Genome-wide analysis of genes encoding core components of the ubiquitin system during cerebral cortex development

The data obtained from the genome-scale profiling of gene expression are organized into two parts: the first part covers the ubiquitination process, and the second part is devoted to the genes encoding DUBs, a class of enzymes participating in the novo synthesis of Ub and are responsible of the deubiquitination of substrates.

Ubiquitination

This first section is subdivided into 4 sections covering the following topics: (1) genes involved in the synthesis of Ub and Ub-like proteins, followed by the genes encoding (2) Ub-activating (E1) enzymes; (3) Ub-conjugating (E2) enzymes; and (4) Ub ligases (E3).

Ubiquitin genes

In mammals, two classes of genes are involved in de novo synthesis of Ub: the monomeric Ub-ribosomal fusion genes Uba52 and Rps27a (Uba80) and the stress-inducible poly-Ub genes Ubb and Ubc [16]. In the immature cerebral cortex, Ubb and Rps27a were the most highly expressed Ub genes (Fig. 1A). Ubb transcripts accounted for 30–40% of the total Ub transcripts, which is actually in close agreement with previously reported data [17], indicating a high abundance of total Ubb transcripts in the brain. Ubb and Rps27a followed dissimilar expression patterns: transcripts of Rps27a predominated at the onset of corticogenesis (E11–13) before their number significantly decreased, whereas the Ubb gene was the most highly Ub gene expressed at the end of corticogenesis (E17-PN1). Notably, the number of transcripts (expressed in transcripts per million, TPM) was on the order of 700–1700, reflecting a very high abundance of Ubb, Rps27a and Uba52 transcripts at all stages (Fig. 1A). In comparison, the TPM values of H2afz (encoding histone 2A, member z), one of the most abundant cellular proteins ubiquitously expressed [18], were 1740 (at E11) and 400 (on PN1). H2af was the 66th most highly expressed gene on E11, whereas Rps27a was the 68th mostly highly expressed. On PN1, Ubb, the most prominent Ub gene (with TPM values of 1400) was the 60th most highly expressed gene in the immature cerebral cortex. These data are in line with the known high abundance of the Ub protein in biological samples, in which it has been shown to comprise up to 5% of total protein [19]. These 4 genes generate single (Uba52 and Rps27a) or multiple copies (9 for Ubc, and 3 for Ubb) of Ub. Therefore, to better assess the contribution of these genes to the total pool of Ub, we calculated the theoretical production of Ub molecules assuming a similar translation efficiency for the four transcripts [20]. The Ubc gene accounted for nearly 65% of the total pool of Ub in the embryonic cortex, followed by Ubb, Rps27a and Uba52, accounting for 18%, 11% and 6%, respectively. These data are in line with a previous report showing that UBC accounts for 64% of the total Ub pool in HeLa cells [20]. Thus, the poly-ubiquitin gene Ubc is the major cellular contributor of Ub molecules.

Fig. 1figure 1

Expression of genes involved in the synthesis of Ub and Ub-like proteins and the genes encoding E1 and E2 enzymes. For this report, the data shown were extracted from datasets derived by genome-wide transcriptome sequencing (RNA-Seq) [13]. The original database is freely available on the GEO repository (www.ncbi.nlm.nih.gov/geo/) with the accession number GSE154677. The analysis covers the temporal pattern of expression of the major genes in the Ub system. RNA was extracted from the cerebral cortex of mice in four stages: embryonic Days 11 (E11), 13 (E13), and 17 (E17) and postnatal Day 1 (PN1). RNA abundance is reported in transcripts per million (TPM), and only genes with TPM values ≥ 2 were considered to be actively transcribed, following the criteria presented in [25]. Figure shows the expression of genes involved in the synthesis of Ub (A), Ub-like proteins (B), and genes encoding Ub-activating E1 enzymes (C) and Ub-conjugating E2 enzymes (D, E)

In the mouse brain, 60% of the Ub pool is in the free form (i.e., not attached to target substrates) [19]. The level of free Ub is important to neuronal functions and survival [16, 19]. The morphology, neurite outgrowth and synaptic development are impaired in cultured neurons isolated from the brains of Ubb-knockout mice [21].

A recent study reported 52 Ub pseudogenes in humans [22]. Moreover, some of these genes, such as human Ubb pseudogene 4 (Ubbp4), Rps27a pseudogene 16 (Rps27ap16), and Uba52 pseudogene 8, encode proteins [22]. Here, the expression of the following murine Ub pseudogenes was examined: Gm1821 (Ubb-pseudo gene or Ubb-ps), Rps27a-ps1, Rps27a-ps2 and Gm7866 (Uba52-ps). Only transcripts of the pseudogene Gm1821 were found with TPM values of ⁓ 15, which was nearly ⁓ 100-fold less abundant than Ubb transcripts. In conclusion, our data indicated a high expression level of the three Ub-encoding genes Ubb, Rps27a and Uba52. In comparison, the expression of Ub pseudogenes was negligible.

Ubiquitin-like genes

Similar to Ub, various Ub-like proteins can be covalently conjugated to target substrates via an enzymatic cascade involving E1, E2, and E3 enzymes [23], [24]. The expression of this set of genes was analysed, and the results are reported in Fig. 1B. The following 10 genes were identified: Sumo1-3, Nedd8, Isg15, Ubd (Fat10), Ufm1, Atg8 (Map1lc3b), Atg12, and Urm1. No transcript for Sumo2 or Ubd was found (TPM < 2) [25]. All the other genes were, however, significantly expressed, with Atg8, Sumo3 and Nedd8 displaying the highest levels of expression. For this set of genes, the TPM ranged from 70 to ⁓ 200 (Fig. 1B), a number that is approximately tenfold lower than that of Ub genes (Fig. 1A). Atg8 was the major gene, and its expression was highly upregulated during development, with a number of transcripts showing expression increases by a factor of 3 between E11 and E17, suggesting activation of Atg8-dependent physiological processes, such as autophagy activation, at the end of cortical development (Fig. 1B). A recent proteomic analysis showed that the levels of conjugated and free NEDD8 (or ISG15) in the mouse brain were at least 60- and 20-fold lower than those of Ub [6], which is consistent with the profoundly lower gene expression levels of these genes, particularly Isg15, whose expression was negligible.

Ub-activating (E1) enzymes

Ubiquitination is a three-step enzymatic reaction. During the initial step, Ub is activated in an adenosine triphosphate-dependent manner by the Ub-activating (E1) enzyme before being transferred to a Ub-conjugating (E2) enzyme [2, 26]. Figure 1C shows the expression profile of the two genes Uba1 and Uba6 (Ube1l2) encoding mammalian Ub-activating E1 enzymes and seven genes encoding Ub-like proteins that activate E1 enzymes (Uba2-3, Uba5, Uba7, Nae1, and Sae1) [24]. The Uba1 gene was by far the most prominently expressed E1 gene in the cerebral cortex (Fig. 1C). Its TPM values were ⁓ 200 on E11 and ⁓ 390 on E17, thus showing a nearly twofold increase in transcript abundance during embryonic development. Abundant expression of UBA1 may be a common feature of many cellular types, as the UBA1 protein is among the top 2% of the most highly expressed proteins in HeLa cells [18], reflecting the crucial requirement of this E1 enzyme in Ub-dependent cell processes. In the cerebral cortex, Uba1 was the 597th and 343th most highly expressed gene on E11 and PN1, respectively, which confirms the relatively high abundance of Uba1 transcripts. The Uba1 gene product is abundant in the nucleus and cytoplasm [27], whereas the other mammalian E1 Uba6 gene product is found only in the cytoplasm, which may ensure much more specific and restricted functions. Uba6 was expressed at very low levels, with TPM values ranging from ⁓ 9 on E11 to ⁓ 4 on PN1. In the cerebral cortex, the ratios of Uba1:Uba6 transcript abundance were > 20:1 and 90:1 on E11 and PN1, respectively. This differential expression was consistent with proteomic data showing that the relative abundance ratio of the UBA1 and UBA6 proteins is > 10:1 in HeLa cells [18], further suggesting a restricted function for UBA6 compared to that of UBA1. Collectively, the expression profile data of Uba1 showed that it is the primary E1 gene in the cerebral cortex of mice. Due to its central role in Ub homeostasis, UBA1 is likely to regulate a wide range of neurobiological processes [28].

Far below the level of expression observed for Uba1, the expression of a set of six genes encoding Ub- and Ub-like proteins activating E1 enzymes (Uba2-3, Uba5, Uba7, Nae1, and Sae1) exhibited TPM values < 100. Uba2 encodes an E1 enzyme specific for the Ub-like molecule SUMO. This Uba2 gene product is thought to form heterodimers with SAE1 [24, 26]. Interestingly, the expression of both genes (Uba2 and Sae1) was downregulated during development, with an abundance of transcripts reduced by nearly 50% between E11 and PN1 (Fig. 1C). Notably, no transcript for the Ub-like E1 gene Atg7 was detected. Despite its low level of expression in the developing cortex, Uba6 plays important roles in neuronal development, dendritic spine architecture, and mouse behaviour, and its deficiency is lethal [29]. Moreover, Uba6 is required for neuronal viability in primary hippocampal neuronal cultures. Collectively, our data led to the identification of the highly regulated Uba1 and Uba2 genes as the major E1 and E1-like enzyme genes, respectively, expressed during cortical brain development.

Ub-conjugating (E2) enzymes

The analysis of the genes encoding Ub- and Ub-like protein-conjugating E2 enzymes [30, 31] is shown in Fig. 1D, E. Transcripts for Ube2d4, Ube2e2, Cdc34b, Atg10 and Ube2u were not found. This observation reinforces the validity of our results since Ube2u transcripts were detected specifically in tissues of the urogenital tract [32]. All the other genes were expressed at significant levels (35 of 40 genes), particularly Ube2m (Ubc12), encoding a Nedd8-conjugating enzyme, with TPM values increasing from ⁓270 on E11 to ⁓320 on PN1 (Fig. 1D). The most important expressed genes encoding Ub-conjugating E2 enzymes were Ube2c and Ube2r1 (cdc34) (Fig. 1D) and, to a lesser extent, Ube2q1, Ube2ql1, and Ube2z (Use1) (Fig. 1E). Expression of Ube2c was inhibited during embryonic development, with high levels of transcripts evident in the neurogenic period (E11–E13) and a marked decrease from E17 and later. Overall, TPM values of the Ube2c gene decreased by a factor of 25 between E11 (> 150 TPM) and PN1 (⁓ 7 TPM) (Fig. 1D). This decline represented the most downregulated genes in the Ub pathway during corticogenesis. The UBE2C protein is an exclusive partner of APC/C E3 ligases and controls cell cycle progression. Its mRNA and protein levels were low in quiescent cells but greatly increased and peaked during mitosis [31, 33]. Ube2c mRNA was thought to be barely detectable in tissues except under oncogenic conditions, with high levels in various cancers, such as brain and breast cancers [33]. The data presented in Fig. 1D show that high levels of Ube2c mRNA were found in nontumorous tissue under conditions not related to cancer onset or progression, similar to many developmental-specific genes whose re-expression is associated with carcinogenesis. Further studies are required to verify whether the UBE2C protein is a marker of neurogenesis in the brain.

Ube2r1 was another prominently expressed Ub-conjugating E2 enzyme-encoding gene. Similar to that of Ube2c, the expression of Ube2r1 was repressed, with an abundance of transcripts reduced by a factor of 4 between E11 and PN1 (from 126 to 34 TPM) (Fig. 1D). The E2 enzyme CDC34 (encoded by Ube2r1) is the primary E2 for cullin-RING E3 ligases (CRLs). Two members of the Ube2q gene family were expressed at moderate levels: Ube2q1 and Ube2ql1. The expression of the Ube2q1 gene was constant, showing no sign of developmental regulation. Western blot and immunohistochemical experiments showed the presence of UBE2Q1 proteins in the rat brain cortex, mainly in neurons [34]. UBE2Q1has been postulated to play an anti-apoptotic role, at least in pathological states such as traumatic brain injury [34]. The expression of Ube2ql1 was not detected before E13, and it peaked in the E17-PN1 period, with TPM values increasing from ⁓8 to 85 TPM from E13 to E17. This increase represented an 11-fold increase in transcript abundance, making Ube2ql1 the second most induced gene (in the Ub system) during embryonic development. In HeLa cells, UBE2QL1 exhibited a dual function: it is required for the efficient clearance of damaged lysosomes by lysophagy and maintains lysosome integrity [35].

The expression of minor genes (TPM values < 40) encoding Ub- and Ub-like proteins-conjugating E2 enzymes is reported in Additional file 1: Fig. S1A, B. The vast majority of these genes were expressed at constant levels, except Ube2g2, Ube2s, Ube2t and Ube2l6, which were downregulated. In particular, the expression Ube2l6 was profoundly repressed, with TPM values decreasing from ⁓ 18 to ⁓ 2 (a ⁓ninefold reduction in transcript abundance) (Additional file 1: Fig. S1B), making it one of the most important downregulated genes observed in this study. Mutations in the Ube2a gene lead to neurodevelopmental disorders such as X-linked syndromic mental retardation. The precise roles Ube2a plays in brain formation are unknown. In the rodent brain, the Ube2a gene was expressed at low levels, with few transcripts evident throughout cortical development (Additional file 1: Fig. S1A). It has been proposed that some of the cellular effects of UBE2A involve the E3 Ub ligase Parkin. This might be the case in adults, but this enzyme was not expressed during embryonic development of the cerebral cortex, suggesting the involvement of other E3 partners (see below, “RING E3 Ub ligases”). UBE2A exerts some of its actions via the E3 Ub ligases RAD18 and RNF20. The UBE2A/RAD18 complex is at least partially responsible for the pathogenesis of mental retardation (in association with the proliferating cell nuclear antigen, PCNA [12]). The rad18 gene was expressed uniquely on E11 and E13, but the abundance of transcripts was very low (< 6 TPM), suggesting that its presence is required for brain development exclusively during neurogenesis.

This transcriptomic analysis provides a detailed overview of the expression patterns of E2 genes that are central players in the Ub pathway. Overall, these genes were expressed at low levels during embryonic development of the mouse cerebral cortex. As in HeLa cells or Swiss 3T3 cells, Ube2m (encoding UBE2M/UBC12 and involved in neddylation) was the prominent E2. However, the pattern of expression of the Ub- and Ub-like protein-conjugating E2 genes in the cerebral cortex did not completely overlap with that reported in cell lines. For instance, UBE2I/UBC9 (encoding SUMO) and UBE2N were abundant E2 proteins in cell lines [18]. Together with UBE2V1, UBE2N represents > 50% of the Ub-dedicated E2 enzymes in HeLa cells, and it is associated with UBE2V2 in Swiss 3T3 cells [18]. UBE2L3 is another abundant E2 that is expressed at levels twofold higher that all HECT and RBR E3 ligase genes in HeLa cells combined [18]. This expression pattern is profoundly different than that of the cerebral cortex, where the Ube2l3 and Ube2n genes were expressed at moderate levels (TPM values of 20–30) (Additional file 1: Fig. S1). The E2 enzyme UBE2L3 works in concert with the E3 ligase Ube3a (also known as E6-associated protein or E6-AP) [36]. Mutations and genetic defects in the Ube3a gene are associated with the Angelman syndrome, a neurodevelopmental disease [37]. The profile of E2 enzyme expression in nontumorous tissue is likely to differ from that in immortalized cells. Our data clearly illustrate that the pattern of E2 gene expression was temporally regulated. This pattern may, however, differ from one brain area to another.

Ub ligases (E3)

Ub ligase (E3) enzymes exert two crucial functions: they target a specific type of ubiquitinated substrate and enable the final transfer of Ub (to the substrate) [38]. In this study, E3 Ub ligases are grouped into 3 families according to [39]: the HECT (homologous to the E6-AP carboxyl terminus), RBR (RING-in-between-RING), and RING (really interesting new gene) E3 families. Depending on the E3 ligase, the transfer of Ub from the E2 enzyme to the target substrate can occur directly or via a 2 step process. For instance, RING E3 ligases enable a direct transfer of Ub from E2 to the target whereas the ubiquitination involving HECT develops via a 2 step process with Ub carried by the E2 enzyme binds first to a HECT domain before being transferred to the target protein [38, 39]. Although RBR E3 ligases have two RING domains and could be categorised as a sub-class of RING-type ligases, they are described as RING-HECT hybrids catalysing ubiquitination not directly like RING-type ligases but via a two-step reaction like HECT-type ligases during which Ub is transferred to the RING2 domain and then to the target [38,39,40,41].

HECT Ub ligases Genes were subdivided into 3 groups: Nedd, Herc and other HECT ligases [42]. Transcripts of twenty-four HECT genes were found (Fig. 2), with four genes (Ube2cbp, Hace1, Herc6, Hecw2) below the detection limit. The group of HECT E3 Ub ligases was dominated by the high abundance of Nedd4 transcripts. The TPM values decreased from 249 (on E11) to 84 (on PN1), reflecting a ⁓ threefold reduction in transcript abundance throughout corticogenesis. Nedd4 was the most highly expressed HECT E3 gene during neurogenesis (E11–E13) and the second most highly expressed gene at the end of corticogenesis (E17-PN1), after Hectd3, the other prominent HECT gene. Our data were in line with the first study reporting the isolation of both a set of Nedd4 cDNA clones and the corresponding mRNA expression [43]. This later study also showed a gradual mRNA decrease during embryonic development in the brain. Nedd4l, which is closely related to Nedd4, the founding and most ancient member of the Nedd4 family, was expressed on E13 and onwards at a very low level (TPM values < 9). These data illustrate the temporal regulation of these E3 Ub ligases, which play essential roles in neuronal cell fate determination and survival, neurite outgrowth, axon guidance and branching [44].

Fig. 2figure 2

Expression of the genes encoding HECT Ub E3 ligases. The figure shows the abundance of three groups of transcripts: Nedd, Herc and other HECT

Compared to HECT, E3 Ub ligases in the Herc group were expressed at much lower levels, with only Herc1 and Herc2 showing a TPM reaching a maximum of 23, on E17 (Fig. 2). In the third category (i.e., the HECT E3 Ub ligases that differ from Nedd and Herc), Hectd3 was the predominant gene, with highly regulated expression during corticogenesis, with TPM values increasing by a factor of 3 from E11 to PN1 (from ⁓ 30 to ⁓ 94 TPM) (Fig. 2). Huwe1 was the third most highly expressed gene of this subfamily of HECT E3 ligases, with TPM values of 43–60. Interestingly, knocking down HUWE1 expression in cortical tissue with a siRNA resulted in an increase in the fraction of proliferating cells in the developing brain and blockade of neuronal differentiation [45]. These results show that HUWE1 controls neural differentiation and proliferation. All the other genes in this subgroup were expressed at low levels, with TPM values < 30 (Fig. 2). The HECT E3 Ub ligase UBE3A has been well characterized because mutations in the Ube3a gene cause Angelman syndrome, a neurodevelopmental disease [37]. However, Ube3a was expressed at low levels throughout the cortex (TPM values of 16–25, Fig. 2). Notably, the highest abundance of HECT Ub ligase gene transcripts was noted at the peak of neurogenesis (E13) and then declined.

RING E3 Ub ligases RING E3 ligases constitute the largest family of E3 Ub ligases [8] [38]. For instance, a previous analysis of the mouse genome identified 398 putative E3 enzymes [46]. In the following sections, RING E3 Ub ligase genes are classified into three main subgroups: (1) single subunit, (2) multiple subunit RING E3 and (3) U-box RING E3 ligases.

A.

Single subunit RING E3:

A list compiled by [47] was used for the analysis of the major E3 ligase subgroups: Cbl, Deltex, Goliath, IAP, Listerin, Makorin, MARCH, Neuralized, Pellino, Pex, Polycomb, Praja, RBR, Siah, Traf, Trim and Ubr. Notable heterogeneity in expression levels was observed among these genes. The most highly expressed subgroup genes included Deltex, Goliath, Makorin, March, Neuralized, Praja, Polycomb, Traf (Fig. 3) and Trim genes (Fig. 4). The expression levels of the other minor gene groups (Cbl, IAP, Listerin, Pellino, Pex, Siah, and Ubr) are presented in Table 1. The TPM values of all the genes in this set were < 37, except the Ubr7 gene, for which the TPM value was ⁓ 60 on E11 and E13.

Deltex E3 Ub ligases Dtx3 and Dtx4 were the major deltex genes, with TPM values increasing from 179 to 351 (Dtx3) and from 40 to 91 (Dtx4) (Fig. 3A), revealing strong positive regulation during corticogenesis. However, the most highly regulated gene in this group was Dtx1: no transcripts were detected on E11, but it was clearly strongly induced later, with TPM values increasing from 9 (on E13) to 60 (on PN1), a nearly sevenfold increase (Fig. 3A). Deltex E3 has been principally studied in the context of tumorigenesis and tumour cell invasion [48], but very little is known about the roles played by Deltex E3 Ub ligases in the developing or adult brain in mammals. The marked enhancement of deltex gene expression supports the notion suggesting key roles in neuronal growth and differentiation in mammals.

Goliath E3 Ub ligases Twenty-nine orthologous genes of the Drosophila Goliath E3 Ub ligases were identified in mice (https://flybase.org/reports/FBgg0000104.html). However, nine of these genes were not expressed (Rnf43, Rnf128 (Grail), Rnf133, Rnf148, Rnf150, Znrf3, Znrf4, Zswim2 and 4930595M18Rik). In the expressed gene subgroup, Rnf44 (68–115 TPM), Rnf167 (72–125 TPM), and Rnf126 (46–70 TPM) were the major genes (Fig. 3B). Notably, Rnf215 was the most highly regulated gene, with the abundance of its transcripts decreasing from 74 to 27 TPM from E11 to PN1, a 2.7-fold decrease during corticogenesis. The contribution of RNF44 to brain formation and functions is unknown. The E3 ligase RNF167 plays important roles in neuronal cells. Although principally found in lysosomes, a fraction of RNF167 is present at the cell surface, where it participates in the ubiquitination of AMPA receptors. Ubiquitination modulates the number of AMPA receptors at the cell surface as well as synaptic currents. Therefore, RNF167 is an important physiological modulator of glutamatergic neurotransmission [49]. This RNF167-dependent ubiquitination of AMPA receptors was recently shown to be mediated by the E2 enzymes Ube2D1 and Ube2N [50]. RNF126, another prominent factor in this subgroup, has been shown to be involved in Friedreich ataxia, a severe genetic neurodegenerative disease characterized by reduced expression of the essential mitochondrial protein frataxin. The E3 Ub ligase RNF126 specifically mediates frataxin ubiquitination, which induces its degradation [51]. Our results point towards a role played by Goliath E3 Ub ligases in neuronal function from early embryonic stages.

Makorin E3 Ub ligases All three makorin genes (Mkrn1-3) were expressed in the immature cerebral cortex (Fig. 3A). Mkrn1 was the predominant gene, with TPM values increasing from 32 (on E11) to 116 (on E17). Consistent with our findings, Mkrn1 had been originally identified as a highly expressed gene during mouse embryonic development, with a high level of mRNA expression in the developing brain [52]. Low levels of Mkrn1 proteins were found in the brain despite its relatively high mRNA abundance due to the autoubiquitination properties of this E3 Ub ligase, which induces its own proteasomal degradation [53]. Experiments performed with Xenopus embryos showed that Mkrn2 proteins inhibit neurogenesis by acting downstream of phosphatidylinositol 3-kinase (PI3K) and Akt [54]. Thus, Mkrn proteins clearly play major roles in the developing nervous system.

MARCH E3 Ub ligases The family of proteins of the membrane-associated RING-CH (MARCH) comprises eleven E3 Ub ligases (MARCH-1 to -11) [55]. Four March genes of the eleven analysed in this study were not expressed: March-1, -3, -10 and -11. The major gene in the group was March 9. Its TPM value was approximately 60 on E13, which was profoundly increased on E17 (192 TPM) and PN1 (217 TPM), corresponding to a > 3.5-fold increase (Fig. 3A). On E17 and PN1, March 9 transcripts represented more than 50% of all March transcripts. In dendritic cells, MARCH-9 proteins localize to the trans-Golgi network (TGN) and controls a TGN‐to‐endosome transport step [56]. In previous studies, MARCH-9 expression had been mainly found in immune cells and organs su

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