Activating Signal Cointegrator Research Articles

ASCC1 structures and bioinformatics reveal a novel helix-clasp-helix RNA-binding motif linked to a two-histidine phosphodiesterase

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE) (associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high resolution details and features conserved over 500 million years of evolution. Extending understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence–specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1 roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

Extended DNA threading through a dual-engine motor module of the activating signal co-integrator 1 complex

Activating signal co-integrator 1 complex (ASCC) subunit 3 (ASCC3) supports diverse genome maintenance and gene expression processes, and contains tandem Ski2-like NTPase/helicase cassettes crucial for these functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. We present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. TRIP4 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning an ASC-1 homology domain next to the C-terminal helicase cassette of ASCC3, likely supporting substrate engagement and assisting the DNA exit. TRIP4 binds ASCC3 mutually exclusively with the DNA/RNA dealkylase, ALKBH3, directing ASCC3 for specific processes. Our findings define ASCC3-TRIP4 as a tunable motor module of ASCC that encompasses two cooperating NTPase/helicase units functionally expanded by TRIP4.

Comparative Analysis of Mesophilic YqfB-Type Amidohydrolases.

The widespread superfamily of the human activating signal cointegrator homology (ASCH) domain was identified almost 20 years ago; however, the amount of experimental data regarding the biological function of the domain is scarce. With this study, we aimed to determine the putative cellular functions of four hypothetical ASCH domain-containing amidohydrolase YqfB analogues by investigating their activity towards various N-acylated cytosine derivatives, including potential nucleoside-derived prodrugs, as well as their ability to bind/degrade nucleic acids in vitro. According to determined kinetic parameters, N4-acetylcytidine is assumed to be the primary substrate for amidohydrolases. Despite the similarity to the proteins containing the PUA domain, no nucleic acid binding activity was detected for YqfB-like proteins, suggesting that, in vivo, these enzymes are a part of the pyrimidine salvage pathway. We also demonstrate the possibility of the expression of YqfB-type amidohydrolases in both prokaryotic and eukaryotic hosts. The small protein size and remarkable halotolerance of YqfB-type amidohydrolases are of great interest for further fundamental research and biotechnological applications

Contrasting role of ASCC3 and ALKBH3 in determining genomic alterations in Glioblastoma Multiforme

Glioblastoma Multiforme (GBM) is the most malignant brain tumor with the highest fraction of genome alterations (FGA), manifesting poor disease-free status (DFS) and overall survival (OS). Methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) is the most promising biomarker known to have better therapy prediction and prognosis. However, MGMT’s predictive abilities are effective in only 50-60% GBM patients, suggesting a need to identify new biomarkers. We explored The Cancer Genome Atlas (TCGA) and cBioportal public dataset- Firehose legacy GBM to study DNA repair genes Activating Signal Cointegrator 1 Complex Subunit 3 (ASCC3) and Alpha-Ketoglutarate-Dependent Dioxygenase AlkB Homolog 3 (ALKBH3). To test our hypothesis that these genes have correlations with FGA and can better determine prognosis and survival, we sorted the dataset to arrive at 254 patients. Analyzing using RStudio, both ASCC3 and ALKBH3 demonstrated hypomethylation in 82.3% and 61.8% of patients, respectively. Interestingly, low mRNA expression was observed in both these genes. We further conducted correlation tests between both methylation and mRNA expression of these genes with FGA. ASCC3 was found to be negatively correlated, while ALKBH3 was found to be positively correlated, potentially indicating contrasting dysregulation of these two genes. Prognostic analysis showed the following: ASCC3 hypomethylation is significant with DFS and high ASCC3 mRNA expression to be significant with OS, demonstrating ASCC3’s potential as disease prediction marker. Further research using in vitro studies and mechanistic analysis of ASCC3 and ALKBH3 is needed to better understand their roles in causing genetic alterations in GBM.

Immunoaffinity Purification of Epitope-Tagged DNA Repair Complexes from Human Cells.

Immunoaffinity purification allows for the purification of epitope-tagged proteins and their associated multisubunit complexes from mammalian cells. Subsequent identification of the proteins by proteomic analysis enables unbiased biochemical characterization of their associated partners, potentially revealing the physiological or functional context of any given protein. Here, we use immunoaffinity isolation of the Activating Signal Co-integrator Complex (ASCC) from human cells as an example, demonstrating the utility of the approach in revealing protein complexes involved in genotoxic stress responses.

The ASCC2 CUE domain in the ALKBH3–ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3–Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.

Inherited Defects of the ASC-1 Complex in Congenital Neuromuscular Diseases.

Defects in transcriptional and cell cycle regulation have emerged as novel pathophysiological mechanisms in congenital neuromuscular disease with the recent identification of mutations in the TRIP4 and ASCC1 genes, encoding, respectively, ASC-1 and ASCC1, two subunits of the ASC-1 (Activating Signal Cointegrator-1) complex. This complex is a poorly known transcriptional coregulator involved in transcriptional, post-transcriptional or translational activities. Inherited defects in components of the ASC-1 complex have been associated with several autosomal recessive phenotypes, including severe and mild forms of striated muscle disease (congenital myopathy with or without myocardial involvement), but also cases diagnosed of motor neuron disease (spinal muscular atrophy). Additionally, antenatal bone fractures were present in the reported patients with ASCC1 mutations. Functional studies revealed that the ASC-1 subunit is a novel regulator of cell cycle, proliferation and growth in muscle and non-muscular cells. In this review, we summarize and discuss the available data on the clinical and histopathological phenotypes associated with inherited defects of the ASC-1 complex proteins, the known genotype–phenotype correlations, the ASC-1 pathophysiological role, the puzzling question of motoneuron versus primary muscle involvement and potential future research avenues, illustrating the study of rare monogenic disorders as an interesting model paradigm to understand major physiological processes.

Discovery of a neuromuscular syndrome caused by biallelic variants in ASCC3.

SummaryActivating Signal Cointegrator 1 Complex, Subunit 3 (ASCC3) is part of the four-part ASC-1 transcriptional cointegrator complex. This complex includes ASCC1 (associated with spinal muscular atrophy with congenital bone fractures 2), TRIP4 (associated with spinal muscular atrophy with congenital bone fractures 1), and ASCC2 (not yet associated with human disease.) ASCC3 encodes a DNA helicase responsible for generating single-stranded DNA as part of the DNA damage response. Interestingly, ASCC3 expresses coding and non-coding isoforms, which act in opposition to balance the recovery of gene transcription after UV-induced DNA damage. Here we report the discovery of ASCC3 as the cause of a neuromuscular syndrome in seven unreported individuals from six unrelated families and updates on the one previously reported family. All the individuals share a neurologic phenotype that ranges from severe developmental delay to muscle fatigue. There appears to be genotype-phenotype correlation, as the most mildly affected individual is homozygous for a rare missense variant, while the more severely affected individuals are compound heterozygotes for a missense and a presumed loss-of-function (LOF) variant. There are no individuals with biallelic presumed LOF variants in our cohort or in gnomAD, as this genotype may not be compatible with life. In summary we report a syndrome in these eleven individuals from seven families with biallelic variants in ASCC3.

The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the α-ketoglutarate-dependent dioxygenase AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2–ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2–ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2–ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.

Glycogen synthase kinase-3β inhibition alleviates activation of the NLRP3 inflammasome in myocardial infarction

Inflammasome-promoted sterile inflammation following cardiac damage is critically implicated in heart dysfunction after myocardial infarction (MI). Glycogen synthase kinase-3 (GSK-3β) is a prominent mediator of the inflammatory response, and high GSK-3 activity is associated with various heart diseases. We investigated the regulatory mechanisms of GSK-3β in activation of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in a rat model with successful induction of MI on days 2–28. An in vitro investigation was performed using newborn rat/human cardiomyocytes and fibroblast cultures under typical inflammasome stimulation and hypoxia treatment. GSK-3β inhibition markedly improved myocardial dysfunction and prevented remodeling, with parallel reduction in the parameters of NLRP3 inflammasome activation after MI. GSK-3β inhibition reduced NLRP3 inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes. GSK-3β's interaction with activating signal cointegrator (ASC) as well as GSK-3β inhibition reduced ASC phosphorylation and oligomerization at the tissues and cellular levels. Taken together, these data show that GSK-3β directly mediates NLRP3 inflammasome activation, causing cardiac dysfunction in MI.

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

In this study, the development of a rapid, high-throughput method for the selection of amide-hydrolysing enzymes from the metagenome is described. This method is based on uridine auxotrophic Escherichia coli strain DH10B ∆pyrFEC and the use of N4-benzoyl-2’-deoxycytidine as a sole source of uridine in the minimal microbial M9 medium. The approach described here permits the selection of unique biocatalysts, e.g., a novel amidohydrolase from the activating signal cointegrator homology (ASCH) family and a polyethylene terephthalate hydrolase (PETase)-related enzyme.

Identification of a novel trigger complex that facilitates ribosome-associated quality control in mammalian cells

Ribosome stalling triggers the ribosome-associated quality control (RQC) pathway, which targets collided ribosomes and leads to subunit dissociation, followed by proteasomal degradation of the nascent peptide. In yeast, RQC is triggered by Hel2-dependent ubiquitination of uS10, followed by subunit dissociation mediated by the RQC-trigger (RQT) complex. In mammals, ZNF598-dependent ubiquitination of collided ribosomes is required for RQC, and activating signal cointegrator 3 (ASCC3), a component of the ASCC complex, facilitates RQC. However, the roles of other components and associated factors of the ASCC complex remain unknown. Here, we demonstrate that the human RQC-trigger (hRQT) complex, an ortholog of the yeast RQT complex, plays crucial roles in RQC. The hRQT complex is composed of ASCC3, ASCC2, and TRIP4, which are orthologs of the RNA helicase Slh1(Rqt2), ubiquitin-binding protein Cue3(Rqt3), and zinc-finger type protein yKR023W(Rqt4), respectively. The ATPase activity of ASCC3 and the ubiquitin-binding activity of ASCC2 are crucial for triggering RQC. Given the proposed function of the RQT complex in yeast, we propose that the hRQT complex recognizes the ubiquitinated stalled ribosome and induces subunit dissociation to facilitate RQC.

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (kcat/Km up to 2.8 × 106 M−1 s−1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases.

Crystal Structure of Human EOLA1 Implies Its Possibility of RNA Binding.

Human endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) has been suggested to regulate inflammatory responses in endothelial cells by controlling expression of proteins, interleukin-6 and vascular cell adhesion molecule-1, and by preventing apoptosis. To elucidate the structural basis of the EOLA1 function, we determined its crystal structure at 1.71 Å resolution and found that EOLA1 is structurally similar to an activating signal cointegrator-1 homology (ASCH) domain with a characteristic β-barrel fold surrounded by α-helices. Despite its low sequence identity with other ASCH domains, EOLA1 retains a conserved ‘GxKxxExR’ motif in its cavity structure. The cavity harbors aromatic and polar residues, which are speculated to accommodate nucleotide molecules as do YT521-B homology (YTH) proteins. Additionally, EOLA1 exhibits a positively charged cleft, similar to those observed in YTH proteins and the ASCH protein from Zymomonas mobilis that exerts ribonuclease activity. This implies that the positively charged cleft in EOLA1 could stabilize the binding of RNA molecules. Taken together, we suggest that EOLA1 controls protein expression through RNA binding to play protective roles against endothelial cell injuries resulting from lipopolysaccharide (LPS)-induced inflammation responses.

Data-independent acquisition of the proteomics of spleens from chickens infected by avian leukosis virus.

Immunosuppression caused by avian leukemia virus J subgroup (ALV-J) infection includes atrophy or regeneration disorders of the lymphoid organs, decreased immune response, and termination of B lymphocyte maturation process and inhibition of T-lymphocyte development. The regulatory mechanism of the related resistance genes and protein expression is not clear. While searching for a molecular marker for the immune response to ALV-J infection, we detected differentially expressed proteins (DEPs) of spleens from chicken infected by ALV-J at 15th day and 30th day by the data-independent acquisition technique. Approximately 220 DEPs from the spleens of chickens infected by ALV-J were detected. To find a relatively stable biomarker molecule, we summarized the DEPs at two timepoints and selected activating signal cointegrator 1 complex subunit 3 (ASCC3), TBC1 domain family member 2 (TBC1D2), MHC class II beta chain 1 (BLB2), ensconsin (MAP7), complement component 1 Q subcomponent B chain (C1QB), and Follistatin-like 1 (FSTL1) from both comparisons for protein interaction network analysis. ASCC3, BLB2, C1QB, and FSTL1 were potential biomarkers for the complex infection mechanism of ALV-J and the dynamic immune mechanism of the body.

TRIP4 promotes tumor growth and metastasis and regulates radiosensitivity of cervical cancer by activating MAPK, PI3K/AKT, and hTERT signaling.

Thyroid hormone receptor interactor 4 (TRIP4), a subunit of the tetrameric nuclear activating signal co-integrator 1 (ASC-1) complex, exerts pro-tumorigenic effects. The role for TRIP4 in the regulation of cervical cancer growth and radiation resistance is presently unknown. In this study, TRIP4 was found to be highly expressed in cervical cancer cells and tumor tissues. Knockdown of TRIP4 significantly suppressed cervical cancer cell proliferation and epithelial-mesenchymal transition (EMT), accompanied by inactivation of PI3K/AKT and MAPK/ERK signaling. TRIP4 was also found to target hTERT signaling by regulating its binding to the hTERT promoter. Moreover, the knockdown of TRIP4 increased cell sensitivity to radiation, concomitant with downregulation of Rad51 and p-H2AX. We also demonstrated in an in vivo study that the knockdown of TRIP4 effectively suppressed cervical cancer growth and progression in a xenograft tumor model, and these effects were concomitant with the downregulation of p-AKT, p-ERK, p-MEK1/2, MMP-9 and hTERT expression. Immunohistochemical analysis of tumor tissue microarrays showed that TRIP4 overexpression predicted poor prognosis in patients with cervical cancer. Collectively, these results show that TRIP4 plays an essential role in cervical cancer growth and survival.

RNA ligase-like domain in activating signal cointegrator 1 complex subunit 1 (ASCC1) regulates ASCC complex function during alkylation damage

Multiple DNA damage response (DDR) pathways have evolved to sense the presence of damage and recruit the proper repair factors. We recently reported a signaling pathway induced upon alkylation damage to recruit the AlkB homolog 3, α-ketoglutarate-dependent dioxygenase (ALKBH3)-activating signal cointegrator 1 complex subunit 3 (ASCC3) dealkylase-helicase repair complex. As in other DDR pathways, the recruitment of these repair factors is mediated through a ubiquitin-dependent mechanism. However, the machinery that coordinates the proper assembly of this repair complex and controls its recruitment is still poorly defined. Here, we demonstrate that the ASCC1 accessory subunit is important for the regulation of ASCC complex function. ASCC1 interacts with the ASCC complex through the ASCC3 helicase subunit. We find that ASCC1 is present at nuclear speckle foci prior to damage, but leaves the foci in response to alkylation. Strikingly, ASCC1 loss significantly increases ASCC3 foci formation during alkylation damage, yet most of these foci lack ASCC2. These results suggest that ASCC1 coordinates the proper recruitment of the ASCC complex during alkylation, a function that appears to depend on a putative RNA-binding motif near the ASCC1 C terminus. Consistent with its role in alkylation damage signaling and repair, ASCC1 knockout through a CRISPR/Cas9 approach results in alkylation damage sensitivity in a manner epistatic with ASCC3. Together, our results identify a critical regulator of the ALKBH3-ASCC alkylation damage signaling pathway and suggest a potential role for RNA-interacting domains in the alkylation damage response.

A ubiquitin-dependent signalling axis specific for ALKBH-mediated DNA dealkylation repair.

SummaryDNA repair is essential to prevent the cytotoxic or mutagenic effects of various types of DNA lesions, which are sensed by distinct pathways to recruit repair factors specific to the damage type. Although biochemical mechanisms for repairing several forms of genomic insults are well understood, the upstream signaling pathways that trigger repair are established for only certain types of damage, such as double-stranded breaks and interstrand crosslinks1–3. Understanding the upstream signaling events that mediate recognition and repair of DNA alkylation damage is particularly important, since alkylation chemotherapy is one of the most widely used systemic modalities for cancer treatment and because environmental chemicals may trigger DNA alkylation4–6. Here, we demonstrate that human cells have a previously unrecognized signaling mechanism for sensing damage induced by alkylation. We find that the ASCC alkylation repair complex7 relocalizes to distinct nuclear foci specifically upon exposure of cells to alkylating agents. These foci associate with alkylated nucleotides, and coincide spatially with elongating RNA polymerase II and splicing components. Proper recruitment of the repair complex requires recognition of K63-linked polyubiquitin by the CUE domain of ASCC2. Loss of this subunit impedes alkylation adduct repair kinetics and increases sensitivity to alkylating agents, but not other forms of DNA damage. We identify RNF113A as the E3 ligase responsible for upstream ubiquitin signaling in the ASCC pathway. Cells from patients with X-linked trichothiodystrophy (TTD), which harbor a mutation in RNF113A, are defective in ASCC foci formation and are hypersensitive to alkylating agents. Together, our work reveals a heretofore unrecognized ubiquitin-dependent pathway induced specifically to repair alkylation damage, shedding light on the molecular mechanism of X-linked TTD.

Essential role of Ufm1 conjugation in the hematopoietic system.

Protein modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins plays a pivotal role in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. Despite its discovery more than a decade ago, its biological functions and working mechanism remains poorly understood. Recent genetic studies using knockout mouse models provide unambiguous evidence for the indispensable role of the Ufm1 system in animal development and hematopoiesis, especially erythroid development. In this short review, we summarize the recent progress on this important protein modification system and highlight potential challenges ahead. Further elucidation of the function and working mechanism of the ufmylation pathway would provide insight into disease pathogenesis and novel therapeutic targets for blood-related diseases such as anemia.