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Williams–Beuren syndrome (WBS) is a neurodevelopmental and multisystemic disease that results from hemizygosity of approximately 25 genes mapping to chromosomal region 7q11.23. We report here the preliminary description of eight novel genes mapping within the WBS critical region and/or its syntenic mouse region. Three of these genes, TRIM50, TRIM73 and TRIM74, belong to the TRIpartite motif gene family, members of which were shown to be associated to several human genetic diseases. We describe the preliminary functional characterization of these genes and show that Trim50 encodes an E3 ubiquitin ligase, opening the interesting hypothesis that the ubiquitin-mediated proteasome pathway might be involved in the WBS phenotype.
The Williams–Beuren syndrome (OMIM#194050) is characterized by mental retardation with unique cognitive and personality profile and multiple dysmorphic and metabolic features.1-5 WBS is caused by a microdeletion of a 1.55 or 1.84 Mb region mapping to chromosome band 7q11.23,6 that contains about two dozen of genes.7-11 The hemizygosity of this region is almost exclusively due to sporadic de novo deletions.1,3,12 Mouse models of LIMK1, CYLN2 and GTF2IRD1 suggest that hemizygosity of these genes might play a role in some aspects of this phenotype and/or in craniofacial anomalies13-15 although no absolute association was established. On the contrary, the hemizygosity of the ELN gene was unequivocally linked to the supravalvular aortic stenosis phenotype.16 Further genotype–phenotype correlations have been proposed following diagnosis of atypical patients presenting smaller or ‘shifted’ deletions.13,17-26 Nevertheless further understanding of the molecular pathogenesis of WBS requires the identification and functional characterization of all the genes mapping within the 7q11.23 WBS region.
The TRIpartite motif (TRIM) proteins (also known as RBCC proteins) harbor from their N- to their C-terminal end, a RING (R), one or two B-boxes (B) and a predicted Coiled coil (CC).27-30 The TRIM motif is usually followed by either one or two C-terminal domains, which are specific for each member of the family. To date, more than 68 genes encoding TRIM proteins have been identified in the human genome.31 Alteration of these proteins causes pathological conditions that range from Mendelian diseases to cancer development and viral infection.32-40 In this study, we report the preliminary description of eight mammalian genes mapping in the WBS critical region (WBSCR) and/or the mouse syntenic 5G1 band and experimental evidence showing that one of those genes, TRIM50 encodes an E3 ubiquitin ligase.
A fully detailed description of the procedure followed for gene identification and sequencing can be viewed in the Supplementary online section. Briefly, we screened the human and mouse dbEST databases to create clusters of ESTs mapping to the genomic interval of interest. These contigs were repeatedly extended by rescreening of dbEST until no new matching ESTs were found.41,42 To identify the 5′-end of the TRIM50-like genes 5′-rapid amplification of cDNA ends was performed on polyA+ RNAs from 12 human tissues as described.43
Mouse northern RNA blot-12 major tissues (Origene) were hybridized following the manufacturers' recommendations with the inserts of IMAGE clones 3484221 (Wbscr24), 602660 (Wbscr25), 336334 (Wbscr26), 4950109 (Wbscr27), with the 1452 bp Trim50 ORF fragment, or with the 464 bp Wbscr28 partial ORF fragment. Sample loading was assessed using an actin probe (not shown).
The full-length ORF of mouse Trim50 was cloned into pCDNA3 vectors modified to include either an myc-EGFP or an HA-tag.30 Trim50 deletion mutants were created using appropriate oligonucleotides and amplification followed by in-frame insertion into the above vectors. The Trim50 A251X that correspond to a murine Trim73-like cDNA was engineered with the Quick-change directed mutagenesis kit (Stratagene).
293T, COS-7, HeLa, U2OS and HepG2 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% antibiotics. Fugene 6 (Roche) was used for transfection according to the manufacturers' instructions.
Co-immunoprecipitation experiments were performed as described previously.44,45 Complexes were analyzed by western blotting using appropriate antibodies (anti-GFP and anti-myc (Santa Cruz Biotechnology), anti-HA (Covance) and anti-FLAG (Sigma). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies (GE Healthcare) and the ECL chemiluminescence system (GE Healthcare) were used for detection. Where indicated, the MG132 proteasome inhibitor (Santa Cruz Biotechnology) was added at 10 μm for 4 h.
Interaction mating is a variation of the classic two-hybrid screening. The bait plasmids express the cDNA directionally fused to the first 202 residues of LexA under the control of the constitutive ADH promoter. Prey plasmids express cDNAs fused to the B42 activation domain, the SV40 T nuclear localization signal (NLS) and an HA tag under the control of the inducible GAL1 promoter. The two plasmids are introduced separately in the two yeast haploid strains EGY48 and EGY42. The pSH18-34 vector with 6 LexA-operators lacZ and a genome integrated 4 LexA-operators LEU2 are used as reporters. EGY48/EGY42 diploids for every pairwise combination were generated by mating.46
Lysates of U20S cells transfected with myc-EGFP-Trim50 were incubated with varying concentrations (0, 0.2, 0.4, 0.8, 2.0 mm) of glutaraldehyde (Sigma) at room temperature for 5 min, followed by the addition of an excess of glycine to quench the reaction. The cross-linked lysates were then subjected to western blot using anti-GFP antibody. Transfected U20S with myc-EGFP-Trim50 were treated with 100 μg/ml of cycloheximide at different time points and cell lysates containing equal amount of total proteins were subjected to western blot using anti-GFP and GAPDH antibodies.
Transfected cells were fixed in 4% formaldehyde for 15 min at room temperature, washed, permeabilized with 0.1% Triton X-100 and blocked 1 h with 1% bovine serum albumin. The anti-HA antibody (Covance) and the secondary anti-mouse AlexaFluor 568 (Molecular Probes) were used at 1:1000 and 1:500 dilutions, respectively. Cells were incubated for 1 h with the primary antibody at room temperature. After extensive washing, cells were incubated with the secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI (0.5 μg/ml). MitoTracker, ER-Tracker, LysoTracker and SelectFX 488 Peroxisome Labeling Kit (all Molecular Probes) and ADLP (Chemicon) were used for staining of mitochondria, endoplasmic reticulum, lysosomes, PMP70 and ADLP peroxisomal proteins, respectively.
To refine the WBSCR annotation, we identified, cloned and sequenced the human WBSCR24, WBSCR26, WBSCR27, WBSCR28, TRIM50, TRIM73 and TRIM74 genes and the murine Wbscr24, Wbscr25, Wbscr26, Wbscr27, Wbscr28 and Trim50 genes (nomenclature committee approved symbols, ie, WBSCR transcript 24–28, TRIM gene 50, 73 and 74). A schematic representation of the region is depicted in Figure 1. All sequences were deposited in GenBank (AF498998–999, AK008014, AY081947–954, AY352519, AY354925–928, AY369079-080, AY372053–054 and BM008899). Mouse tissue northern blots were performed to confirm these as bona fide genes (Figure 2). The size of the Wbscr24-, Wbscr25-, Wbscr26-, Wbscr27- and Wbscr28-deposited sequences is analogous to the estimated sizes determined by northern blotting for these transcripts, suggesting that we have identified and sequenced the entire mouse cDNAs (Supplementary Table S1), while we confirmed the entire Trim50 ORF by RT–PCR followed by sequencing (see ‘Materials and methods’).
We identified three TRIM50-like copies on the human genome, TRIM50 (also known as TRIM50A), TRIM73 (TRIM50B) and TRIM74 (TRIM50C). All three are expressed, as demonstrated by the identification of spliced ESTs specific to each transcript. TRIM50 maps between WBSCR20A and FKBP6 within repeat BLOCK C-mid, TRIM73 between WBSCR20B and FKBP6T2 in BLOCK C-tel, while TRIM74 maps to the BLOCK C-cen interval, between WBSCR20C and FKBP6T1 (Figure 1).8,10,47 WBS patients are therefore hemizygotes for TRIM50, but not for TRIM73 and TRIM74 (Figure 1).6 TRIM50, TRIM73 and TRIM74 share five orthologous exons. Stronger sequence conservation is observed between the telomeric and centromeric TRIM50-like copies (TRIM73 and TRIM74, 947/949 bp identity (99.8%)), than between these copies and the central copy (TRIM50, 913/949 (96.2%) and 914/949 bp (96.3%) identity, respectively) (Figure 1). The sequences of the three TRIM50-like copies diverge after exon 5, as exons 6 and 7 are absent from BLOCK C-cen and BLOCK C-tel.
The TRIM50 transcript encodes a putative protein of 487 residues 88% identical to its murine orthologue (Trim50). The presence of a single copy of TRIM50-like genes in rodents is consistent with the absence of duplications in the WBS-corresponding region of mouse chromosome 5.11,48 TRIM50 and Trim50 harbor, from their N- to their C-terminal end, a RING, a B-box type 2, a CC and an RFP-like/B30.2 domain. TRIM73 and TRIM74 encode almost identical putative proteins of 250 residues (99.6% identical, 249/250) containing only the R, B2 and CC domains. These peptides are 93 (233/250) and 94% (234/250) identical to the first 250 amino-acid residues encoded by TRIM50. They show 80% identity to Trim50 (201/250), in a region where TRIM50 and Trim50 are 85% identical (212/250), an observation consistent with the notion that speciation preceded duplication.
Because recent data suggest that some TRIM proteins act as E3 ubiquitin ligase31,49-55 we investigated whether Trim50 retains this ability. First, we co-transfected HEK293T cells with HA-tagged Trim50 (HA-Trim50) and myc-tagged ubiquitin-expressing plasmids or corresponding empty vectors. Total cell lysates were immunoprecipitated with anti-HA antibody, separated by gel electrophoresis and subjected to western blot analysis with anti-myc antibody (Figure 3a). The precipitated HA-Trim50 is polyubiquitinated at varying degree. These polypeptides are detected as a high-molecular weight smear. Specificity of this signal is assessed by transfection with the myc-tag vector only (compare lane 2 with lane 1 and lane 4 with lane 8). The formation of the Trim50-Ub polypeptides is increased in presence of UbcH8 (also known as UBE2L6), an E2 ubiquitin-conjugating enzyme (compare lanes 3 and 2). Further intensification of the signal is achieved through inactivation of the proteasome with the MG132 inhibitor (compare lanes 4 and 3). Similar results were obtained using transfected COS-7 cells (data not shown).
E3 ubiquitin ligase enzymes directly interact with E2 ubiquitin-conjugating enzymes and their substrate.56 To further assess the E3 ubiquitin ligase activity of Trim50, we therefore investigated if this protein was able to interact with some E2 ubiquitin-conjugating enzymes. We found that Trim50 preferentially binds to ubiquitin-conjugating enzyme UbcH8 (UBE2L6) rather than UbcH7 (UBE2L3; Figure 3b) in immunoprecipitation experiments. Moreover, we took advantage of the yeast interaction-mating technology46 to determine if Trim50 was able to bind to other E2 enzymes. The cDNAs encoding Trim50 and several E2 enzymes were expressed as fusion proteins with the LexA-DNA-binding domain (baits) and tested for interaction in a binary assay with the same proteins fused to the B42-acidic moiety (preys). Strong binding was observed with UbcH6 (UBE2E1) and UbcH9 (UBE2E3; Figure 4a). Weak binding was detected with an additional E2 enzyme, UbcH5B (UBE2D2), while no interaction was detected with UbcH2 (UBE2H) (data not shown). We then sought to investigate whether, as reported for other RING-containing E3 ubiquitin ligases, the RING domain of Trim50 was responsible of the specific interaction with the E2-conjugating enzyme.57 To assess the contribution of each TRIM domain we created a set of Trim50 deletion mutants (Figure 4b) and tested their ability to retain the binding with the E2 ubiquitin-conjugating enzymes in interaction-mating and co-immunoprecipitation experiments. We confirmed that the RING domain is necessary and sufficient for binding to E2 enzymes (Figures (Figures3c3c and and4a4a).
Together these results indicate that Trim50 is a novel E3 ubiquitin ligase that through the RING domain catalyzes ubiquitination in presence of UbcH8 and possibly other E2s and that Trim50 is itself ubiquitinated.
TRIM proteins were shown to define specific subcellular compartments as a consequence of their propensity to form higher-order molecular weight structures and oligomerization seems to be a common feature of cytoplasmic TRIM family members.30,58,59 To investigate whether also Trim50 has this propensity, we first performed yeast interaction-mating assays and showed that Trim50 is able to homodimerize (data not shown). In addition lysates from U2OS cells transfected with myc-EGFP-Trim50 were incubated with increasing concentration of glutaraldehyde and analyzed by western blotting. As reported in Figure 5a, Trim50 is cross-linked into gel-stable complexes with high molecular weight of approximately 250 kDa consistent with the formation of trimers.
Next, we examined the half-life of Trim50 in U20S-transfected cell lines treated with cycloheximide and assayed for protein expression level at different points. These experiments demonstrate that Trim50 is a stable protein also 6 h after treatment (Figure 5b).
We investigated the subcellular localization of Trim50 in mammalian cell lines. Ectopically expressed Trim50 localizes into discrete cytoplasmic structures in COS-7, HeLa, HepG2 and 293T cells (Figure 6; data not shown). Next, we investigated the contribution of the TRIM domains to this localization (Figures (Figures4b4b and and6).6). Independent expression of the RING and the RFP domain shows a diffuse nuclear and a vast perinuclear subcellular distribution, respectively (Figures 6g and m), suggesting that these domains include an NLS and a nuclear export signal, respectively. Consistently, deletion of the RFP domain induces relocalization of the mutant protein to discrete nuclear structures, while the RING domain deletion mutant localizes like the full-length protein (Figures 6j and p). The expression of a construct carrying only the B-box and the CC domains defines the same discrete cytoplasmic structures as the full-length and the RING domain-deleted protein (Figure 6s). This construct also shows a weak diffuse signal in the nucleus, suggestive of a decreased capacity of this protein to be exported outside of the nucleus. These results confirm that the central region of the tripartite proteins (B-box and CC domains) is indispensable for the proper localization to subcellular compartments,30 while the RING and RFP domains cooperate to this localization.
Next, we compared the localization of myc-EGFP-Trim50 with compartment-specific cellular markers. No co-localization of Trim50 staining was observed with endoplasmic reticulum (Figures 7p–r), mitochondria (Figures 7m–o) and lysosomes (Figures 7g–i). Interestingly, we observed a partial colocalization with peroxisomes (Figures 7a–f and j–l). These results are in agreement with the notion that TRIM proteins mainly define novel subcellular compartments.30
To identify candidate genes for the multiple WBS phenotypes and contribute to the update of the WBSCR annotation, we analyzed ESTs and cDNAs corresponding to previously identified.11 and novel transcription units. Our analysis allowed the mapping and sequencing of eight mammalian genes mapping to 7q11.23 (WBSCR24, WBSCR26, WBSCR27, WBSCR28, TRIM50, TRIM73, TRIM74) or the mouse syntenic 5G1 band (Wbscr24, Wbscr25, Wbscr26, Wbscr27, Wbscr28, Trim50). They encode TRIM-containing proteins (TRIM50, TRIM73, TRIM74 and Trim50), proteins containing a ‘modifier of rudimentary’ (WBSCR24 and Wbscr24) or a methyltransferase domain (WBSCR27 and Wbscr27). The role of these novel genes in the pathology of WBS remains to be determined; however we present here the preliminary genomic and functional characterization of Trim50.
Trim50 specifically interacts with E2 ubiquitin-conjugating enzymes and autoubiquitinates showing that it can act as an E3 ubiquitin ligase. This enzymatic activity was already reported for other TRIM protein members, namely TRIM5δ, TRIM18/MID1, TRIM23/ARD1, TRIM25/Efp and TRIM32/HT2A, TRIM37 targeting the degradation of proteins such as phosphatase 2A and 14-3-3σ,49-51,53-55 The interaction of Trim50 with the E2 enzymes is mediated by the RING domain as already reported for other RING-containing proteins.54,55,57,60 This E3 enzyme localizes to cytoplasmic aggregates that do not correspond to organelles such as the endoplasmic reticulum, mitochondria, lysosomes, although a very partial localization was shown with peroxisomes (Figure 7). This localization is dependent on the integrity of a central region that includes the B-box type 2 and the CC domains (Figure 6). Many other TRIM family proteins showed co-localization with cytoplasmic aggregates similar to those observed for Trim50.30 For example, TRIM20/pyrin, defective in familial Mediterranean fever, forms perinuclear aggregates when transiently expressed in cell culture.61 Similarly aggregates were reported for TRIM37, defective in mulibrey nanism,50 supporting the notion that their formation is a common propensity of TRIM proteins.31 These structures, named aggresomes, are defined as perinuclear regions where misfolded and aggregated proteins are sequestered for proteasomal degradation.62 Hence, we can speculate that the TRIM E3 ubiquitin ligases localize to these sites to control the ubiquitination and eventually the degradation of specific targets.
WBS patients are hemizygous for TRIM50, but not for the paralogous TRIM73 and TRIM74 copies (Figure 1)6 and the expression of this gene appears to be reduced accordingly in lymphoblastoid cell lines established with patient blood.63
Thus hemizygosity of the TRIM50 E3 ubiquitin ligase possibly plays a role in the WBS phenotype as the result of accumulation of specific TRIM50 target substrates.
Considering the myriad of substrates that can be targeted by an E3 ligase, it is not surprising that mutations in these genes result in several pathological conditions. Consistently, several syndromes associated with mental impairment such as Opitz syndrome (OMIM #300000), mulibrey nanism (OMIM #253250), Bardet–Biedl syndrome (OMIM #209900) and limb-girdle muscular dystrophy type 2H (OMIM #254110) are caused by mutations of TRIM family E3 ubiquitin ligases.34-36,40 For example, TRIM18/MID1, the gene mutated in Opitz syndrome, targets the catalytic subunit (PP2Ac) of the microtubule-associated phosphatase PP2A for degradation. Mutations in its B30.2 domain abolish microtubule binding leading to accumulation of PP2Ac and hypophosphorylation of microtubule-associated proteins.34,49
It is unclear how a defective TRIM50 E3 ligase activity could influence some of the clinical manifestations of WBS. The specific expression of TRIM50 and Trim50 in stomach, intestine, liver and brain (Figure 2; L Micale et al, unpublished data) suggest a possible involvement of TRIM50 haploinsufficiency in the gastrointestinal pathologies and/or the cognitive profile of WBS patients. The identification of TRIM50-interacting proteins, as well as its substrates should provide a better understanding of the biological function of this E3 ligase, as potentially an insight into the molecular pathogenesis of WBS.
It is likely, although not experimentally verified in this study, that both TRIM73 and TRIM74 proteins retain their ability to act as E3 ligase because they have the CC and RING domains. However, these proteins might not be fully redundant with TRIM50, because the RFP-like domain (also known as B30.2), that is absent in TRIM73 and TRIM74, was suggested to be important for interaction with proteasome subunits.64 Furthermore, our Trim73-like construct, which mimics TRIM73 and TRIM74, also shows a nuclear staining absent in Trim50 (Figure 4b; Supplementary Figure S1). Consistently, analysis of MID1 (TRIM18) mutations detected in Opitz syndrome patients demonstrated the importance of the RFP-like domain in the proper localization of the protein.58,65
In conclusion, the present report increases the number of identified genes mapping within the region commonly deleted in WBS patients and thus putatively involved in phenotype determination. Having shown that one of these novel genes, TRIM50, acts as an E3 ubiquitin ligase opens the interesting hypothesis of a direct involvement of ubiquitination in the WBS pathology.
We thank Satoshi Inoue for reagents and Stylianos E Antonarakis and Samuel Deutsch for discussion. This work was supported by grants from the Jérôme Lejeune Foundation, the Désirée and Niels Yde Foundation, the Novartis Foundation, the Swiss National Science Foundation and European commission (grant 037627) to AR, grants from the Italian Ministry of Health (RC 2006–2007), the Fondazione Banca del Monte di Foggia ‘Domenico Siniscalco Ceci’, Italian Telethon Foundation (Grant N. GGP06122) and the Jérôme Lejeune Foundation to GiM, and the Italian Telethon Foundation to GeM.
DDBJ/EMBL/GenBank accession nos.: AF498998–999; AK008014; AY081947–954; AY352519; AY354925–928; AY369079–080; AY372053–054; BM008899.
Conflict of interest