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
) or the mouse syntenic 5G1 band (Wbscr24
). They encode TRIM-containing proteins (TRIM50
), proteins containing a ‘modifier of rudimentary’ (WBSCR24
) or a methyltransferase domain (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 (). This localization is dependent on the integrity of a central region that includes the B-box type 2 and the CC domains (). 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 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 (; 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 (; 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.