UBR-5, a Conserved HECT-Type E3 Ubiquitin Ligase, Negatively Regulates Notch-Type Signaling in Caenorhabditis elegans

Notch-type signaling mediates cell−cell interactions important for animal development. In humans, reduced or inappropriate Notch signaling activity is associated with various developmental defects and disease states, including cancers. Caenorhabditis elegans expresses two Notch-type receptors, GLP-1 and LIN-12. GLP-1 mediates several cell-signaling events in the embryo and promotes germline proliferation in the developing and adult gonad. LIN-12 acts redundantly with GLP-1 in certain inductive events in the embryo and mediates several cell−cell interactions during larval development. Recovery of genetic suppressors and enhancers of glp-1 or lin-12 loss- or gain-of-function mutations has identified numerous regulators of GLP-1 and LIN-12 signaling activity. Here, we report the molecular identification of sog-1, a gene identified in screens for recessive suppressors of conditional glp-1 loss-of-function mutations. The sog-1 gene encodes UBR-5, the sole C. elegans member of the UBR5/Hyd family of HECT-type E3 ubiquitin ligases. Molecular and genetic analyses indicate that the loss of ubr-5 function suppresses defects caused by reduced signaling via GLP-1 or LIN-12. In contrast, ubr-5 mutations do not suppress embryonic or larval lethality associated with mutations in a downstream transcription factor, LAG-1. In the gonad, ubr-5 acts in the receiving cells (germ cells) to limit GLP-1 signaling activity. SEL-10 is the F-box component of SCFSEL-10 E3 ubiquitin–ligase complex that promotes turnover of Notch intracellular domain. UBR-5 acts redundantly with SEL-10 to limit Notch signaling in certain tissues. We hypothesize that UBR-5 activity limits Notch-type signaling by promoting turnover of receptor or limiting its interaction with pathway components.

The NICD/CSL activator complex up-regulates transcription of target genes whose identity depends on cell type.
Notch-type signaling is modulated by a variety of mechanisms (Louvi and Artavanis-Tsakonas 2012;Yamamoto et al. 2014). In the signaling cell, endocytosis of the ligand2receptor complex causes an essential conformational change that allows the S2 cleavage to occur. This process requires components of the endosomal trafficking machinery and mono-ubiquination of DSL intracellular domain by E3 ubiquitin ligases. In some signaling contexts, endocytosis also plays an earlier role in activation of ligand. In the receiving cell, newly synthesized Notch-type receptor receives a variety of post-translational modifications that modulate its activity prior to reaching the cell surface. As is the case for signaling activity in general, it is important that Notch-type signaling not be continuous, and mechanisms are in place to down-regulate NICD activity (reviewed by Baron 2012; Barth and Kohler 2014;Le Bras et al. 2011;Kandachar and Roegiers 2012;Weinmaster and Fischer 2011). For example, NICD activity is limited by ubiquitin-mediated targeting to the proteasome for degradation (reviewed by Lai 2002;Wang et al. 2011). In this process, ubiquitin is covalently linked to ubiquitin-activating enzyme (E1), transferred to ubiquitin-conjugating enzyme (E2), and finally transferred to the protein substrate (e.g., NICD) via the activity of a ubiquitin-protein ligase (E3) (reviewed by Kipreos 2005;Hershko and Ciechanover 1998). Target specificity is conferred by the E3 ligase, and SEL-10/Fbw7 is a conserved E3 ligase component that limits Notch signaling activity in many species (Hubbard et al. 1997; see reviews by Lai 2002;Wang et al. 2011). In addition to promoting protein turnover, E3-mediated ubiquitination can modulate proteinprotein interactions and impact processes such as nuclear import (Rodriguez 2014). Therefore, E3 ligase activity may also regulate Notch signaling by modulating interactions between signaling components.
Two Notch isoforms are present in Caenorhabditis elegans: GLP-1 (germline proliferation defective-1) and LIN-12 (lineage defective-12) (Greenwald and Kovall 2013). GLP-1 and/or LIN-12 mediate numerous cell-signaling events throughout development and in the adult gonad (reviewed by Priess 2005;Sternberg 2005; Greenwald and Kovall 2013). GLP-1 is active in cell2cell interactions in the embryo and in soma-to-germline signaling in the larval and adult gonad. LIN-12 is redundant with GLP-1 for some signaling events in the late embryo and later mediates signaling events in many different somatic cells during Figure 1 sog-1 encodes UBR-5, a HECT-type E3 ubiquitin ligase. (A) Working model for GLP-1/Notch signaling in the adult C. elegans germline. Interaction of transmembrane LAG-2 and/or APX-1 ligand and GLP-1 heterodimer triggers proteolytic cleavage of GLP-1. The S2 cleavage requires an ADAM family protease and releases the GLP-1 ectodomain (bound to LAG-2); the S3 cleavage requires g-secretase and releases the GLP-1 intracellular domain (ICD) for transport to the nucleus. Nuclear GLP-1 ICD interacts with the CSL-1-type transcription factor, LAG-1, and the coactivator SEL-8/LAG-3 (M), and displaces the LAG-1-bound corepressor complex (R). Signaling "strength" is modulated by numerous processes, as described in the text. (B) Diagram represents ubr-5 gene structure; mutant lesions associated with suppression of glp-1 are indicated. Nucleotide coordinates refer to genome version WS240. (C) Diagram represents UBR-5 protein structure. Conserved domains are indicated, as are amino acid substitutions and deletions/insertions associated with mutant alleles. The UB domain is variably referred to as the EDD or E3 domain in the literature. See Table S2 for details of sequence insertions and deletions in ok1108 and om2. (D) Domain architecture of human UBR5. Many UBR5 family members contain a poly(A) binding protein (PABP) motif (aka MLLE motif) just upstream of the HECT domain. larval development. Numerous modulators of Notch signalingas well as core components of the pathwayhave been identified in extensive genetic screens for suppressors or enhancers of loss-or gain-of-function mutations in glp-1, lin-12, or components of the g-secretase complex (reviewed by Greenwald and Kovall 2013). Most of these modulators also regulate additional developmental processes not known to involve Notch signaling.
GLP-1-mediated inductive signaling from the somatic gonad to the germline is essential for germ cell proliferation in the larva and adult (reviewed by Hansen and Schedl 2013;Kimble and Seidel 2013). All germ cells are proliferative in early larval development; later, proximal germ cells enter meiosis, and germline proliferation becomes restricted to the distal region of the gonad arm. During the early proliferative phase, several somatic gonadal cells signal to the germline via GLP-1; in the later phase, only the distal tip cell signals to the germline via GLP-1. Proliferative germ cells are not maintained if signaling is reduced or abolished by mutation in a GLP-1 pathway component or by ablation of somatic signaling cells; instead, germ cells prematurely exit mitosis, enter meiosis, and form gametes. Additional factors promote a wildtype level of germ cell proliferation, including the distal sheath cells (Killian and Hubbard 2005), gap junctions between the somatic gonad and germline (Starich et al. 2014), and nutritional factors, among others (Hubbard et al. 2013).
We previously recovered suppressor of glp-1 (sog) mutations in genetic screens for suppressors of the glp-1 temperature-sensitive (ts) phenotype (Maine and Kimble 1993). The sog mutations partially suppressed glp-1 maternal effect embryonic lethality and germline proliferation defects. Here, we report the molecular characterization of sog-1. We demonstrate that sog-1 encodes UBR-5, a member of the HECT (homologous to the E6-AP carboxyl terminus)-type E3 proteinubiquitin ligase family whose closest mammalian relative is UBR5 (Ubiquitin protein ligase E3 component n-recognin 5; also called EDD, E3 identified by differential display). We find that loss of UBR-5 activity causes an increase in both GLP-1 and LIN-12 signaling activity in sensitized genetic backgrounds where the receptor carries a conditional mutation. Genetic analysis suggests that UBR-5 acts in the receiving cell. The SCF SEL-10 E3 ubiquitin2ligase complex is known to limit LIN-12 activity and, to a minor extent, GLP-1 activity by binding to and promoting turnover of the intracellular domain (Sundaram and Greenwald 1993;Hubbard et al. 1997). Our genetic analysis suggests that UBR-5 and SEL-10 function in concert to limit Notch signaling in some tissues.

Mutant strains
C. elegans strains were cultured using standard methods (Epstein and Shakes 1995). All strains were derived from the Bristol strain, N2.
Whole genome sequencing Genomic DNA isolation from strains JK946 [carrying sog-1(q303)] and JK952 [carrying sog-1(q308)], library construction, whole genome sequencing (WGS), and bioinformatics analysis were performed as described (Flibotte et al. 2010;Thompson et al. 2013). For analysis of other sog-1 strains, the F36A2.13 gene region was recovered by DNA amplification and sequenced using standard methods. The sog-1 mutations were originally mapped to the cluster on LGI, and we particularly focused on WGS data from this region.
In the course of this study, we also performed WGS analysis of JK953, a strain previously reported to carry a sog-1 mutation called q309. Unlike other sog-1 alleles, which were recovered in F2 screens for recessive suppressors of glp-1, q309 was recovered in the course of a dominant suppressor screen as described (Maine and Kimble 1993). WGS analysis revealed that JK953 is wild-type for glp-1 and does not contain a mutation in F36A2.13. We conclude that the reduced brood size and temperature sensitivity associated with JK953 result from a combination of other mutations in the genetic background.

Phenotypic analysis
Brood size assays were carried out using standard methods as follows. All sog-1;unc-32 glp-1(231) strains were maintained at 20°. The unc-32 glp-1(q231ts) control strain was maintained at 15°, and animals to be used for brood size experiments were shifted to 20°as late-stage embryos or newly hatched L1 larvae. Broods were assayed by placing individual L4 larvae onto single plates; once they became gravid adults, they were moved to a fresh plate daily until they no longer produced embryos. Embryos were counted immediately after the mother was moved; viable L4 larvae were counted 223 d later.
The anchor cell (AC)-ventral uterine (VU) precursor phenotype was assayed by examining late L2 stage larvae with differential interference contrast and epifluorescence microscopy using a Zeiss Axioscope. Anchor cells were identified based on morphology, position within the gonad primordium, and expression of a cdh-3::gfp transgene included in the strain.
Proliferative zone size was analyzed in adults labeled with DAPI using standard methods (e.g., Qiao et al. 1995). L4 stage hermaphrodites were picked to a fresh culture plate, aged 24225 hr, fixed for 15 min with 220°methanol, stained for 15 min with 0.2 mg/ml DAPI, and mounted in Vectashield (Vector Laboratories) for epifluorescence analysis. To assay the proliferative region of each specimen, we counted the number of rows of germ cells distal to the leptotene2zygotene "transition" zone.
RNAi assays RNA interference (RNAi) was performed by the feeding method (Timmons et al. 2001). L4 larvae were placed onto culture plates seeded with E. coli expressing sog-1 double-stranded RNA (dsRNA), and their F1 progeny were assayed in the first day of adulthood for the presence/ absence of embryos. Animals were removed from culture plates as they were counted. Non-RNAi controls were performed by culturing each strain [glp-1(ar202) or rrf-1(pk1417);glp-1(ar202)] on the standard OP50 E. coli food source. Presence of the rrf-1(pk1417) deletion was verified by DNA amplification. In addition, the sensitivity of rrf-1(pk1417);glp-1(ar202) animals to RNAi of various somatic genes was tested to confirm the presence of the RNAi defect as described by Kumsta and Hansen (2012). As a control for an effect of RNAi per se on the glp-1(ar202) phenotype, RNAi was performed with empty L4440 vector, which produces a short dsRNA that does not correspond to C. elegans genomic sequence.

Data availability
Strains are available upon request. Whole genome sequence data are provided in Supplemental Material, Table S1.

RESULTS
sog-1 encodes a HECT-type E3 ubiquitin ligase Mutations in sog-1 were recovered in a genetic screen for recessive suppressors of glp-1(q224ts) and glp-1(q231ts) (Maine and Kimble 1993). Both of these glp-1(ts) alleles have a Glp-1 null phenotype at 25°and a partial loss-of-function (lf) phenotype at 20° (Maine and Kimble 1989). Consequently, when L1 stage glp-1(ts) larvae are shifted from permissive temperature to 25°, their germ cells exit mitosis, enter meiosis, and undergo spermatogenesis. In contrast, when L1 stage glp-1(ts) larvae are shifted from permissive temperature to 20°, their germ cells proliferate for a period of time before prematurely entering meiosis and undergoing gametogenesis; in most cases, a full complement of sperm and some oocytes form and some embryos are generated. These embryos die due to defects in GLP-1 signaling during embryogenesis. The sog-1 mutations partially suppress the Glp-1(ts) defects at 20°, but not at 25°, and therefore do not bypass the requirement for GLP-1 activity (Maine and Kimble 1993).
We initiated a molecular study of sog-1 by performing whole genome sequence analysis of two sog-1 mutant strains: JK946, containing sog-1(q303); and JK952, containing sog-1(q308) (see Materials and Methods). When we compared the JK946 and JK952 sequence data to the reference C. elegans genome, we identified numerous common mutations that presumably were present in the original unc-32(e189) glp-1(q231ts) strain prior to mutagenesis (Table S1). In addition, we identified a number of mutations unique to either JK946 or JK952. Of note, JK946 and JK952 contain distinct mutations in a common open reading frame (ORF), F36A2.13 (Table S1, Figure 1B). F36A2.13 is predicted to encode a member of the HECT family of E3 ubiquitin ligases ( Figure 1). Its closest mammalian relative is UBR5/EDD (Callaghan et al. 1998;Tasaki et al. 2005), and consequently F36A2.13 is listed in Wormbase as ubr-5 (UBR E3 ubiquitin ligase homolog -5) (www.wormbase.org).
We confirmed that ubr-5 and sog-1 are the same gene by amplifying and sequencing the F36A2.13 genomic region from five additional strains carrying sog-1 alleles recovered following mutagenesis with EMS (q295, q298, q305, q345; Maine and Kimble 1993) or UV (om2; see Materials and Methods). Each strain contained a mutation in F36A2.13 ( Figure 1, Table S2). In addition, we obtained an F36A2.13 deletion allele, ok1108, from the Caenorhabditis Genetics Center and tested its ability to suppress glp-1(q231ts). At 20°, ok1108 partially suppresses glp-1(q231ts) ( Table 1). We conclude that sog-1 and F36A2.13 are the same gene. Although sog-1 is the original published gene name, "ubr-5" better denotes the gene product. Therefore, we will refer to F36A2.13 as ubr-5 for the rest of this article.
Previous analysis of ubr-5 mutants indicated that they were superficially normal (Maine and Kimble 1993). We reevaluated this question with the deletion alleles, ubr-5(om2) and ubr-5(ok1108). These mutants likewise do not have obvious developmental defects in a glp-1(+) background under laboratory conditions. In particular, we considered that ubr-5 mutants might impact the germline stem cell pool. As a measure of proliferative zone size, we counted the number of rows of germ cell nuclei from the distal end of the somatic gonad to the start of the leptotene/zygotene region in animals raised at 20°. When we compared the number of rows of nuclei in the mitotic zone in glp-1(q231ts) and ubr-5(om2); glp-1(q231ts) animals at 24 hr post-L4 stage, we observed an increase in mitotic zone size from an average of four rows in glp-1(q231ts) to an average of 11 rows in ubr-5(om2); glp-1(q231ts) ( Table 2). Therefore, the loss of UBR-5 activity leads to increased germ cell proliferation in the sensitized GLP-1(ts) background. In contrast, we did not observe an increase in the number of rows of mitotic germ cells in ubr-5 mutants compared with wild-type at 24 hr post-L4 (Table 2). Hence, the loss of UBR-5 activity did not impact the length of the mitotic region in germlines with wild-type GLP-1 function.

Figure 2
Genetic and physical map of the ubr-5 region. Genetic mapping previously placed sog-1 between unc-13 and lin-10. Our molecular studies reported here indicate that sog-1 corresponds to F36A2.13/ubr-5, located between lin-10 and unc-29. The positions shown for deficiencies ozDf5 and nDf25 reflect their ability to uncover mutations in genes in the region.

UBR-5 negatively regulates GLP-1 activity
The predicted ubr-5 product is a 2944 amino acid protein with domain architecture characteristic of the UBR5/EDD HECT protein subfamily. It contains three conserved motifs: a UB (also called E3) domain located near the N terminus, a zinc-finger domain located in the middle of the protein (the UBR motif), and a C-terminal HECTc domain ( Figure 1C). Many members of this protein family also contain a PABP (poly A binding protein; also called MLLE) domain within 100 amino acids of the HECT domain (Callaghan et al. 1998;Tasaki et al. 2005;Scheffner and Kumar 2014) ( Figure 1D). C. elegans UBR-5 appears to lack this domain. Among the eight ubr-5 alleles we characterized, three contain a premature stop codon. The ubr-5(om2) allele is likely to be null; it contains a deletion of 105 nucleotides in the center of the gene just downstream of the zinc-finger domain that is predicted to shift the ORF, inserting 45 amino acids and deleting the C-terminal half of the protein, including the entire HECT domain (Figure 1, Table S2). Similarly, ubr-5(q345) may be null as it contains a single nucleotide change that converts residue 1026 to a stop codon (Figure 1, Table S2). It is predicted to encode a truncated protein lacking the zinc-finger and HECT domains. ubr-5(ok1108) is a complex mutation with a 1360 nucleotide deletion and 73 nucleotide insertion; most of the HECT domain is deleted as well as the 39 UTR and some downstream sequence ( Figure 1, Table S2). The net result of the mutation is to insert nine amino acids downstream of residue 2631. This allele is expected to lack E3 ligase activity, as well. The nature of these alleles suggests that a reduction in ubr-5 function suppresses the loss of glp-1 activity, and therefore UBR-5 is a negative regulator of GLP-1. The other ubr-5 alleles we characterized contain missense mutations predicted to cause single amino acid substitutions as follows: q308 just upstream of the E3 domain; q298 just upstream of the HECT domain; and q295, q303, and q305 within the HECT domain ( Figure 1, Table S2).
We characterized glp-1 suppression by the ubr-5 deletion alleles, om2 and ok1108 (Table 1), and compared the results with data obtained previously for other alleles (Maine and Kimble 1993). We evaluated the total number of progeny (viable and nonviable) produced and the number of progeny that hatched and developed to adulthood. As observed previously (Maine and Kimble 1993), we do not see a simple relationship between suppression of the brood size defect and embryonic lethality, and the likely null alleles do not show identical suppression. We suspect that suppression is influenced by other, unique mutations present in different ubr-5 strains. For example, WGS data revealed a suite of shared mutations and a number of unique mutations in ubr-5(q303); unc-32(e189) glp-1(q231) and ubr-5(q308); unc-32(e189) glp-1(q231) (Table S1). We hypothesize that some of these mutations may influence the degree of suppression by ubr-5 in one or more tissues. Indeed, it was noted during previous three-factor mapping experiments that suppression by ubr-5 is abrogated to a large extent in the presence of certain marker mutations (Maine and Kimble 1993). As detailed in the Materials and Methods, data obtained in the course of our studies allow us to make corrections to the literature with respect to (i) gene dosage requirements and (ii) the identity of a previously reported allele, q309.
To confirm further that the ubr-5 mutations are loss-of-function, we knocked down UBR-5 protein in a glp-1 gain-of-function background using RNAi and examined the consequences for GLP-1 signaling. Gain-of-function glp-1 mutations have elevated GLP-1 signaling, resulting in germline over-proliferation and eventual formation of a germline tumor (Berry et al. 1997;Pepper et al. 2003). The glp-1-(ar202gf) allele is temperature sensitive, producing a more elevated level of GLP-1 signaling at higher temperatures (Pepper et al. 2003). At 25°, germ cells form a tumor relatively early in development, and the animal does not produce oocytes. At lower (semipermissive) temperatures, overproliferation occurs more slowly, and a sizable proportion of glp-1(ar202) mutants are fertile (Pepper et al. 2003).

ubr-5 mutations do not suppress lag-1(ts) lethality
We hypothesize that UBR-5, as an E3 ligase, may limit GLP-1 signaling by promoting turnover of a pathway component, e.g., full-length GLP-1 or processed GLP-1 intracellular domain, or a positive modulator of pathway activity. By this scenario, an elevated level of the partially active GLP-1(q231) protein would accumulate in ubr-5 mutants and GLP-1 signaling activity would increase. To investigate potential UBR-5 targets, we tested whether ubr-5 mutations can suppress a ts mutation in the Notch pathway transcription factor, LAG-1. The lag-1(om13ts) mutation strongly enhances the glp-1(bn18ts) germline proliferation defect at semipermissive temperatures and, in a glp-1(+) background, causes embryonic and early larval lethality (Qiao et al. 1995). Larval lethality occurs at the L1 stage shortly after hatching and is characteristic of the Lag phenotype (Lambie and Kimble 1991). CSL-type transcription factors also function independently of Notch (reviewed by Johnson and MacDonald 2011;Ghai and Gaudet 2008), and we hypothesize that at least some aspects of the lag-1(om13ts) phenotype may be independent of GLP-1 and LIN-12 signaling.
In the course of our work, we observed that the sel-10(ok1632) strain we obtained from the Caenorhabditis Genetics Center, RB1432, has highly penetrant defects not reported for sel-10 in the literature, including substantial embryonic lethality and a reduced brood size of 160 (Table 5). When we examined the ubr-5(om2);sel-10(ok1632) double mutant phenotype, we noted that embryonic viability and brood size approximated that of ubr-5(om2) single mutants (Table 5). To determine whether ubr-5(om2) suppressed these defects or, alternatively, they might be caused by mutations in the RB1432 strain background and were eliminated when we constructed the ubr-5(om2);sel-10(ok1632) double, we reisolated the sel-10(ok1632) allele from the ubr-5(om2);sel-10(ok1632) strain and examined the phenotype. We found that the brood size and % viability of the reisolated sel-10(ok1632) single mutant were essentially the same as the ubr-5(om2); sel-10(ok1632) double (Table 5). We conclude that strain RB1432 indeed carries one or more additional mutation(s) distinct from sel-10(ok1632) causing embryonic lethality and reduced brood size.

DISCUSSION
Here, we demonstrate that the sog-1 gene, previously shown to interact genetically with glp-1, encodes UBR-5, the sole C. elegans member of a conserved E3 ubiquitin ligase family. Mammalian UBR5 and the Drosophila ortholog, hyperplastic discs (hyd), function in diverse aspects of development including cell proliferation (reviewed by Shearer et al. 2015;Mansfield et al. 1994). Our data indicate that UBR-5 activity limits GLP-1/Notch signaling in the embryo and larval/adult gonad and LIN-12/Notch signaling in the AC/VU decision. Moreover, UBR-5 acts autonomously to repress germline proliferation, suggesting its primary impact on Notch signaling is within the receiving cell. In contrast, UBR-5 activity does not suppress the brood size or viability defects associated with reduced function of LAG-1, the CSL-type transcription factor component of the GLP-1 signaling pathway in the germline. Given these findings, we hypothesize that UBR-5 functions in the turnover of GLP-1/LIN-12 receptor and/or other proteins responsible for receptor production. Alternatively, UBR-5-mediated ubiquitination may modulate signaling by reducing the ability of GLP-1/LIN-12 to interact with other pathway components, e.g., LAG-1 or LAG-3. Additional experiments will be required to determine if UBR-5 restricts Notch-type signaling in all tissues.
n Tests were performed at 20°. Most nonviable larvae died at early L1 stage, as is characteristic of the Lag phenotype (Lambie and Kimble 1991). n, number of full broods counted.
Extensive genetic and molecular analysis has revealed that the outcome of Notch-type signaling is extremely sensitive to the level ("strength") of signaling activity in a wide variety of contexts (Louvi and Artavanis-Tsakonas 2012;Yamamoto et al. 2014). Our data are consistent with these previous observations. Eliminating just one negative regulator of GLP-1 and LIN-12 activity, UBR-5, increased signaling activity sufficiently to rescue moderately severe loss-of-function phenotypes and strongly enhance a weak gain-of-function phenotype.
The relationship between UBR-5 and SEL-10 Our data suggest a complex relationship between UBR-5 and SEL-10 with respect to Notch signaling. These findings may reflect, in part, the equivalent receptor activity of GLP-1 and LIN-12 (Fitzgerald et al. 1993) and the fact that both GLP-1 and LIN-12 are active in some tissues, e.g., in the embryo, whereas only GLP-1 or LIN-12 is active in other tissues, e.g., GLP-1 in the gonad. Whereas UBR-5 has a major role in limiting Notch signaling in the embryo and gonad, SEL-10 appears to have a larger role in limiting Notch signaling in the embryo than in the gonad. Our observation of a synergistic genetic interaction between ubr-5 and sel-10 with respect to suppression of the glp-1(q231ts) embryonic lethality indicates that UBR-5 and SEL-10 do not function in a simple linear pathway, at least in embryonic tissues. One model for the relationship between these factors in the embryo is that UBR-5 restricts GLP-1 activity broadly during embryogenesis, whereas SEL-10 restricts GLP-1 primarily in the late embryowhere it also restricts LIN-12 ( Figure 4B). Only rarely would development of the glp-1(q231ts) embryo be rescued by sel-10(ok1632) alone. An alternative hypothesis is that SEL-10 primarily restricts LIN-12 signaling activity and has little impact on GLP-1 signaling activity. This would explain the very minor role of SEL-10 in the gonad, where LIN-12 signaling is not active. One way to think about these data is that UBR-5 may primarily limit GLP-1 signaling, SEL-10 may primarily limit LIN-12 signaling, and reduced UBR-5 or SEL-10 activity may better suppress reduced glp-1 or lin-12 activity in those cells or tissues where increased activity of one receptor can compensate for reduced activity of the other, e.g., in the embryo ( Figure 4A).
UBR5 activity in development and disease UBR5 proteins in mammals and Drosophila have been implicated in numerous developmental processes (reviewed by Shearer et al. 2015). Vertebrate UBR5 regulates cell cycle progression, and misregulation of UBR5 activity is linked to cancer in many tissues (Scheffner and Kumar 2014;Shearer et al. 2015). UBR5 activity appears to promote cell proliferation in some tissues and limit it in others, as the loss of UBR5 activity promotes cancer in some tissues whereas UBR5 overexpression promotes cancer in other tissues. Presumably these differences reflect the multitude of UBR5 targets that may contribute to oncogenesis. The Drosophila ortholog, Hyperplastic discs (Hyd), regulates cell proliferation in developing imaginal discs and promotes development of other tissues, as well (Mansfield et al. 1994).
UBR5 appears to contribute to cell proliferation control in a number of ways. One role for UBR5 is in modulating activity of the mitotic spindle assembly checkpoint (SAC) mechanism, thereby impacting the ability of cells to enter anaphase. SAC activity ensures cells remain in metaphase until all chromosomes have attached to the mitotic spindle; once chromosomes have done so, then SAC activity must be reduced in order to allow anaphase entry. Evidence suggests that UBR5 functions both to promote SAC activity when needed, e.g., if microtubules are disrupted , and to reduce SAC activity once chromosomes have attached to the mitotic spindle (Jiang et al. 2015). UBR5 targets different SAC components in these different situations, and its subcellular localization changes during the cell cycle Jiang et al. 2015).
n Table 5 Tests for redundancy between UBR-5 and SEL-10
Another function of UBR5 is in modulating stability of nuclear myosin 1 (NM1), a factor required for RNA polymerase I transcriptional activity. NM1 is stabilized by GSK3b-mediated phosphorylation, which prevents UBR5-mediated ubiquitination and subsequent degradation (Sarshad et al. 2014). Progression beyond G1 stage requires NM1 activity, and hence modulation of NM1 is one means by which UBR5 limits cell cycle progression. Antagonistic activity of GSK3b vs.
Hyd/UBR5 appears to be a general regulatory mechanism as it also modulates Hedgehog signaling in Drosophila (Lee et al. 2002;Moncrieff et al. 2015). As we observed for Notch signaling, Hedgehog signaling is limited by Hyd/UBR5 activity. Interestingly, mammalian FBW7/SEL10 is recruited to certain targets upon GSK3b2mediated phosphorylation (Flugel et al. 2012).
Other studies have implicated UBR5 in modulating the DNA damage response (DDR), where it participates at several steps, and in regulation of certain transcription factors (Shearer et al. 2015). One aspect of UBR5 activity is that it limits activity of the DDR machinery to sites of double-strand breaks and prevents inappropriate/ unnecessary activity elsewhere on the chromosome. In regulating transcription factor activity, UBR5 functions in many cell types, positively regulating certain transcription factors and negatively regulating others. We present the first evidence, to our knowledge, of a UBR5 family E3 ligase modulating Notch signaling activity. In the future, it will be informative to identify the targets of UBR-5 activity.