CCK-8 and colony formation assays showed that the knockdown of Eg5 significantly inhibited cell proliferation in HeLa cells. This inhibitory effect was rescued by the co-expression of Eg5-WT, whereas the co-expression of Eg5-K564R failed to produce the same rescue effect (Fig. 5 D-F). Collectively, these data demonstrated that Eg5 UFMylation plays a critical role in promoting spindle organization and facilitating cell proliferation. DISCUSSION In our previous research, we demonstrated that a deficiency in UFMylation significantly disrupts the process of mitosis(22). The present study introduces Eg5 as a novel substrate, emerging as a highly plausible candidate based on our and other proteomics data(25, 27, 30, 31). This finding established a direct link between UFMylation and the regulation of mitosis. Similar to other ubiquitin-like proteins, UFMylation influences the stability or subcellular localization of substrate proteins(25–27, 32–34). Our study revealed that UFMylation impacted the localization of Eg5, without altering its protein stability. The function and localization of Eg5 undergo dynamic changes during different mitotic phases. In prophase, Eg5 accumulates at the centrosomes, facilitating centrosome separation. A failure in this process results in the formation of a monopolar spindle. In the metaphase stage, Eg5 mainly localizes to spindle microtubules, maintaining spindle assembly and stabilizing a bipolar structure(23, 24). Remarkably, upon knockdown of UFL1 or DDRGK1, Eg5 exhibited a reduced distribution along the spindle, yet its centrosomal positioning remained unchanged. This led to the formation of a shorter bipolar spindle rather than a monopolar structure. Thus, our findings emphasized the pivotal role of Eg5 UFMylation in the metaphase stage, particularly in modulating its spindle localization to facilitate the assembly and maintenance of a bipolar spindle. Although UFMylation was discovered about two decades ago, the majority of its substrates have only been identified in recent years(10, 35). Research into these substrates has revealed a broad spectrum of functions associated with this modification. For example, UFMylation of RPL26, RPN1, CYB5R3, and HRD1 maintains the ER proteins during ER stress(31, 34, 36–38). UFMylation regulates tumorigenesis through ASC1, SLC7A11, and PD-L1(39–41). P4HB UFMylation impacts mitochondrial oxidative stress(27). Atg9 UFMylation protects the nerves(42). UFMylation of MRE11, histone H4, and P53 triggers DNA damage response following double-strand breaks(25, 32, 33). DNA damage can occur at different stages of mitosis and may contribute to mitotic abnormalities. The occurrence of DNA damage during the G1, S, or G2 phases activates the DNA damage checkpoint, resulting in the arrest of the cell cycle in interphase. If damage arises during prophase and metaphase, cells pass through anaphase and telophase without arrest, resulting in activation of the DNA damage checkpoint in the subsequent G1 phase(43, 44). Nonetheless, our observations indicate that the knockdown of either UFL1 or DDRGK1 predominantly arrests the cell cycle at metaphase, suggesting that DNA damage may not be the direct cause of mitotic defects upon UFMylation deficiency. Instead, our findings reveal that the disruption of Eg5 UFMylation led to the formation of shortened or asymmetric metaphase spindles, demonstrating its importance as a primary substrate of UFMylation during the mitotic process. During mitosis, the localization and function of Eg5 during mitosis are intricately regulated by post-translational modifications. Phosphorylation, acetylation, and monoubiquitination of Eg5 are known to play roles in spindle assembly and centrosomal dynamics(29, 45–49). Our data reveal that UFMylation at the K564 site is crucial for ensuring the correct localization of Eg5 on the spindle, a step essential for the assembly and maintenance of the spindle. Additionally, impaired UFMylation was found to decrease the monoubiquitination levels of Eg5, suggesting a complex interplay among various post-translational modifications. A similar phenomenon has been observed in p53, another substrate for UFMylation. UFMylation at the K351, K357, K370, and K373 sites of p53 can inhibit its polyubiquitination, thereby enhancing its protein stability. Furthermore, these sites may also undergo acetylation or ubiquitination modifications, but the regulatory mechanisms between these modifications remain elusive(25). In conclusion, the precise post-translational modifications of Eg5 ensure its optimal function throughout the complexities of mitosis. Our study elucidates that UFMylation of Eg5 promotes spindle assembly during mitosis, providing insights into the molecular pathogenesis of microcephaly. It's noteworthy that individuals with mutations in Eg5 typically exhibit severe microcephaly and developmental delays(50–52), which closely resemble the phenotypes observed in patients with mutations in UBA5, UFM1, or UFC1. According to the gnomAD database, the allele frequency of c.1111G > A (p.A371T) in UBA5 is reported as 0.0027, with a prevalence of over 60% in patients carrying this variant. Future research could explore precision therapies, similar to those developed for various CFTR mutations(53), which may help maintain UFMylation modification on critical mitotic substrates like Eg5 or even other substrates, thereby facilitating the development of effective treatment strategies. Declarations ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (82071273, 82271906 and 82101958) and the Hunan Science and Technology major project of Birth Defect Cooperative Control (2019SK1010). AUTHOR CONTRIBUTIONS Ranhui Duan and Guangxu Li conceived, designed, and performed the experiments, analyzed the data, and wrote the manuscript. Yuanjiang Huang contributed to mass spectrometry analysis and protein purification. Liyi Wei and Hongjing Huang assisted with plasmid construction. Qiao Xiao conducted confocal imaging. Zujia Wang managed cell culture. Yingbao Zhu and Wen Huang provided experimental support and contributed to manuscript revisions. All authors reviewed and approved the manuscript. COMPETING INTERESTS The authors declare no competing interests. References Komatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, Okazaki N, et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J. [Comparative Study; Journal Article; Research Support, Non-U.S. Gov't]. 2004 2004-05-05;23(9):1977-86. Gerakis Y, Quintero M, Li H, Hetz C. The UFMylation System in Proteostasis and Beyond. TRENDS CELL BIOL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Review]. 2019 2019-12-01;29(12):974 − 86. Banerjee S, Kumar M, Wiener R. Decrypting UFMylation: How Proteins Are Modified with UFM1. BIOMOLECULES. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2020 2020-10-14;10(10). Kang SH, Kim GR, Seong M, Baek SH, Seol JH, Bang OS, et al. Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't]. 2007 2007-02-23;282(8):5256-62. Bacik JP, Walker JR, Ali M, Schimmer AD, Dhe-Paganon S. Crystal structure of the human ubiquitin-activating enzyme 5 (UBA5) bound to ATP: mechanistic insights into a minimalistic E1 enzyme. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2010 2010-06-25;285(26):20273-80. Liu G, Forouhar F, Eletsky A, Atreya HS, Aramini JM, Xiao R, et al. NMR and X-RAY structures of human E2-like ubiquitin-fold modifier conjugating enzyme 1 (UFC1) reveal structural and functional conservation in the metazoan UFM1-UBA5-UFC1 ubiquination pathway. J Struct Funct Genomics. [Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.]. 2009 2009-04-01;10(2):127 − 36. Tatsumi K, Sou YS, Tada N, Nakamura E, Iemura S, Natsume T, et al. A novel type of E3 ligase for the Ufm1 conjugation system. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't]. 2010 2010-02-19;285(8):5417-27. Wu J, Lei G, Mei M, Tang Y, Li H. A novel C53/LZAP-interacting protein regulates stability of C53/LZAP and DDRGK domain-containing Protein 1 (DDRGK1) and modulates NF-kappaB signaling. J BIOL CHEM. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2010 2010-05-14;285(20):15126-36. Witting KF, Mulder M. Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma. BIOMOLECULES. [Journal Article; Review]. 2021 2021-02-10;11(2). Zhou X, Mahdizadeh SJ, Le Gallo M, Eriksson LA, Chevet E, Lafont E. UFMylation: a ubiquitin-like modification. TRENDS BIOCHEM SCI. [Journal Article; Review]. 2023 2023-11-07. Chung CH, Yoo HM. Emerging role of protein modification by UFM1 in cancer. BIOCHEM BIOPH RES CO. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2022 2022-12-10;633:61 − 3. Cheng Y, Niu Z, Cai Y, Zhang W. Emerging role of UFMylation in secretory cells involved in the endocrine system by maintaining ER proteostasis. FRONT ENDOCRINOL. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2022 2022-01-20;13:1085408. Duan R, Shi Y, Yu L, Zhang G, Li J, Lin Y, et al. UBA5 Mutations Cause a New Form of Autosomal Recessive Cerebellar Ataxia. PLOS ONE. [Journal Article; Research Support, Non-U.S. Gov't]. 2016 2016-01-20;11(2):e149039. Colin E, Daniel J, Ziegler A, Wakim J, Scrivo A, Haack TB, et al. Biallelic Variants in UBA5 Reveal that Disruption of the UFM1 Cascade Can Result in Early-Onset Encephalopathy. AM J HUM GENET. [Journal Article]. 2016 2016-09-01;99(3):695–703. Muona M, Ishimura R, Laari A, Ichimura Y, Linnankivi T, Keski-Filppula R, et al. Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy. AM J HUM GENET. [Journal Article]. 2016 2016-09-01;99(3):683 − 94. Arnadottir GA, Jensson BO, Marelsson SE, Sulem G, Oddsson A, Kristjansson RP, et al. Compound heterozygous mutations in UBA5 causing early-onset epileptic encephalopathy in two sisters. BMC MED GENET. [Case Reports; Journal Article]. 2017 2017-10-02;18(1):103. Tumiene B, Maver A, Writzl K, Hodzic A, Cuturilo G, Kuzmanic-Samija R, et al. Diagnostic exome sequencing of syndromic epilepsy patients in clinical practice. CLIN GENET. [Journal Article]. 2018 2018-05-01;93(5):1057-62. Daida A, Hamano SI, Ikemoto S, Matsuura R, Nakashima M, Matsumoto N, et al. Biallelic loss-of-function UBA5 mutations in a patient with intractable West syndrome and profound failure to thrive. EPILEPTIC DISORD. [Case Reports; Journal Article]. 2018 2018-08-01;20(4):313-8. Low KJ, Baptista J, Babiker M, Caswell R, King C, Ellard S, et al. Hemizygous UBA5 missense mutation unmasks recessive disorder in a patient with infantile-onset encephalopathy, acquired microcephaly, small cerebellum, movement disorder and severe neurodevelopmental delay. EUR J MED GENET. [Case Reports; Journal Article]. 2019 2019-02-01;62(2):97–102. Hamilton E, Bertini E, Kalaydjieva L, Morar B, Dojcakova D, Liu J, et al. UFM1 founder mutation in the Roma population causes recessive variant of H-ABC. NEUROLOGY. [Journal Article]. 2017 2017-10-24;89(17):1821-8. Nahorski MS, Maddirevula S, Ishimura R, Alsahli S, Brady AF, Begemann A, et al. Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development. BRAIN. [Journal Article; Research Support, Non-U.S. Gov't]. 2018 2018-07-01;141(7):1934-45. Yu L, Li G, Deng J, Jiang X, Xue J, Zhu Y, et al. The UFM1 cascade times mitosis entry associated with microcephaly. FASEB J. [Journal Article; Research Support, Non-U.S. Gov't]. 2020 2020-01-01;34(1):1319-30. Liu M, Ran J, Zhou J. Non-canonical functions of the mitotic kinesin Eg5. THORAC CANCER. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2018 2018-08-01;9(8):904 − 10. Mann BJ, Wadsworth P. Kinesin-5 Regulation and Function in Mitosis. TRENDS CELL BIOL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Review]. 2019 2019-01-01;29(1):66–79. Liu J, Guan D, Dong M, Yang J, Wei H, Liang Q, et al. UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination. NAT CELL BIOL. [Journal Article; Research Support, Non-U.S. Gov't]. 2020 2020-09-01;22(9):1056-63. Yoo HM, Park JH, Kim JY, Chung CH. Modification of ERalpha by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development. MOL CELLS. [Journal Article]. 2022 2022-06-30;45(6):425 − 34. Zhu J, Ma X, Jing Y, Zhang G, Zhang D, Mao Z, et al. P4HB UFMylation regulates mitochondrial function and oxidative stress. FREE RADICAL BIO MED. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-08-01;188:277 − 86. Blangy A, Lane HA, D'Herin P, Harper M, Kress M, Nigg EA. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. CELL. [Journal Article; Research Support, Non-U.S. Gov't]. 1995 1995-12-29;83(7):1159-69. Zheng J, Tan Y, Liu X, Zhang C, Su K, Jiang Y, et al. NAT10 regulates mitotic cell fate by acetylating Eg5 to control bipolar spindle assembly and chromosome segregation. CELL DEATH DIFFER. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-04-01;29(4):846 − 60. Pirone L, Xolalpa W, Sigurethsson JO, Ramirez J, Perez C, Gonzalez M, et al. A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation. SCI REP-UK. [Journal Article; Research Support, Non-U.S. Gov't]. 2017 2017-01-18;7:40756. Ishimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo M, et al. The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3. NAT COMMUN. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-12-21;13(1):7857. Qin B, Yu J, Nowsheen S, Wang M, Tu X, Liu T, et al. UFL1 promotes histone H4 ufmylation and ATM activation. NAT COMMUN. [Journal Article; Research Support, N.I.H., Extramural]. 2019 2019-03-18;10(1):1242. Wang Z, Gong Y, Peng B, Shi R, Fan D, Zhao H, et al. MRE11 UFMylation promotes ATM activation. NUCLEIC ACIDS RES. [Journal Article; Research Support, Non-U.S. Gov't]. 2019 2019-05-07;47(8):4124-35. Luo H, Jiao QB, Shen CB, Gong WY, Yuan JH, Liu YY, et al. UFMylation of HRD1 regulates endoplasmic reticulum homeostasis. FASEB J. [Journal Article; Research Support, Non-U.S. Gov't]. 2023 2023-11-01;37(11):e23221. Wang X, Xu X, Wang Z. The Post-Translational Role of UFMylation in Physiology and Disease. CELLS-BASEL. [Journal Article; Review]. 2023 2023-10-29;12(21). Walczak CP, Leto DE, Zhang L, Riepe C, Muller RY, DaRosa PA, et al. Ribosomal protein RPL26 is the principal target of UFMylation. P NATL ACAD SCI USA. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2019 2019-01-22;116(4):1299 − 308. Wang L, Xu Y, Rogers H, Saidi L, Noguchi CT, Li H, et al. UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis. CELL RES. [Journal Article; Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural]. 2020 2020-01-01;30(1):5–20. Liang JR, Lingeman E, Luong T, Ahmed S, Muhar M, Nguyen T, et al. A Genome-wide ER-phagy Screen Highlights Key Roles of Mitochondrial Metabolism and ER-Resident UFMylation. CELL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2020 2020-03-19;180(6):1160-77. Yoo HM, Kang SH, Kim JY, Lee JE, Seong MW, Lee SW, et al. Modification of ASC1 by UFM1 is crucial for ERalpha transactivation and breast cancer development. MOL CELL. [Journal Article; Research Support, Non-U.S. Gov't]. 2014 2014-10-23;56(2):261 − 74. Yang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J EXP CLIN CANC RES. [Journal Article]. 2021 2021-06-23;40(1):206. Zhou J, Ma X, He X, Chen B, Yuan J, Jin Z, et al. Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target. P NATL ACAD SCI USA. [Journal Article; Research Support, Non-U.S. Gov't]. 2023 2023-03-14;120(11):e2079235176. Li H, Yu Z, Niu Z, Cheng Y, Wei Z, Cai Y, et al. A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila. CELL MOL LIFE SCI. [Journal Article]. 2023 2023-04-22;80(5):129. Hayashi MT, Karlseder J. DNA damage associated with mitosis and cytokinesis failure. ONCOGENE. [Journal Article; Research Support, N.I.H., Extramural; Review]. 2013 2013-09-26;32(39):4593 − 601. Heijink AM, Krajewska M, van Vugt MA. The DNA damage response during mitosis. MUTAT RES-FUND MOL M. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2013 2013-10-01;750(1–2):45–55. Rapley J, Nicolas M, Groen A, Regue L, Bertran MT, Caelles C, et al. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation. J CELL SCI. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2008 2008-12-01;121(Pt 23):3912-21. Smith E, Hégarat N, Vesely C, Roseboom I, Larch C, Streicher H, et al. Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1. EMBO J. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2011 2011-06-01;30(11):2233-45. Duan Y, Huo D, Gao J, Wu H, Ye Z, Liu Z, et al. Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5. NAT COMMUN. [Journal Article; Research Support, Non-U.S. Gov't]. 2016 2016-08-25;7:12648. Bickel KG, Mann BJ, Waitzman JS, Poor TA, Rice SE, Wadsworth P. Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5. CYTOSKELETON. [Journal Article]. 2017 2017-09-01;74(9):317 − 30. Muretta JM, Reddy B, Scarabelli G, Thompson AF, Jariwala S, Major J, et al. A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function. P NATL ACAD SCI USA. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2018 2018-02-20;115(8):E1779-88. Hazan F, Ostergaard P, Ozturk T, Kantekin E, Atlihan F, Jeffery S, et al. A novel KIF11 mutation in a Turkish patient with microcephaly, lymphedema, and chorioretinal dysplasia from a consanguineous family. AM J MED GENET A. [Case Reports; Journal Article]. 2012 2012-07-01;158A(7):1686-9. Mirzaa GM, Enyedi L, Parsons G, Collins S, Medne L, Adams C, et al. Congenital microcephaly and chorioretinopathy due to de novo heterozygous KIF11 mutations: five novel mutations and review of the literature. AM J MED GENET A. [Case Reports; Journal Article; Research Support, N.I.H., Extramural; Review]. 2014 2014-11-01;164A(11):2879-86. Jones GE, Ostergaard P, Moore AT, Connell FC, Williams D, Quarrell O, et al. Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR): review of phenotype associated with KIF11 mutations. EUR J HUM GENET. [Journal Article; Research Support, Non-U.S. Gov't]. 2014 2014-07-01;22(7):881-7. Lopes-Pacheco M. CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine. FRONT PHARMACOL. [Journal Article; Review]. 2019 2019-01-20;10:1662. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3754446","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267459843,"identity":"87dde8eb-768f-4231-ad36-90e320c9cbbb","order_by":0,"name":"Ranhui Duan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYDCCAxAqgYGZ+QBjQwGYY0CsFrYExgYDkrQw8BgQp4XvRvKzxxU1d/IMjvN8/jjDYJtdA3vzNgmGmjs4tUjeSDM3PHPsWbHBYd5tkhsMbic38Bwrk2A49gynFoMbCWaSDWyHEzcAtTA+AGphkMgxk2BsOIxHS/o3yYZ/IC08jz+Ctci/IaQlx0yysQ2shQHkMDsGCR78WiTPvCmTbOw7nDjzMJuZ5AyD2wlsPGnFFgnHcGvhO56+TbLh2+HEvvOHH3/sqbhtz89+eOONDzW4tWCAxDYQmUC8BgYGe1IUj4JRMApGwcgAAB3HXaWJsEATAAAAAElFTkSuQmCC","orcid":"","institution":"Central South University","correspondingAuthor":true,"prefix":"","firstName":"Ranhui","middleName":"","lastName":"Duan","suffix":""},{"id":267459844,"identity":"ffedf588-ae15-42e1-be65-0455fd68f51f","order_by":1,"name":"Guangxu Li","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Guangxu","middleName":"","lastName":"Li","suffix":""},{"id":267459845,"identity":"30455473-abc2-42ef-9997-3b5fee1f96b6","order_by":2,"name":"Yuanjiang Huang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yuanjiang","middleName":"","lastName":"Huang","suffix":""},{"id":267459846,"identity":"760d6a40-7630-4aaa-8e39-b62861f3f5c8","order_by":3,"name":"Liyi Wei","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Liyi","middleName":"","lastName":"Wei","suffix":""},{"id":267459847,"identity":"910bd3fb-5877-4700-a8a5-d21aa45e7c57","order_by":4,"name":"Hongjing Huang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Hongjing","middleName":"","lastName":"Huang","suffix":""},{"id":267459848,"identity":"dc763cfb-de15-4fa9-897c-044a40745379","order_by":5,"name":"Yingbao Zhu","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yingbao","middleName":"","lastName":"Zhu","suffix":""},{"id":267459849,"identity":"e6f7b286-409b-421e-8344-510221f4abf2","order_by":6,"name":"Qiao Xiao","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Qiao","middleName":"","lastName":"Xiao","suffix":""},{"id":267459850,"identity":"05f044bd-3c7f-4a8a-b8fb-f5f29900125e","order_by":7,"name":"Wang Zujia","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Zujia","suffix":""},{"id":267459851,"identity":"51731a14-75bb-4545-8124-e30499e18794","order_by":8,"name":"Huang Wen","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Huang","middleName":"","lastName":"Wen","suffix":""}],"badges":[],"createdAt":"2023-12-14 16:06:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3754446/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3754446/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-06934-w","type":"published","date":"2024-07-31T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49802374,"identity":"52f28e2b-9b50-4384-b2af-cc005ff04f66","added_by":"auto","created_at":"2024-01-18 09:13:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1202386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEg5 interacts with UFL1 and DDRGK1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Strategy for identification of targets for UFMylation. (B) Proteins eluted from anti-Flag M2 beads were subjected to SDS-PAGE followed by coomassie blue staining. (C-E) Flag-Eg5, HA-UFL1 or HA-DDRGK1 were expressed in HEK293T cells, respectively. Cell lysates were subjected to immunoprecipitation with anti-Flag or anti-HA beads followed by Western blot analysis with the indicated antibodies. (F, G) HEK293T cell lysates were subject to immunoprecipitation with anti-Eg5, anti-UFL1 or anti-DDRGK1 antibody followed by Western blot analysis with the indicated antibodies. IgG was used as a control. (H) \u003cem\u003eIn vitro\u003c/em\u003e binding assay. Purified His-UFL1 and His-DDRGK1 were incubated with GST or GST-tagged Eg5, followed by GST pulldown assay. The samples were then subjected to Western blot analysis with anti-His antibody.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/1fae60a896d0944c4b94a2ff.png"},{"id":49802969,"identity":"f29ee621-2ee6-45cb-ba85-e8669a5a83e5","added_by":"auto","created_at":"2024-01-18 09:21:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1738430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEg5 is a target substrate for UFMylation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Eg5 is UFMylated \u003cem\u003ein vivo\u003c/em\u003e. UFMylation system components (HA-UBA5, HA-UFC1, HA-UFL1, HA-DDRGK1, HA-UFM1) and Flag-Eg5 were expressed in HEK293T cells. Cell lysates were subjected to immunoprecipitation with anti-Flag beads followed by Western blot analysis with the indicated antibodies. (B) UBA5, UFC1, UFL1 and DDRGK1 are required for Eg5 UFMylation. Flag-Eg5 and HA-UFM1-ΔC2 were expressed in UFSP2 knockout HEK293T cells with si-NC, si-UBA5, si-UFC1, si-UFL1 or si-DDRGK1, respectively. Cell lysates were subjected to the UFMylation assay. (C) Bacterially produced UFMylation components (His-UBA5, His-UFC1, His-UFL1, His-UFM1-ΔC2) were subjected to Coomassie brilliant blue or Western blot analysis with anti-His antibody. (D) Eg5 is UFMylated \u003cem\u003ein vitro\u003c/em\u003e. Purified UFMylation components and GST-Eg5 were incubated in UFMylation buffer. The reaction was terminated by adding SDS sample buffer, and the samples were subjected to Western blot with the indicated antibodies.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/4df7abb18ef23aa038f28874.png"},{"id":49802376,"identity":"e9258710-6cec-4fa6-80a4-b8c6e3c52705","added_by":"auto","created_at":"2024-01-18 09:13:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1350029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eK564 is the essential UFMylation site in Eg5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Identification of UFMylation region. A series of deletion constructs of Flag-Eg5 were generated as indicated, and expressed in UFSP2 knockout HEK293T cells with HA-UFM1-ΔC2. Cell lysates were subjected to the UFMylation assay. (C) Identification of the UFMylation site (s). The six Lys residues in the amino acid sequence of 552–641 were replaced by Arg, respectively, and the UFMylation assay was performed in UFSP2 knockout HEK293T cells. (D) In vitro UFMylation assay of Eg5 and its mutants, as described in Figure 2D.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/786e6a841cb5857e0f6e19cc.png"},{"id":49802375,"identity":"556a6a0d-cf07-457a-9199-9836e973d25f","added_by":"auto","created_at":"2024-01-18 09:13:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1157757,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUFMylation is required for Eg5 monoubiquitination.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Eg5 stability was examined by western blot in HeLa cells with UFL1 or DDRGK1 knockdown. The cells were treated with 100 µg ml\u003csup\u003e−1\u003c/sup\u003e cycloheximide (CHX) for the indicated times, and the Eg5 protein levels were quantified. The mean ± SD from three independent experiments is shown. The \u003cem\u003eP \u003c/em\u003evalues were determined by one-way ANOVA. ns, not significant. (B) HA-Ub and Flag-Eg5 were expressed in HeLa cells with si-NC, si-UFL1 or si-DDRGK1, respectively. Cell lysates were subjected to immunoprecipitation with anti-Flag beads followed by Western blot analysis with the indicated antibodies.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/0cc58e7ac817775fa22361c4.png"},{"id":49802378,"identity":"8a198601-b861-4322-bc9d-7d9383b80b77","added_by":"auto","created_at":"2024-01-18 09:13:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2312310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-localization between UFM1 and Eg5 at the centrosome and spindle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe localization of UFM1 and Eg5 was detected by immunofluorescence staining using anti-UFM1 and anti-Eg5 antibodies in HeLa cells at interphase, prophase, metaphase, anaphase and telophase. The cell nuclei was stained with DAPI. Scale bar, 5 μm.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/6f35f56f94e92ff63718f5c4.png"},{"id":49802382,"identity":"be7375e8-42f7-4195-a8ee-2508e9333830","added_by":"auto","created_at":"2024-01-18 09:13:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2594198,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDefective Eg5 UFMylation impairs distribution of Eg5 on the mitotic spindle.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The localization of Eg5 at the centrosome was detected by immunofluorescence staining using anti-Eg5 and anti-γ-tubulin in HeLa cells. Scale bar, 5 μm. The mean ± SD from at least 45 mitotic cells is shown. The \u003cem\u003eP \u003c/em\u003evalues were determined by one-way ANOVA. ns, not significant. (B) Sum intensity of Eg5 in the prophase centrosome region. (C) The localization of Eg5 at the spindle was detected by immunofluorescence staining using anti-Eg5 and anti-α-tubulin in HeLa cells. (D-F) Sum intensity of Eg5, spindle area, and mean intensity of Eg5 in the metaphase spindle region. Scale bar, 5 μm. The mean ± SD from at least 80 mitotic cells is shown. The \u003cem\u003eP \u003c/em\u003evalues were determined by one-way ANOVA. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/b778a5fd3679c946ffa28cf9.png"},{"id":49802381,"identity":"ab6346e9-dced-4c85-8d4e-ea423f9eee6d","added_by":"auto","created_at":"2024-01-18 09:13:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2772687,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEg5 UFMylation is required for the spindle assembly and cell proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHela cells were transfected with indicated siRNAs or plasmids. (A) The phenotypes of mitotic spindle (monopole, muitipole or disorganized) were determined by immunofluorescence staining using anti-α-tubulin and DAPI in HeLa cells. Transfected cells were identified by Flag positivity. (B) Cell lysates were subjected to Western blot analysis with anti-Flag or anti-Eg5 antibody to determine Eg5 expression. (C) The percent of mitotic phenotypes were calculated (n = 25 cells). The mean ± SD from three independent experiments is shown. The \u003cem\u003eP \u003c/em\u003evalues were determined by two-way ANOVA. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. (D) CCK-8 assay. (E) Colony formation assay. HeLa cells were cultured and stained with crystal violet. (F) The number of colonies in each condition was counted. The mean ± SD from three independent experiments is shown. The \u003cem\u003eP \u003c/em\u003evalues were determined by one-way ANOVA. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/17ef1fb112f43b291ab44743.png"},{"id":61554911,"identity":"33f13591-63b6-4f02-9398-ad52bfd877bf","added_by":"auto","created_at":"2024-08-01 07:19:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17636129,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/4919c498-8810-4359-a751-093bfbfa480b.pdf"},{"id":49802379,"identity":"45352d13-6fe9-4c3b-bfed-751256795aa1","added_by":"auto","created_at":"2024-01-18 09:13:40","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":286720,"visible":true,"origin":"","legend":"Table sup 1","description":"","filename":"Tablesup1.xls","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/9d17785054350d6eec986b0f.xls"},{"id":49802383,"identity":"0b21a4d6-bd43-464e-bb5c-8b3160afcc09","added_by":"auto","created_at":"2024-01-18 09:13:42","extension":"doc","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":18517793,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterical.doc","url":"https://assets-eu.researchsquare.com/files/rs-3754446/v1/fdc3bfb679b47470e5e23960.doc"}],"financialInterests":"(Not answered)","formattedTitle":"Eg5 UFMylation promotes spindle organization during mitosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eUbiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like protein (UBL) that remains less well-understood compared to other UBLs(1). UFM1 is highly conserved across evolutionary lines, with orthologs found in both Metazoa and plants(2). Even rice shares over 66.27% nucleotide sequence similarity with humans. Although UFM1 shares only 16% amino acid sequence identity with ubiquitin, its tertiary structure closely resembles the ubiquitin fold, featuring specific β-sheets and an α-helix(3). The UFM1 precursor consists of 85 amino acids and is processed to its mature form by specific cysteine proteases, UFSP1 and UFSP2, which cleave the C-terminal dipeptide Ser-Cys, exposing the conserved Gly83 conjugating residue(4). The activation of UFM1 involves the UFM1-specific E1 enzyme, UBA5, through processes of adenylation and thioesterification(5). The UFM1-specific E2 enzyme, UFC1, accepts the activated UFM1 in a trans-esterification reaction(6). The UFM1-specific E3 enzyme, UFL1, catalyzes the covalent binding of UFM1 to substrate lysine residues(7). The DDRGK domain-containing protein 1 (DDRGK1, also known as UFBP1) serves as an E3 ligase adaptor, regulating the UFMylation of substrates(8). UFMylation is a reversible process, as UFM1 can be cleaved from its target proteins by UFSP1 and UFSP2(9).\u003c/p\u003e \u003cp\u003eUFMylation regulates various cellular functions, such as hematopoiesis, endoplasmic reticulum (ER) proteostasis, DNA damage response, autophagy, transcriptional regulation, and signaling pathways(2, 10\u0026ndash;12). In 2016, we reported for the first time that compound heterozygous variants of UBA5 lead to neurodevelopmental disorders characterized by cerebellar atrophy and developmental delays(13). Subsequently, a series of biallelic variants of UBA5 were discovered in patients, causing severe infantile-onset epileptic encephalopathy primarily characterized by microcephaly(14\u0026ndash;19). Furthermore, biallelic mutations in UFM1 or UFC1 have revealed similar microcephalic traits(20, 21). Genes implicated in microcephaly typically play key roles during mitosis. Our recent study in Drosophila demonstrate that knockdown of UFMylation results in reduced brain size, partial embryonic lethality before gastrulation, and mitotic defects in spindle assembly and chromosome separation(22). However, the underlying mechanisms remain to be elucidated, due to the limited identification of relevant substrates.\u003c/p\u003e \u003cp\u003eHere, we employed mass spectrometry and cross-referenced other reliable proteomic datasets to identify various substrates, among which Kinesin-5 (Eg5, also referred to as KIF11) is included. Functionally, Eg5 acts as a microtubule-oriented motor protein that moves towards the plus-end, facilitating bipolar spindle assembly during mitosis(23). Inhibition of Eg5 leads to a monopolar spindle, causing cell mitotic arrest and subsequent cell death(24).\u003c/p\u003e \u003cp\u003eIn this study, we confirmed Eg5 as a novel substrate for UFMylation and pinpointed Lys564 as the crucial UFMylation site among its 90 lysine residues. Eg5 directly interacted with the E3 ligases UFL1 and DDRGK1, and exhibited modification by UFM1 both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. During mitosis, Eg5 and UFM1 co-localized at the centrosomes and spindle, and defective UFMylation resulted in reduced spindle localization of Eg5. The UFMylation-defective Eg5 mutant (K564R) displayed shortened or asymmetrical spindles during metaphase, and inhibited cell proliferation in HeLa cells. Our findings suggest that Eg5 UFMylation is significant for understanding the pathogenic mechanisms involving cell cycle dysregulation.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHEK-293T and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), at 37℃ in a 5% CO2 atmosphere. The cells used in this study were verified to be free from mycoplasma contamination, and the identity of HeLa cells was verified through STR profiling.\u003c/p\u003e \u003cp\u003eTo generate \u003cem\u003eUFSP2\u003c/em\u003e knockout cell lines, the guide RNA targeting \u003cem\u003eUFSP2\u003c/em\u003e (5\u0026prime;-AGCAGTGACATAAACACC-3\u0026prime;) was cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (62988, Addgene, Watertown, MA, USA). HEK293T cells were transfected with PX459 and selected with puromycin (1 \u0026micro;g/ml). After selecting for 5 days, cells were cloned using a limiting dilution and screened for knockout by Sanger sequencing and Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003esiRNA and plasmid transfection\u003c/h2\u003e \u003cp\u003eThe siRNA duplexes were synthesized by GenePharma (Shanghai, China), and the corresponding sequences were as follows:\u003c/p\u003e \u003cp\u003esi-NC: UUCUUCGAACGUGUCACGU; si-\u003cem\u003eUBA5\u003c/em\u003e: GGUUAUACAAGAAGAGGAA;\u003c/p\u003e \u003cp\u003esi-\u003cem\u003eUFC1\u003c/em\u003e: GAAAGACAGCAAAGAUGUA; si-\u003cem\u003eUFL1\u003c/em\u003e-1: GGAACUUGUUAAUAGCGGA;\u003c/p\u003e \u003cp\u003esi-\u003cem\u003eUFL1\u003c/em\u003e-2: GAGGAGUAAUUUUUACGGA; si-\u003cem\u003eDDRGK1\u003c/em\u003e-1: GAAAAUUGGAGCUAAGAAA;\u003c/p\u003e \u003cp\u003esi-\u003cem\u003eDDRGK1\u003c/em\u003e-2: CCAUAAAUCGCAUCCAGGA; si-\u003cem\u003eEg5\u003c/em\u003e-1: CAGAUUGAUGUUUACCGAA;\u003c/p\u003e \u003cp\u003esi-\u003cem\u003eEg5\u003c/em\u003e-2: CUGGAUAUCCCAACAGGUA; si-\u003cem\u003eEg5\u003c/em\u003e-3: CGAUGAGUUUAGUGUGUAAAG.\u003c/p\u003e \u003cp\u003e \u003cem\u003eUBA5\u003c/em\u003e, \u003cem\u003eUFC1\u003c/em\u003e, \u003cem\u003eUFL1\u003c/em\u003e, \u003cem\u003eDDRGK1\u003c/em\u003e and \u003cem\u003eUFM1\u003c/em\u003e cDNAs were inserted into the pRK5-HA vector. UFM1 cDNA variants with two or three amino acids deleted in the C-terminal (UFM1-ΔC2 or UFM1ΔC3) were cloned into both the pRK5-HA vector and the pcDNA3.1-Flag vector. Eg5 cDNA was cloned into the pRK5-Flag vector. Truncations and point mutations of Eg5 were generated using the MutExpress II Fast Mutagenesis Kit V2 (C214-02, Vazyme, Nanjing, China). Bacteria expressing His-tagged UBA5, UFC1, UFL1, DDRGK1, UFM1-ΔC2 and GST-tagged Eg5 were generated using the pET-28a and pGEX-4T-1 systems, respectively. All plasmids were validated through Sanger sequencing. RNA interference was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), and plasmid transfection was performed with Lipofectamine 3000 (Invitrogen), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry\u003c/h2\u003e \u003cp\u003e \u003cem\u003eUFSP2\u003c/em\u003e knockout HEK293T cells were transfected with Flag-tagged UFM1-ΔC2 and Flag-tagged UFM1ΔC3, respectively. for 36 h. The cells were then lysed with NP-40 lysis buffer containing 50mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40 and 1\u0026times;protease inhibitor cocktail (P8340, Sigma, St. Louis, MO, USA). Subsequently, Flag-UFM1-ΔC2 or Flag-UFM1ΔC3 was immunoprecipitated by incubation the lysates with anti-FLAG M2 beads (M8823, Sigma) overnight at 4\u0026deg;C. The protein precipitates were subjected to SDS-PAGE and mass spectrometry. Mass spectrometric analysis of peptide samples was carried out using a Q Exactive Mass Spectrometer (Thermo Fisher, Waltham, MA, USA) at Applied Protein Technology Company (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells were lysed with RIPA lysis buffer consisting of 50mM Tris (pH 7.4), 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1\u0026times;protease inhibitor cocktail. Whole cell lysates with equal protein were resolved by SDS-PAGE and subsequently transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 1\u0026times;TBST (TBS with 0.1% Tween 20), supplemented with 5% nonfat milk for 1 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4℃ and with the secondary antibodies for 1 h at room temperature.\u003c/p\u003e \u003cp\u003ePrimary antibodies were as follows: anti-Flag (F1804, 1:5000, Sigma), anti-HA (C29F4, 1:2000, Cell Signaling, Danvers, MA, USA), anti-UFL1 (PA5-56501, Thermo Fisher, 1:1000), anti-DDRGK1 (21445-1-AP, Proteintech, Hubei, China, 1:2000), anti-Eg5 (23333-1-AP Proteintech, 1:2000), anti-UBA5 (ab177478, Abcam, Cambridge, UK, 1:1000), anti-UFC1 (ab189251, Abcam, 1:1000), anti-UFM1 (15883-1-AP, Proteintech, 1:2000), anti-UFSP2 (ab185965, Abcam, 1:1000), anti-His (ab18184, Abcam, 1:5000), anti-α-tubulin (ab18251, Abcam, 1:5000), and anti-GAPDH (10494-1-AP, Proteintech, 1:500). Secondary antibodies were as follows: HRP-conjugated goat anti-mouse IgG (115-035-166, Jackson ImmunoResearch, West Grove, PA, USA, 1:10000) or HRP-conjugated goat anti-rabbit IgG (111-035-144, Jackson ImmunoResearch; 1:10000). The protein bands were visualized using a ECL-chemiluminescent kit .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eUFMylation assays\u003c/h2\u003e \u003cp\u003eFor the \u003cem\u003ein vivo\u003c/em\u003e UFMylation assays, cells transfected with appropriate plasmids were cultured for 36 h. To prepare cell lysates, the cells were boiled in a buffer containing 150 mM Tris (pH 8.0), 5% SDS, and 30% glycerol for 8 min. Subsequently, the lysates were diluted 20-fold with NP-40 lysis buffer and subjected to immunoprecipitation using anti-FLAG M2 beads overnight at 4\u0026deg;C. The samples were then subjected to SDS-PAGE and Western blot analysis.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e UFMylation assays were performed following previous reported method. Recombinant GST-Eg5 were expressed in BL21 cells and purified using the GST-tag Protein Purification Kit (P2262, Beyotime, Jiangsu, China). Additionally, His-UBA5, His-UFC1, His-UFL1, His-DDRGK1 and His-UFM1-ΔC2 were expressed in BL21 cells and purified using the His-tag Protein Purification Kit (P2226, Beyotime). His-UFM1-ΔC2 (5 ng), His-UBA5 (5 ng), His-UFC1 (5 ng), His-UFL1 (5 ng), His-DDRGK1 (5 ng) and GST-Eg5 (10 ng) were mixed in the reaction buffer containing 5 mM ATP and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, and incubated at 30\u0026deg;C for 90 min. The mixtures were boiled with the addition of loading buffer containing 5% mercaptoethanol for 5 min.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoprecipitation and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003ebinding assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor immunoprecipitation, cells were lysed with NP-40 lysis buffer and the lysates were then incubated with 1 \u0026micro;g of the indicated antibodies and 10 \u0026micro;l of Protein A/G Magnetic Beads (B23201, Biomake, Shanghai, China) overnight at 4\u0026deg;C. For the \u003cem\u003ein vitro\u003c/em\u003e binding assays, purified His-UFL1 and His-DDRGK1 were incubated with GST or GST-Eg5 in PBS with 0.2% NP-40 for 2 h at 4\u0026deg;C, followed by pull-down with GSH-resin. The samples were subjected to SDS-PAGE and Western blot analysis. 5% of each supernatant was used as input control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eHeLa cells transfected with the indicated siRNAs or plasmids were cultured for 48 h. The cells were fixed in 4% paraformaldehyde for 30 min, washed in 0.2% PBST (PBS with 0.2% Triton X-100), and blocked in 5% normal goat serum for 1 h at room temperature.Cells were incubated overnight at 4\u0026deg;C with primary antibodies: rabbit anti-UFM1 (ab109305, Abcam, 1:200), mouse anti-Eg5 (ab51976, Abcam, 1:500), rabbit anti-Eg5 (23333-1-AP Proteintech, 1:200), rabbit anti-α-tubulin (ab18251, Abcam, 1:500), or mouse anti-γ-tubulin (ab11316, Abcam, 1:500). Secondary antibodies were as follows: Alexa Fluor 488-conjugated goat anti-mouse IgG (115-545-003, Jackson ImmunoResearch, 1:200) or Cy3-conjugated goat anti-rabbit IgG (111-165-003, Jackson ImmunoResearch, 1:200). The cells were then stained with DAPI and mounted with Fluoromount mounting medium (F4680, Sigma). The image was acquired using the LSM 880 (Zeiss, Oberkochen, Germany) or the SP8 (Leica, Wetzlar, Germany) confocal system, and fluorescence intensities were calculated using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and colony formation assays\u003c/h2\u003e \u003cp\u003eFor the cell viability assays, 5000 HeLa cells were seeded into 96-well plates and transfected with the indicated siRNAs or plasmids. After culturing for 48h, the cells were treated with a CCK-8 solution (C0037, Beyotime) and incubated for 2 h. The absorbance values at a wavelength of 450 nm (OD450) were measured using an elx800 reader (BioTek, Winooski, VT, USA).\u003c/p\u003e \u003cp\u003eFor the colony formation assays, HeLa cells were placed in 6-well plates at a density of 500 cells per well. Every three days, the cells were transfected with the indicated siRNAs or plasmids. After 10 days of culturing, during which the clones became visible, the cells were fixed using a 4% paraformaldehyde and stained with 0.1% crystal violet. The colonies were then photographed and quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical software GraphPad Prism 8 was used to carry out unpaired Student's t test to compare two specific datasets and one-way ANOVA for multiple comparisons. The data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three independent replicates. The values of *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 were considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of candidate substrates for UFMylation\u003c/h2\u003e \u003cp\u003eTo identify target proteins for UFMylation, we overexpressed Flag-tagged mature UFM1 (UFM1-ΔC2) in \u003cem\u003eUFSP2\u003c/em\u003e knockout HEK293T cells to facilitate UFM1 conjugate formation. In addition, we employed a Flag-tagged conjugation-defective UFM1 lacking Gly83 (UFM1-ΔC3) as a negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). After verifying the expression levels of Flag-UFM1 (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), we immunoprecipitated the cell lysates with anti-Flag M2 beads, and the precipitated proteins were subjected to SDS-PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and mass spectrometry. We screened for UFMylation substrates by identifying proteins that specifically bound to Flag-UFM1-ΔC2 (Supplementary Table\u0026nbsp;1). Among these candidates, we selected Eg5 for further investigation due to its pivotal role in the dynamic assembly and function of the mitotic spindle through cross-linking and sliding adjacent microtubules(24).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEg5 interacts with UFL1 and DDRGK1\u003c/h2\u003e \u003cp\u003eTo determine whether Eg5 is a target protein for UFMylation, we examined its ability to interact with UFL1, the UFM1 E3 ligase, and DDRGK1, a critical regulatory factor for UFMylation. Immunoprecipitation analysis demonstrated that Eg5 is capable of binding to both UFL1 and DDRGK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-E). Moreover, endogenous Eg5, UFL1 and DDRGK1 exhibited mutual interactions in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). Furthermore, \u003cem\u003ein vitro\u003c/em\u003e binding assays showed that Eg5 directly interacted with UFL1 and DDRGK 1(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH), suggesting that Eg5 could be a bona fide substrate of UFMylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEg5 is a target substrate for UFMylation\u003c/h2\u003e \u003cp\u003eTo confirm whether Eg5 can undergo UFMylation, we co-expressed Flag-Eg5 with the UFMylation components UBA5, UFC1, UFL1, UFM1 and DDRGK1 in HEK293T cells. \u003cem\u003eIn vivo\u003c/em\u003e UFMylation assays demonstrated that Eg5 can be UFMylated by both the wild-type UFM1 (UFM1-WT) and mature UFM1 (UFM1-ΔC2), but not by the conjugation-defective UFM1 (UFM1-ΔC3) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, we observed that individually knocking down UBA5, UFC1, UFL1, or DDRGK1 resulted in a decrease in the UFMylation levels of Eg5 in \u003cem\u003eUFSP2\u003c/em\u003e knockout HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), indicating their crucial involvement in the Eg5 UFMylation process. Furthermore, the UFMylation assays validated Eg5 as a target substrate for UFMylation \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings unequivocally establish Eg5 as a novel target substrate for UFMylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the essential UFMylation site in Eg5\u003c/h2\u003e \u003cp\u003eTo identify the essential sites for Eg5 UFMylation, we employed three deletion constructs (ΔN, ΔM and ΔC) and transfected them into \u003cem\u003eUFSP2\u003c/em\u003e knockout HEK293T cells, along with the HA-UFM1-ΔC2 plasmid. Among these constructs, UFMylation was specifically observed in the ΔM construct of Eg5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To further investigate the UFMylation sites, we generated additional deletion constructs targeting specific segments within the ΔM region. Notably, the deletion constructs lacking amino acids 552\u0026ndash;641 (ΔD) showed a significant reduction in UFMylation levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As there are six Lys residues in this region, we individually substituted each Lys residues with Arg in wild-type Eg5. Among these substitutions, the replacement of Lys564 by Arg (referred to as K564R) resulted in a diminished Eg5 UFMylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In addition, the K564R mutation significantly reduced Eg5 UFMylation \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Collectively, these data demonstrated that K564 is a essential UFMylation site in Eg5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eUFMylation deficiency leads to decreased monoubiquitination of Eg5\u003c/h2\u003e \u003cp\u003ePrevious studies have shown that UFMylation is related to substrate stability and ubiquitination(25\u0026ndash;27). To determine whether UFMylation influences the stability of Eg5, we suppressed the expression of UFL1 or DDRGK1 in HeLa cells using siRNAs (Figure S2). We treated the cells with cycloheximide and found that Eg5 protein stability remained unaffected after knockdown of UFL1 or DDRGK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Interestingly, the monoubiquitination of Eg5 was significantly decreased by the knockdown of UFL1 or DDRGK1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, we did not extensively explore how UFMylation of Eg5 affects its monoubiquitination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eUFM1 and Eg5 exhibit co-localization at the centrosome and spindle\u003c/h2\u003e \u003cp\u003eTo gain functional insight into the association between UFMylation and Eg5, we investigated the cellular localization of UFM1 and Eg5. Immunofluorescence staining revealed that during interphase, UFM1 was present in both the nucleus and the cytoplasm, while Eg5 predominantly localized in the cytoplasm. As cells entered prophase, UFM1 and Eg5 start to accumulated at the centrosomes, displaying an overlapping pattern. During metaphase, UFM1 and Eg5 were distributed throughout the spindle apparatus. Subsequently, UFM1 and Eg5 were observed at the spindle poles and midbody in anaphase and telophase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The dynamic co-localization of UFM1 and Eg5 throughout different stages of the cell cycle suggests their potential roles in mitotic processes and spindle organization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eUFMylation maintains Eg5 interaction with the mitotic spindle\u003c/h2\u003e \u003cp\u003eThe spatial and temporal dynamics of Eg5 are crucial for its motor function in driving centrosome separation, spindle assembly, and chromosome segregation during mitosis(24). Given the co-localization of UFM1 and Eg5 at the centrosome and spindle, we were intrigued to explore whether UFMylation affects the spatial relationship between Eg5 and the mitotic centrosome or spindle. Indeed, knockdown of UFL1 or DDRGK1 had no influence on the localization of Eg5 at the centrosome during prophase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). However, the knockdown of UFL1 or DDRGK1 lead to a smaller spindle area and a reduction in both sun and mean EG5 intensity at the spindle during metaphase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F). These results suggested that Eg5 UFMylation is crucial for maintaining its interaction with the mitotic spindle.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eUFMylation promotes spindle organization and cell proliferation\u003c/h2\u003e \u003cp\u003eGiven the crucial role of Eg5 in mitotic spindle assembly, we proceeded to explore the effects of Eg5 UFMylation on mitotic spindle morphology. We employed siRNAs to suppress Eg5 expression in HeLa cells and observed a significant increase in mitotic arrest, characterized by condensed chromosomes arranged around a monopolar spindle (Figure S3), a phenomenon also observed in other studies(28, 29). Next we co-transfected Flag-Eg5-WT or Flag-Eg5-K564R plasmids to rescue the effects of Eg5 siRNA-3, which targets the 3'-UTR sequence without affecting the expression of exogenous Eg5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Remarkably, spindle bipolarity was restored when HeLa cells were co-transfected with Eg5 siRNA and Eg5-WT. In contrast, co-transfection of cells with Eg5 siRNA and Eg5-K564R resulted in a significant increase in disorganized spindle defects, such as shorter length and asymmetric morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). Additionally, CCK-8 and colony formation assays showed that the knockdown of Eg5 significantly inhibited cell proliferation in HeLa cells. This inhibitory effect was rescued by the co-expression of Eg5-WT, whereas the co-expression of Eg5-K564R failed to produce the same rescue effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F). Collectively, these data demonstrated that Eg5 UFMylation plays a critical role in promoting spindle organization and facilitating cell proliferation.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn our previous research, we demonstrated that a deficiency in UFMylation significantly disrupts the process of mitosis(22). The present study introduces Eg5 as a novel substrate, emerging as a highly plausible candidate based on our and other proteomics data(25, 27, 30, 31). This finding established a direct link between UFMylation and the regulation of mitosis.\u003c/p\u003e \u003cp\u003eSimilar to other ubiquitin-like proteins, UFMylation influences the stability or subcellular localization of substrate proteins(25\u0026ndash;27, 32\u0026ndash;34). Our study revealed that UFMylation impacted the localization of Eg5, without altering its protein stability. The function and localization of Eg5 undergo dynamic changes during different mitotic phases. In prophase, Eg5 accumulates at the centrosomes, facilitating centrosome separation. A failure in this process results in the formation of a monopolar spindle. In the metaphase stage, Eg5 mainly localizes to spindle microtubules, maintaining spindle assembly and stabilizing a bipolar structure(23, 24). Remarkably, upon knockdown of UFL1 or DDRGK1, Eg5 exhibited a reduced distribution along the spindle, yet its centrosomal positioning remained unchanged. This led to the formation of a shorter bipolar spindle rather than a monopolar structure. Thus, our findings emphasized the pivotal role of Eg5 UFMylation in the metaphase stage, particularly in modulating its spindle localization to facilitate the assembly and maintenance of a bipolar spindle.\u003c/p\u003e \u003cp\u003eAlthough UFMylation was discovered about two decades ago, the majority of its substrates have only been identified in recent years(10, 35). Research into these substrates has revealed a broad spectrum of functions associated with this modification. For example, UFMylation of RPL26, RPN1, CYB5R3, and HRD1 maintains the ER proteins during ER stress(31, 34, 36\u0026ndash;38). UFMylation regulates tumorigenesis through ASC1, SLC7A11, and PD-L1(39\u0026ndash;41). P4HB UFMylation impacts mitochondrial oxidative stress(27). Atg9 UFMylation protects the nerves(42). UFMylation of MRE11, histone H4, and P53 triggers DNA damage response following double-strand breaks(25, 32, 33). DNA damage can occur at different stages of mitosis and may contribute to mitotic abnormalities. The occurrence of DNA damage during the G1, S, or G2 phases activates the DNA damage checkpoint, resulting in the arrest of the cell cycle in interphase. If damage arises during prophase and metaphase, cells pass through anaphase and telophase without arrest, resulting in activation of the DNA damage checkpoint in the subsequent G1 phase(43, 44). Nonetheless, our observations indicate that the knockdown of either UFL1 or DDRGK1 predominantly arrests the cell cycle at metaphase, suggesting that DNA damage may not be the direct cause of mitotic defects upon UFMylation deficiency. Instead, our findings reveal that the disruption of Eg5 UFMylation led to the formation of shortened or asymmetric metaphase spindles, demonstrating its importance as a primary substrate of UFMylation during the mitotic process.\u003c/p\u003e \u003cp\u003eDuring mitosis, the localization and function of Eg5 during mitosis are intricately regulated by post-translational modifications. Phosphorylation, acetylation, and monoubiquitination of Eg5 are known to play roles in spindle assembly and centrosomal dynamics(29, 45\u0026ndash;49). Our data reveal that UFMylation at the K564 site is crucial for ensuring the correct localization of Eg5 on the spindle, a step essential for the assembly and maintenance of the spindle. Additionally, impaired UFMylation was found to decrease the monoubiquitination levels of Eg5, suggesting a complex interplay among various post-translational modifications. A similar phenomenon has been observed in p53, another substrate for UFMylation. UFMylation at the K351, K357, K370, and K373 sites of p53 can inhibit its polyubiquitination, thereby enhancing its protein stability. Furthermore, these sites may also undergo acetylation or ubiquitination modifications, but the regulatory mechanisms between these modifications remain elusive(25). In conclusion, the precise post-translational modifications of Eg5 ensure its optimal function throughout the complexities of mitosis.\u003c/p\u003e \u003cp\u003eOur study elucidates that UFMylation of Eg5 promotes spindle assembly during mitosis, providing insights into the molecular pathogenesis of microcephaly. It's noteworthy that individuals with mutations in Eg5 typically exhibit severe microcephaly and developmental delays(50\u0026ndash;52), which closely resemble the phenotypes observed in patients with mutations in UBA5, UFM1, or UFC1. According to the gnomAD database, the allele frequency of c.1111G\u0026thinsp;\u0026gt;\u0026thinsp;A (p.A371T) in UBA5 is reported as 0.0027, with a prevalence of over 60% in patients carrying this variant. Future research could explore precision therapies, similar to those developed for various CFTR mutations(53), which may help maintain UFMylation modification on critical mitotic substrates like Eg5 or even other substrates, thereby facilitating the development of effective treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82071273, 82271906 and 82101958) and the Hunan Science and Technology major project of Birth Defect Cooperative Control (2019SK1010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRanhui Duan and Guangxu Li conceived, designed, and performed the experiments, analyzed the data, and wrote the manuscript. Yuanjiang Huang contributed to mass spectrometry analysis and protein purification. Liyi Wei and Hongjing Huang assisted with plasmid construction. Qiao Xiao conducted confocal imaging. Zujia Wang managed cell culture. Yingbao Zhu and Wen Huang provided experimental support and contributed to manuscript revisions. All authors reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKomatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, Okazaki N, et al. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J. [Comparative Study; Journal Article; Research Support, Non-U.S. Gov't]. 2004 2004-05-05;23(9):1977-86.\u003c/li\u003e\n\u003cli\u003eGerakis Y, Quintero M, Li H, Hetz C. The UFMylation System in Proteostasis and Beyond. TRENDS CELL BIOL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Review]. 2019 2019-12-01;29(12):974\u0026thinsp;\u0026minus;\u0026thinsp;86.\u003c/li\u003e\n\u003cli\u003eBanerjee S, Kumar M, Wiener R. Decrypting UFMylation: How Proteins Are Modified with UFM1. BIOMOLECULES. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2020 2020-10-14;10(10).\u003c/li\u003e\n\u003cli\u003eKang SH, Kim GR, Seong M, Baek SH, Seol JH, Bang OS, et al. Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't]. 2007 2007-02-23;282(8):5256-62.\u003c/li\u003e\n\u003cli\u003eBacik JP, Walker JR, Ali M, Schimmer AD, Dhe-Paganon S. Crystal structure of the human ubiquitin-activating enzyme 5 (UBA5) bound to ATP: mechanistic insights into a minimalistic E1 enzyme. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2010 2010-06-25;285(26):20273-80.\u003c/li\u003e\n\u003cli\u003eLiu G, Forouhar F, Eletsky A, Atreya HS, Aramini JM, Xiao R, et al. NMR and X-RAY structures of human E2-like ubiquitin-fold modifier conjugating enzyme 1 (UFC1) reveal structural and functional conservation in the metazoan UFM1-UBA5-UFC1 ubiquination pathway. J Struct Funct Genomics. [Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.]. 2009 2009-04-01;10(2):127\u0026thinsp;\u0026minus;\u0026thinsp;36.\u003c/li\u003e\n\u003cli\u003eTatsumi K, Sou YS, Tada N, Nakamura E, Iemura S, Natsume T, et al. A novel type of E3 ligase for the Ufm1 conjugation system. J BIOL CHEM. [Journal Article; Research Support, Non-U.S. Gov't]. 2010 2010-02-19;285(8):5417-27.\u003c/li\u003e\n\u003cli\u003eWu J, Lei G, Mei M, Tang Y, Li H. A novel C53/LZAP-interacting protein regulates stability of C53/LZAP and DDRGK domain-containing Protein 1 (DDRGK1) and modulates NF-kappaB signaling. J BIOL CHEM. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2010 2010-05-14;285(20):15126-36.\u003c/li\u003e\n\u003cli\u003eWitting KF, Mulder M. Highly Specialized Ubiquitin-Like Modifications: Shedding Light into the UFM1 Enigma. BIOMOLECULES. [Journal Article; Review]. 2021 2021-02-10;11(2).\u003c/li\u003e\n\u003cli\u003eZhou X, Mahdizadeh SJ, Le Gallo M, Eriksson LA, Chevet E, Lafont E. UFMylation: a ubiquitin-like modification. TRENDS BIOCHEM SCI. [Journal Article; Review]. 2023 2023-11-07.\u003c/li\u003e\n\u003cli\u003eChung CH, Yoo HM. Emerging role of protein modification by UFM1 in cancer. BIOCHEM BIOPH RES CO. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2022 2022-12-10;633:61\u0026thinsp;\u0026minus;\u0026thinsp;3.\u003c/li\u003e\n\u003cli\u003eCheng Y, Niu Z, Cai Y, Zhang W. Emerging role of UFMylation in secretory cells involved in the endocrine system by maintaining ER proteostasis. FRONT ENDOCRINOL. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2022 2022-01-20;13:1085408.\u003c/li\u003e\n\u003cli\u003eDuan R, Shi Y, Yu L, Zhang G, Li J, Lin Y, et al. UBA5 Mutations Cause a New Form of Autosomal Recessive Cerebellar Ataxia. PLOS ONE. [Journal Article; Research Support, Non-U.S. Gov't]. 2016 2016-01-20;11(2):e149039.\u003c/li\u003e\n\u003cli\u003eColin E, Daniel J, Ziegler A, Wakim J, Scrivo A, Haack TB, et al. Biallelic Variants in UBA5 Reveal that Disruption of the UFM1 Cascade Can Result in Early-Onset Encephalopathy. AM J HUM GENET. [Journal Article]. 2016 2016-09-01;99(3):695\u0026ndash;703.\u003c/li\u003e\n\u003cli\u003eMuona M, Ishimura R, Laari A, Ichimura Y, Linnankivi T, Keski-Filppula R, et al. Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy. AM J HUM GENET. [Journal Article]. 2016 2016-09-01;99(3):683\u0026thinsp;\u0026minus;\u0026thinsp;94.\u003c/li\u003e\n\u003cli\u003eArnadottir GA, Jensson BO, Marelsson SE, Sulem G, Oddsson A, Kristjansson RP, et al. Compound heterozygous mutations in UBA5 causing early-onset epileptic encephalopathy in two sisters. BMC MED GENET. [Case Reports; Journal Article]. 2017 2017-10-02;18(1):103.\u003c/li\u003e\n\u003cli\u003eTumiene B, Maver A, Writzl K, Hodzic A, Cuturilo G, Kuzmanic-Samija R, et al. Diagnostic exome sequencing of syndromic epilepsy patients in clinical practice. CLIN GENET. [Journal Article]. 2018 2018-05-01;93(5):1057-62.\u003c/li\u003e\n\u003cli\u003eDaida A, Hamano SI, Ikemoto S, Matsuura R, Nakashima M, Matsumoto N, et al. Biallelic loss-of-function UBA5 mutations in a patient with intractable West syndrome and profound failure to thrive. EPILEPTIC DISORD. [Case Reports; Journal Article]. 2018 2018-08-01;20(4):313-8.\u003c/li\u003e\n\u003cli\u003eLow KJ, Baptista J, Babiker M, Caswell R, King C, Ellard S, et al. Hemizygous UBA5 missense mutation unmasks recessive disorder in a patient with infantile-onset encephalopathy, acquired microcephaly, small cerebellum, movement disorder and severe neurodevelopmental delay. EUR J MED GENET. [Case Reports; Journal Article]. 2019 2019-02-01;62(2):97\u0026ndash;102.\u003c/li\u003e\n\u003cli\u003eHamilton E, Bertini E, Kalaydjieva L, Morar B, Dojcakova D, Liu J, et al. UFM1 founder mutation in the Roma population causes recessive variant of H-ABC. NEUROLOGY. [Journal Article]. 2017 2017-10-24;89(17):1821-8.\u003c/li\u003e\n\u003cli\u003eNahorski MS, Maddirevula S, Ishimura R, Alsahli S, Brady AF, Begemann A, et al. Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development. BRAIN. [Journal Article; Research Support, Non-U.S. Gov't]. 2018 2018-07-01;141(7):1934-45.\u003c/li\u003e\n\u003cli\u003eYu L, Li G, Deng J, Jiang X, Xue J, Zhu Y, et al. The UFM1 cascade times mitosis entry associated with microcephaly. FASEB J. [Journal Article; Research Support, Non-U.S. Gov't]. 2020 2020-01-01;34(1):1319-30.\u003c/li\u003e\n\u003cli\u003eLiu M, Ran J, Zhou J. Non-canonical functions of the mitotic kinesin Eg5. THORAC CANCER. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2018 2018-08-01;9(8):904\u0026thinsp;\u0026minus;\u0026thinsp;10.\u003c/li\u003e\n\u003cli\u003eMann BJ, Wadsworth P. Kinesin-5 Regulation and Function in Mitosis. TRENDS CELL BIOL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.; Review]. 2019 2019-01-01;29(1):66\u0026ndash;79.\u003c/li\u003e\n\u003cli\u003eLiu J, Guan D, Dong M, Yang J, Wei H, Liang Q, et al. UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination. NAT CELL BIOL. [Journal Article; Research Support, Non-U.S. Gov't]. 2020 2020-09-01;22(9):1056-63.\u003c/li\u003e\n\u003cli\u003eYoo HM, Park JH, Kim JY, Chung CH. Modification of ERalpha by UFM1 Increases Its Stability and Transactivity for Breast Cancer Development. MOL CELLS. [Journal Article]. 2022 2022-06-30;45(6):425\u0026thinsp;\u0026minus;\u0026thinsp;34.\u003c/li\u003e\n\u003cli\u003eZhu J, Ma X, Jing Y, Zhang G, Zhang D, Mao Z, et al. P4HB UFMylation regulates mitochondrial function and oxidative stress. FREE RADICAL BIO MED. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-08-01;188:277\u0026thinsp;\u0026minus;\u0026thinsp;86.\u003c/li\u003e\n\u003cli\u003eBlangy A, Lane HA, D'Herin P, Harper M, Kress M, Nigg EA. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. CELL. [Journal Article; Research Support, Non-U.S. Gov't]. 1995 1995-12-29;83(7):1159-69.\u003c/li\u003e\n\u003cli\u003eZheng J, Tan Y, Liu X, Zhang C, Su K, Jiang Y, et al. NAT10 regulates mitotic cell fate by acetylating Eg5 to control bipolar spindle assembly and chromosome segregation. CELL DEATH DIFFER. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-04-01;29(4):846\u0026thinsp;\u0026minus;\u0026thinsp;60.\u003c/li\u003e\n\u003cli\u003ePirone L, Xolalpa W, Sigurethsson JO, Ramirez J, Perez C, Gonzalez M, et al. A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation. SCI REP-UK. [Journal Article; Research Support, Non-U.S. Gov't]. 2017 2017-01-18;7:40756.\u003c/li\u003e\n\u003cli\u003eIshimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo M, et al. The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3. NAT COMMUN. [Journal Article; Research Support, Non-U.S. Gov't]. 2022 2022-12-21;13(1):7857.\u003c/li\u003e\n\u003cli\u003eQin B, Yu J, Nowsheen S, Wang M, Tu X, Liu T, et al. UFL1 promotes histone H4 ufmylation and ATM activation. NAT COMMUN. [Journal Article; Research Support, N.I.H., Extramural]. 2019 2019-03-18;10(1):1242.\u003c/li\u003e\n\u003cli\u003eWang Z, Gong Y, Peng B, Shi R, Fan D, Zhao H, et al. MRE11 UFMylation promotes ATM activation. NUCLEIC ACIDS RES. [Journal Article; Research Support, Non-U.S. Gov't]. 2019 2019-05-07;47(8):4124-35.\u003c/li\u003e\n\u003cli\u003eLuo H, Jiao QB, Shen CB, Gong WY, Yuan JH, Liu YY, et al. UFMylation of HRD1 regulates endoplasmic reticulum homeostasis. FASEB J. [Journal Article; Research Support, Non-U.S. Gov't]. 2023 2023-11-01;37(11):e23221.\u003c/li\u003e\n\u003cli\u003eWang X, Xu X, Wang Z. The Post-Translational Role of UFMylation in Physiology and Disease. CELLS-BASEL. [Journal Article; Review]. 2023 2023-10-29;12(21).\u003c/li\u003e\n\u003cli\u003eWalczak CP, Leto DE, Zhang L, Riepe C, Muller RY, DaRosa PA, et al. Ribosomal protein RPL26 is the principal target of UFMylation. P NATL ACAD SCI USA. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2019 2019-01-22;116(4):1299\u0026thinsp;\u0026minus;\u0026thinsp;308.\u003c/li\u003e\n\u003cli\u003eWang L, Xu Y, Rogers H, Saidi L, Noguchi CT, Li H, et al. UFMylation of RPL26 links translocation-associated quality control to endoplasmic reticulum protein homeostasis. CELL RES. [Journal Article; Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural]. 2020 2020-01-01;30(1):5\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eLiang JR, Lingeman E, Luong T, Ahmed S, Muhar M, Nguyen T, et al. A Genome-wide ER-phagy Screen Highlights Key Roles of Mitochondrial Metabolism and ER-Resident UFMylation. CELL. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.]. 2020 2020-03-19;180(6):1160-77.\u003c/li\u003e\n\u003cli\u003eYoo HM, Kang SH, Kim JY, Lee JE, Seong MW, Lee SW, et al. Modification of ASC1 by UFM1 is crucial for ERalpha transactivation and breast cancer development. MOL CELL. [Journal Article; Research Support, Non-U.S. Gov't]. 2014 2014-10-23;56(2):261\u0026thinsp;\u0026minus;\u0026thinsp;74.\u003c/li\u003e\n\u003cli\u003eYang J, Zhou Y, Xie S, Wang J, Li Z, Chen L, et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J EXP CLIN CANC RES. [Journal Article]. 2021 2021-06-23;40(1):206.\u003c/li\u003e\n\u003cli\u003eZhou J, Ma X, He X, Chen B, Yuan J, Jin Z, et al. Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target. P NATL ACAD SCI USA. [Journal Article; Research Support, Non-U.S. Gov't]. 2023 2023-03-14;120(11):e2079235176.\u003c/li\u003e\n\u003cli\u003eLi H, Yu Z, Niu Z, Cheng Y, Wei Z, Cai Y, et al. A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila. CELL MOL LIFE SCI. [Journal Article]. 2023 2023-04-22;80(5):129.\u003c/li\u003e\n\u003cli\u003eHayashi MT, Karlseder J. DNA damage associated with mitosis and cytokinesis failure. ONCOGENE. [Journal Article; Research Support, N.I.H., Extramural; Review]. 2013 2013-09-26;32(39):4593\u0026thinsp;\u0026minus;\u0026thinsp;601.\u003c/li\u003e\n\u003cli\u003eHeijink AM, Krajewska M, van Vugt MA. The DNA damage response during mitosis. MUTAT RES-FUND MOL M. [Journal Article; Research Support, Non-U.S. Gov't; Review]. 2013 2013-10-01;750(1\u0026ndash;2):45\u0026ndash;55.\u003c/li\u003e\n\u003cli\u003eRapley J, Nicolas M, Groen A, Regue L, Bertran MT, Caelles C, et al. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation. J CELL SCI. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2008 2008-12-01;121(Pt 23):3912-21.\u003c/li\u003e\n\u003cli\u003eSmith E, H\u0026eacute;garat N, Vesely C, Roseboom I, Larch C, Streicher H, et al. Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1. EMBO J. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2011 2011-06-01;30(11):2233-45.\u003c/li\u003e\n\u003cli\u003eDuan Y, Huo D, Gao J, Wu H, Ye Z, Liu Z, et al. Ubiquitin ligase RNF20/40 facilitates spindle assembly and promotes breast carcinogenesis through stabilizing motor protein Eg5. NAT COMMUN. [Journal Article; Research Support, Non-U.S. Gov't]. 2016 2016-08-25;7:12648.\u003c/li\u003e\n\u003cli\u003eBickel KG, Mann BJ, Waitzman JS, Poor TA, Rice SE, Wadsworth P. Src family kinase phosphorylation of the motor domain of the human kinesin-5, Eg5. CYTOSKELETON. [Journal Article]. 2017 2017-09-01;74(9):317\u0026thinsp;\u0026minus;\u0026thinsp;30.\u003c/li\u003e\n\u003cli\u003eMuretta JM, Reddy B, Scarabelli G, Thompson AF, Jariwala S, Major J, et al. A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function. P NATL ACAD SCI USA. [Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't]. 2018 2018-02-20;115(8):E1779-88.\u003c/li\u003e\n\u003cli\u003eHazan F, Ostergaard P, Ozturk T, Kantekin E, Atlihan F, Jeffery S, et al. A novel KIF11 mutation in a Turkish patient with microcephaly, lymphedema, and chorioretinal dysplasia from a consanguineous family. AM J MED GENET A. [Case Reports; Journal Article]. 2012 2012-07-01;158A(7):1686-9.\u003c/li\u003e\n\u003cli\u003eMirzaa GM, Enyedi L, Parsons G, Collins S, Medne L, Adams C, et al. Congenital microcephaly and chorioretinopathy due to de novo heterozygous KIF11 mutations: five novel mutations and review of the literature. AM J MED GENET A. [Case Reports; Journal Article; Research Support, N.I.H., Extramural; Review]. 2014 2014-11-01;164A(11):2879-86.\u003c/li\u003e\n\u003cli\u003eJones GE, Ostergaard P, Moore AT, Connell FC, Williams D, Quarrell O, et al. Microcephaly with or without chorioretinopathy, lymphoedema, or mental retardation (MCLMR): review of phenotype associated with KIF11 mutations. EUR J HUM GENET. [Journal Article; Research Support, Non-U.S. Gov't]. 2014 2014-07-01;22(7):881-7.\u003c/li\u003e\n\u003cli\u003eLopes-Pacheco M. CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine. FRONT PHARMACOL. [Journal Article; Review]. 2019 2019-01-20;10:1662.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3754446/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3754446/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays critical roles in the maintenance of endoplasmic reticulum (ER) proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in \u003cem\u003eUFM1\u003c/em\u003e, as well as in its specific E1 enzyme \u003cem\u003eUBA5\u003c/em\u003e and E2 enzyme \u003cem\u003eUFC1\u003c/em\u003e, have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective mutant of Eg5 (K564R) displayed shortened or asymmetrical spindles, and suppressed cell proliferation in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression and cell proliferation.\u003c/p\u003e","manuscriptTitle":"Eg5 UFMylation promotes spindle organization during mitosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-18 09:13:35","doi":"10.21203/rs.3.rs-3754446/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-02-06T16:41:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-02-06T04:02:58+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-01-24T01:26:46+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-22T09:31:54+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-01-17T03:48:40+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-01-16T17:18:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-12-15T11:10:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-14T16:03:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2023-12-14T16:03:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab8c7bc1-b660-402d-8e97-f3884ac81127","owner":[],"postedDate":"January 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28179307,"name":"Biological sciences/Cell biology/Post-translational modifications"},{"id":28179308,"name":"Biological sciences/Cell biology/Cell division"}],"tags":[],"updatedAt":"2024-08-01T07:18:55+00:00","versionOfRecord":{"articleIdentity":"rs-3754446","link":"https://doi.org/10.1038/s41419-024-06934-w","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-07-31 04:00:00","publishedOnDateReadable":"July 31st, 2024"},"versionCreatedAt":"2024-01-18 09:13:35","video":"","vorDoi":"10.1038/s41419-024-06934-w","vorDoiUrl":"https://doi.org/10.1038/s41419-024-06934-w","workflowStages":[]},"version":"v1","identity":"rs-3754446","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3754446","identity":"rs-3754446","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}