RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis

Abstract

The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. Cell Death and Differentiation (2015) 22, 1922-1934 doi:10.1038/cdd.2015 .51; published online 8 May 2015The Ufm1 (Ubiquitin-fold modifier 1) conjugation system is a novel ubiquitin-like (Ubl) modification system that shares biochemical features with other Ubl systems.1 Ufm1 modifies its target proteins through a biochemical pathway catalyzed by specific E1 (Uba5), E2 (Ufc1) and E3 enzyme(s) even though the identities of E3 ligases remain mostly elusive. Genetic study from Uba5 knockout (KO) mice has shown that Uba5 is indispensable for embryonic erythropoiesis, highlighting the pivotal role of this novel Ubl system in animal development. 2Yet its role in adult erythropoiesis and other developmental processes is largely unexplored and the underlying molecular mechanism remains poorly understood.Regulator of C53 and DDRGK1 (also known as KIAA0776, Ufl1, NLBP and Maxer, referred to as RCAD hereafter) has recently been identified by independent studies as an important regulator of several signaling pathways, including protein ufmylation, NF-κB signaling and unfolded protein response (UPR).3-9 Endogenous RCAD forms a complex with two proteins: C53 (also known as LZAP and Cdk5rap3) 5,6,10 and DDRGK1 (also designated as C20orf116, Dashurin and UFBP1), 3,6,7,11 and regulates the stability of its binding partners. 5,6 Intriguingly, Tatsumi et al. 3 found that Ufl1 (same as RCAD) promoted ufmylation of DDRGK1, suggesting that RCAD may function as an E3 ligase for ufmylation of DDRGK1. In line with its role in ufmylation, knockdown of endogenous RCAD resulted in attenuated ufmylation of endogen...

The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity

“…As part of the ribo-interactome, we identified UFL1, which is the only known enzyme that determines the target specificity for the metazoan-specific PTM, ufmylation (Zhang et al, 2015). Ufmylation is a ubiquitin-like PTM in which Ufm1, an 85-amino acid (9.1 kDa) protein, is conjugated to target proteins via a single enzyme cascade (Figure 4A).…”

“…Ufmylation is a ubiquitin-like PTM in which Ufm1, an 85-amino acid (9.1 kDa) protein, is conjugated to target proteins via a single enzyme cascade (Figure 4A). Although the significance of ufmylation is underlined by its essential roles in embryonic development and erythroid differentiation, research on this modification is still in its infancy (Tatsumi et al, 2011; Yoo et al, 2014; Zhang et al, 2015). By using N-terminally HA tagged UFL1 and an antibody that detects UFL1 at its C-terminus, we find full length UFL1 present in control, RNase, and puromycin treated IPs (Figure 4B).…”

“…Future studies are needed to elucidate whether ufmylation of critical substrates impacts ribosome subunit joining or contributes to transcript-specific translation. Interestingly, the available knockout mouse models for the enzymes of the ufmylation cascade show specific defects in erythrocyte differentiation and result in embryonic lethality (Tatsumi et al, 2011; Zhang et al, 2015). Notably, haploinsufficiency in multiple RPs result in defects in erythrocyte differentiation as a common phenotype, highlighting the sensitivity of hematopoietic cells to defects in protein production (Narla and Ebert, 2010) and raising the question of whether ufmylation of ribosomes plays a causative role in the phenotypes associated with bone marrow failure.…”

Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation

“…Interestingly, conditions that induce ER stress lead to increased expression of components of the UFM1 conjugation system, whereas their downregulation exacerbates ER stress and sensitizes cells to apoptosis (5)(6)(7)(8)(9). Of note, the ufmylation pathway is indispensable for erythro-and megakaryopoiesis, with Uba5 Ϫ/Ϫ , Ufl1 Ϫ/Ϫ , and Ufbp1 Ϫ/Ϫ mice dying in utero because of severe anemia connected to the reduced numbers of functionally differentiated erythrocytes (5,8,10).…”

The UFM1 cascade times mitosis entry associated with microcephaly

“…5 Studies have demonstrated that the knockdown of Uba5 in Drosophila resulted in locomotive defects, a shortened lifespan, as well as aberrant neuromuscular junctions. 13,19 Transgenic expression of UBA5 in the erythroid lineage extended the life span of UBA5-deficient embryos, but only to E18.5, 19 indicating that the UFM1 cascade plays vital roles in not just the hematopoietic system. 18 Mice with knockout of UBA5 or UFL1 died in utero (up to E13.5) with severe anemia.…”