The UFM1 conjugation system in mammalian development

Posttranslational modifications by ubiquitin and ubiquitin‐like proteins are important in regulating cellular protein functions. UFM1 (ubiquitin‐fold modifier 1), first identified almost two decades ago, is a member of the ubiquitin‐like protein family. UFM1 is covalently conjugated to the target proteins in an enzymatic cascade consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. At the molecular level, modification by UFM1 (UFMylation) is an important mediator of the protein function. Dysregulation of the UFM1 system, e.g., the knockout of UFMylation components, disturbs proteome homeostasis and triggers endoplasmic reticulum stress. Such changes are linked to developmental disorders, tumorigenesis, tissue injury, inflammation, and several hereditary neurological syndromes. This review will focus on the role of the UFMylation in animal development and associated congenital disorders. We will cover the hematopoietic system, liver, central nervous system, intestine, heart, kidney, immune, and skeletal system to provide insight into disease pathogenesis and shed light on possible novel therapeutic methods.

Posttranslational modifications (PTM) by ubiquitin (Ub) and ubiquitin-like proteins (Ubls) lead to their covalent conjugation to target proteins, which is essential for many cellular processes. 1 Ubiquitin-fold modifier 1 (UFM1) is a Ubl that was first identified in 2004. 2 Similar to ubiquitin, UFM1 is conjugated to its target proteins by the E1-E2-E3 cascade reaction ( Figure 1). First, the UFM1 precursor pro-UFM1 is cleaved by UFM1-specific proteases (UFSPs) UFSP1 and UFSP2 to expose the C-terminal glycine. 3,4 Next, ubiquitin-like modifieractivating enzyme 5 (UBA5) dimer forms a thioester bond between the UFM1 C-terminus and the catalytic Cys250 of UBA5 via a trans-binding model. UBA5 acting as the E1 recruits and hydrolyzes one molecule of ATP to activate UFM1. 2,5 UBA5 then transfers the activated UFM1 to UFM1-conjugating enzyme 1 (UFC1), which acts as the E2 to form a thioester bond. 6 Finally, UFM1-specific ligase 1 (UFL1) acts as the E3 by recognizing its protein substrates and transferring UFM1 to it. 7 The conjugation of UFM1 to its substrates can be reversed via cleavage by UFSP1 and UFSP2. 3,4 In addition to these key components of the UFM1 system, yeast two-hybrid (Y2H) screening has revealed UFM1 binding protein 1 (known Shuchun Yang and Rui Yang contributed equally to this study. as UFBP1, C20orf116, or DDRGK1) and CDK5 Regulatory Subunit Associated Protein 3 (known as CDK5RAP3, LZAP, or C53) as the binding proteins for UFL1 E3 ligase. 8 Furthermore, an in vitro UFMylation approach with purified components was adopted to study the roles of UFBP1 and CDK5RAP3 in UFMylation. 8 It was revealed that UFL1 forms a functional scaffold-type E3 ligase complex with UFBP1, thereby activating aminolysis by UFC1. [8][9][10] It was also revealed that CDK5RAP3 inhibits UFMylation by preventing the discharge of UFM1 from UFC1. 8 However, some studies show that CDK5RAP3 is required for protein UFMylation, and its deficiency significantly disturbs the protein UFMylation both in vitro and in vivo. 11,12 There have been several UFM1 substrates identified thus far. RPL26, the principal target of UFMylation, is involved in safeguarding co-translational protein translocation into the endoplasmic reticulum (ER) and executing ER-phagy. 12,13 The UFMylated nuclear receptor coactivator ASC1 enhances the recruitment of transcription cofactors p300, SRC1, and itself to estrogen receptor α (ERα) target gene promoters to activate their expression. Transcription initiation factor eIF6 and some ribosomal proteins such as uS3 and uS10 can also be UFMylated, which may coordinate subunit joining and mRNA interactions. 14 In the DNA damage response, the key component of the MRE11-RAD50-NBS1 (MRN) complex, MRE11, is UFMylated, which promotes ATM activation and genome stability. 15 Histone 4 is also UFMylated following DNA damage to maintain genomic integrity. 16,17 P53, P4HB, and ERα can all be UFMylated to antagonize degradation via the ubiquitin-proteasome pathway. 10,18,19 It has been proposed that most UFMylation components reside in the ER. UFL1, UFSP2, UFBP1, and CDK5RAP3 have all been shown to aggregate in a large protein complex at the cytosolic side of the ER membrane. [20][21][22][23] The UFMylation system plays an indispensable role in maintaining ER homeostasis, and loss or mutation of its components results in ER stress. 12,13,[24][25][26][27][28] Other reported cellular functions of the UFMylation system include ER-phagy regulation, protein transactivation, telomere length maintenance, DNA damage response, P53 stability maintenance, autophagy, and apoptosis. 9,10,15,17,21,24,[29][30][31] Animal studies have implicated the UFMylation system in a variety of diseases, including tumorigenesis, neurological disorders, and developmental disorders. 32,33

| THE UFM1 CONJUGATION SYSTEM IN HEMATOPOIESIS
Hematopoiesis is a hierarchically organized process that originates from hematopoietic stem cells (HSCs). 34 The fetal liver is the major hematopoietic organ during mammalian prenatal development, allowing HSCs to expand and differentiate into all mature blood cell lineages. 35 After birth, the bone marrow becomes the predominant site of hematopoiesis. 36 To date, genetic studies have provided solid evidence supporting the essential role of the UFM1 system in murine hematopoiesis, especially erythroid development, in both embryos and adults ( Figure 2 and Table 1). 31 UBA5 is crucial for embryonic development and erythroid differentiation in mice. 37 Uba5-null (Uba5 À/À ) mice die from severe anemia (E12.5-E13.5) due to defective erythropoiesis and megakaryopoiesis. 37 This defective phenotype is rescued in Uba5 À/À mice with transgenic expression of Uba5 in the erythroid and megakaryocytic progenitors, suggesting cell-autonomous impairment of erythropoiesis. 37 UBA5 knockdown in F I G U R E 1 The ubiquitin-fold modifier 1 (UFM1) cascade. Two amino acids (labeled as XX) are cleaved from the precursor of UFM1 (labeled as pro-UFM) to form mature UFM1 and expose glycine by UFSP1/2. Then, the mature UFM1 is adenylated and activated by UBA5. After activation, UFM1 is transferred to UFC1. The ligation of UFM1 to target proteins is mediated by UFM1-specific ligase 1 (UFL1) and its two adaptor proteins, UFBP1 and CDK5RAP3. Finally, the UFM1 is recycled from substrates by UFSP1/2. erythroleukemia K562 cells activates unfolded protein response (UPR), suggesting that UBA5 is involved in regulation of ER homeostasis. 38 The mice carrying homozygous Ufl1 trapped alleles Ufl1 Trap-F/Trap-F (Ufl1 À/À ) also suffer from prenatal lethality. 31 These mice die around E10.5-E11.5, with small body size, small fetal liver, and anemia. 31 Colony formation assays of fetal livers (E11.5) suggest impaired development of erythroid progenitors. 31 Conditional Ufl1 knockout mice Ufl1 F/F ;ROSA26-CreERT2 also display severe anemia and die 3 weeks after tamoxifen administration in adulthood, further suggesting that UFL1 is indispensable in adult hematopoiesis. 31 Competitive repopulation assays reveal a cell-autonomous effect of UFL1 in hematopoiesis. 31 At the cellular level, it is proposed that the apoptosis and cell cycle arrest in Ufl1depleted HSCs are caused by P53 activation triggered by ER stress and attenuation of autophagic degradation. 31 Impaired embryonic development and hematopoiesis are also observed in the knockout mice of two UFMylation adaptors: CDK5RAP3 and UFBP1. 11,38 Constitutive Cdk5rap3 knockout mice Cdk5rap3 tm1b/tm1b (Cdk5rap3 À/À ) die from E16.5 onwards. 11 In E14.5 Cdk5rap3 tm1b/tm1b fetal livers, a decreased number of hematopoietic stem cells (HSCs) is observed. Furthermore, flow cytometry of mouse erythroid progenitors indicates impaired erythropoiesis (unpublished data).
The mice carrying homozygous Ufbp1 trapped alleles Ufbp1 Trap-F/Trap-F (Ufbp1 À/À ) die at E12.5, with both primitive and definitive erythropoiesis compromised. 38 Similar to Ufl1, Ufbp1 F/F ;ROSA26-CreERT2 mice also die 3 weeks after tamoxifen administration. These mice display pancytopenia, indicating the need for UFBP1 in adult hematopoiesis. 38 Competitive repopulation assays reveal that UFBP1 regulates hematopoiesis in a cell-autonomous manner. UFBP1-depeted Lin À Sca-1 + c-Kit + (LSK) cells display elevated ER stress and up-regulated ER stressassociated apoptotic genes. 38 In vitro assays show that the depletion of UBA5, UFBP1, or ASC1 in erythroleukemia K562 cells lead to significant underexpression of GATA-1 and KLF1, suggesting that these proteins play a critical role in the regulation of erythroid transcription programs. 38 Chromatin-immunoprecipitation (ChIP) assay suggests that ASC1 is one of the key downstream effectors of the Ufm1 system that transcriptionally regulates erythroid lineage development. 38

| THE UFM1 CONJUGATION SYSTEM IN LIVER DEVELOPMENT
The liver is critical for organismal homeostasis. 47 In mouse liver development, the liver bud forms between E9.0 and E9.5. 48 The differentiation of bi-potential hepatoblasts into either hepatocytes or biliary epithelial cells happens around E13. 48 Multiple lineage relationships and signaling pathways orchestrate liver development. 49 In a few studies, UFMylation has been implied to regulate liver development ( Figure 2 and Table 1).
Anemia due to compromised erythropoiesis and megakaryopoiesis is proposed to be the main cause of death in Uba5 À/À mice, though severe liver hypoplasia has also been observed. 37 Transgenic expression of Uba5 in the erythroid lineage rescues the Uba5-deficient embryos from anemia. However, the mice still die by E18.5, possibly from the severe liver hypoplasia. 37 This F I G U R E 2 The UFM1 conjugation system regulates the mammalian development, including hematopoietic system, liver, central nervous system, intestine, heart, kidney, and immune and skeletal system. T A B L E 1 UFMylation in mammalian development.

Organ/system
Gene Types of mutation Phenotype Reference

| THE UFM1 CONJUGATION SYSTEM IN THE DEVELOPMENT OF CENTRAL NERVOUS SYSTEM
The central nervous system (CNS) consists of the brain and spinal cord and is one of most complex body systems in mammals. 50 The CNS is asymmetric and divided into multiple regions that collectively regulate human cognition and behavior. 51 The CNS originates from the neural tube formed by embryonic ectoderm on gestational day 24-28 (E9-E9.5 in mice). 52 CNS development continues after birth and is completed in adulthood. 52 There is a substantial literature suggesting that the UFMylation system is crucial for CNS development ( Figure 2 and Table 1). Ufbp1 À/À embryos die at E12.5, with cell death detected in the brain. 38 In CNS-specific Ufm1 knockout (Ufm1 F/F ;Nestin-Cre) mice generated to study UFMylation in the CNS development, the mutant mice survive to birth but die after 1 day. 40 Macroscopic anatomical and histological analyses reveal microcephaly and neuron apoptosis in the occipital region of the neopallium in E18.5 mutant mice. 40 Neuron-specific Ufbp1 knockout (Ufbp1 F/F ;Camka2-Cre) and Ufl1 knockout (Ufl1 F/F ;Cam-ka2-Cre) mice show elevated inflammatory response and remarkable neuronal loss due to cell death, indicating that Ufbp1 and Ufl1 are both essential for mature neuron survival in murine adult brains. 41 Interestingly, seizurelike events are observed in mice with loss of one allele of the Ufbp1 gene. 41 In addition to its role in neurons, UFL1 has been implicated in the non-cell-autonomous pathology of spinocerebellar ataxia type 1. Mutant ataxin-1 (Atx1) down-regulates UFL1 expression, subsequently inhibiting Bergmann glia proliferation during development by regulating the G1/S transition. 23 Abnormal neurological phenotypes are also observed in Uba5knockdown Caenorhabditis elegans, Drosophila, and zebrafish, further supporting the role of UBA5 in neurodevelopment. 40,53,54 A number of gene variants involved in the UFMylation system have been identified in human congenital disorders ( Table 2). A homozygous UFM1 mutation is found in patients with severe early-onset encephalopathy with progressive microcephaly. 55 This UFM1 mutation significantly diminishes the formation of UFM1-UBA5 and UFM1-UFC1 intermediates, which results in impaired protein UFMylation. 55 Additionally, a mutation in the promoter region of UFM1 is present in a diverse population of patients with hypomyelination and atrophy of the basal ganglia and cerebellum. [56][57][58] The deletion of the UFM1 promoter reduces the UFM1 activity in neuroblastoma and astroglioma cell lines. 58 Multiple UBA5 variants are found in patients with neurodevelopment disorders, including autosomal recessive cerebellar ataxia, hypomyelination with atrophy, early-onset encephalopathy, and early myoclonic epilepsy. 40,53,54,[59][60][61] In addition, UFC1 mutation is also present in patients with severe early-onset encephalopathy with progressive microcephaly. 55 This UFC1 mutation impairs the formation of UFM1-UFC1 intermediates, which results in widespread decrease of protein UFMylation. 55 The mutation of UFSP2 is also found in patients with cerebral visual impairment. 62 Recently, a UFSP2 variant has been reported as the pathogenic cause of an autosomal recessive form of pediatric neurodevelopmental anomaly and epilepsy. 63

| ADDITIONAL ROLES OF THE UFM1 CONJUGATION SYSTEM IN MAMMALIAN DEVELOPMENT
The UFM1 conjugation system also functions in intestine, heart, kidney, immune, and skeletal development ( Figure 2 and Table 1). The intestinal epithelium is composed of a single layer of epithelial cells, and the integrity of which is maintained by renewal and differentiation of intestinal stem cells. 66 Intestinal secretory cells, such as goblet and Paneth cells, are crucial in maintaining intestinal homeostasis. Conditional Ufl1 knockout (Ufl1 F/F : ROSA26-CreERT2) results in substantial loss of Paneth and goblet cells in intestinal sections following acute ablation of Ufl1 in adult mice by tamoxifen administration. 42 The specific deletion of Ufbp1 in intestinal epithelial cell model mice (Ufbp1 Δ/ΔIEC ) also causes depletion of Paneth and goblet cells. 42 The mice with Paneth cellspecific deletion of Ufbp1 (Ufbp1 F/F ;D6-Cre) only show a decrease of Paneth cells. 42 At the cellular level, Ufbp1 deficiency triggers ER stress and activates the UPR and cell death programs. 42 During the mouse heart development, UFMylated protein abundance gradually increases over time. 43 A significant reduction in UFL1 expression is observed in the infarct area of mouse hearts subjected to ischemia/reperfusion surgery, suggesting a potential role of UFL1 in regulating cardiomyocyte survival. 43 Cardiomyocyte-restricted Ufl1 knockout mice (Ufl1 F/F ;α MHC-Cre) have been generated to further explore the role of UFL1 in heart development. These mutant mice survive to birth and have no morphological differences from controls. 43 However, the cardiac function of mutant mice starts to deteriorate progressively from 2 to 6 months. 43 ER stress is triggered in the mutant hearts, which subsequently impairs PKR-like ER-resident kinase signaling and aggravates cardiomyocyte cell death. 43 UFL1 is also involved in renal homeostasis. Nephron tubule-specific Ufl1 knockout mice (Ufl1 F/F ;Pax8-Cre) show kidney atrophy and interstitial fibrosis, which may be explained by the activation of UPR and cell apoptosis. 44 The UFM1 conjugate system is also involved in the development of the immune system. Ufl1 F/F ; ROSA26-CreERT2 and Ufbp1 F/F ;ROSA26-CreERT2 mice show decreased counts of lymphocytes, granulocytes, UFBP1 Shohat-type spondyloepimetaphyseal dysplasia NM_023935.3:c.408+1G>A splicing [45] monocytes, and platelet after tamoxifen injection in adulthood. 31,38 In B-cell-specific Ufbp1 knockout mice (Ufbp1 F/F ;CD19-Cre), Ufbp1 promotes naive B-cell differentiation into plasma cells by suppressing the activation of the PERK pathway. 46 In addition, Ufbp1 and the UFMylation system are both induced by the IRE1α/XBP1 axis to expand the ER network in plasma cells. 46 The UFM1 conjugate system also plays a role in regulating the skeletal development. A delay or absence of the early stages of chondrogenesis is observed in Ufbp1 À/À mice. 45 In humans, UFBP1 loss of function is a causative factor in Shohat-type spondyloepimetaphyseal dysplasia, a skeletal dysplasia that affects cartilage development. 45 Additionally, mutations in UFSP2 are found in patients with Beukes hip dysplasia and spondyloepimetaphyseal dysplasia. 64,65 6 | DISCUSSION The UFMylation system has been known for almost two decades, and its functions have been explored extensively. In this review, we summarize the role of the UFM1 conjugation system in the mammalian development. The key components of the UFM1 conjugation system are critical for murine development, including the hematopoietic system, liver, CNS, intestine, heart, kidney, immune system, and skeletal system ( Figure 2). Global knockout of Uba5, Ufl1, Ufbp1, and Cdk5rap3 all lead to prenatal lethality. Severe anemia due to impaired hematopoiesis and liver hypoplasia are common phenotypes in these mice, though liver hypoplasia is only wellcharacterized in Cdk5rap3 À/À mice. Since the fetal liver is the primary hematopoietic organ during the mouse development, the impaired hematopoiesis may be the result of liver hypoplasia. However, both rescue experiments in Uba5 À/À mice and competitive repopulation assays in Ufl1 À/À and Ufbp1 À/À mice indicate that the UFM1 conjugation system impacts hematopoiesis in a cell-autonomous manner. Some components of the UFM1 conjugation system have also been revealed to function in brain, gut, kidney, heart, and skeleton development. So far, Ufm1, Ufc1, Ufsp1, and Ufsp2 global knockout mice have not been reported, and it is necessary to characterize their phenotypes to comprehensively understand the system. Other model organisms have also been used to explore the functions of UFMylation, including Drosophila, Caenorhabditis elegans, and zebrafish. 53,54 The phenotypes from UFMylation deficiency are similar among these different species, indicating that the UFMylation system is highly conservative.
Mammalian developmental studies show that impairment of the UFMylation system carries strong implications for the development of human genetic diseases, especially neurodevelopmental and skeletal development disorders. For instance, CNS-specific Ufm1 knockout mice suffer from microcephaly, a condition that is also observed in humans with UFM1 mutation. 40,55 Delayed limb bud chondrogenic condensation is observed in two types of Ufbp1 À/À mice generated by LoxP-Cre and CRISPR-Cas9 systems. 38,45 Similar pathologic changes are also reported in the skeletons of patients with UFBP1 variants. 45 Although severe erythropoietic, hepatic, and renal phenotypes are reported in mice and zebrafish with the UFMylation gene knockout, similar pathologic changes are not reported in humans with UFMylation variants. It is possible that all reported variants in human are hypomorphic. A recent study linked UFMylation to erythroid differentiation in humans, using an in vitro erythroid differentiation model to show that protein UFMylation promotes the biogenesis of human ER proteins. 13 Considering the crucial role of the UFMylation system in hematopoiesis, it is plausible that some anemia symptoms in humans may result from disturbed protein UFMylation, though the exact relationship remains to be discovered. Since mouse and human development is conserved but divergent, future human developmental studies may benefit from the differentiation of human pluripotent stem cells (hPSCs), including embryonic and induced pluripotent stem cells.
The function of UFMylation components in the mammalian development has been explored by generating global gene knockout and tissue-specific gene knockout mouse. However, the function of protein UFMylation modifications in development is unclear. Some literature has linked UFL1 adaptor CDK5RAP3 deficiency to alteration of the UFMylation profile in the liver development. 11 During mouse heart development, UFMylated protein abundance gradually increases over time. 43 The RPL26 UFMylation level also increases during erythroid differentiation. 13 This indicates that protein UFMylation may be involved in the organ development. In the future, mice with specific amino acids mutations resulting in impaired UFMylation modification, rather than whole gene deletion, can be used to interpret the function of UFMylation in the development more accurately.
UFMylation is involved in the regulation of proteome homeostasis, which is balanced by protein synthesis, protein transport, and protein degradation. 67 The ribosome is the primary organelle of protein synthesis, translating messenger RNA into amino acids. 68 Some ribosome proteins have been identified as UFMylated proteins, including uS3, uS10, and RPL26. 12,14 RPL26 UFMylation is upregulated in ribosome stalling, which signals translational mistakes and triggers the ribosome-associated quality control process during translation. 13,69 The biological effect of ribosome protein UFMylation still needs to be elucidated. For protein degradation, the Ubiquitin-proteasome system and autophagy are the two major pathways used by eukaryotes to degrade most cellular proteins. 70 Protein UFMylations have also been found in autophagy-associated screening studies. 24,71 More importantly, some key components of UFMylation participate in the regulation of autophagy. 72 No evidence has shown the involvement of UFMylation in the ubiquitin-proteasome system. These findings together indicate that UFMylation may mediate the proteome homeostasis through the autophagy pathway.
So far, UFL1 has been the only E3 ligase identified in protein UFMylation. Recent evidence shows that E3 ligase UFL1 forms a complex with its two adaptors, UFBP1 and CDK5RAP3, to cooperatively regulate the UFM1 conjugation of substrates. 8 One study shows that the deletion of Cdk5rap3, the UFL1 substrate adaptor, alters the UFMylation profile in livers. 11 Considering the different levels of CDK5RAP3 and UFBP1 in various organs, 11,21 there may be diversity in substrate UFMylation profiles in different organs under different states. Identification of these UFMylated substrates will be informative in deciphering the molecular mechanism behind the UFM1 conjugation system's regulation of mammalian homeostasis and disease. Moreover, these substrates may serve as potential therapeutic targets for diseases such as cancer, anemia, and organ degeneration.