In recent years a superfamily of ubiquitin-like domains has been identified [30
]. This superfamily can be divided into the ubiquitin-like proteins (UBL's), which consist solely of the ubiquitin-like domain, and ubiquitin domain proteins (UDP's), which are larger proteins containing one or more ubiquitin-like domains. To our knowledge DWNN is the first example of a ubiquitin-like domain that is alternatively expressed both as a UBL and as a UDP.
Ubiquitin-like proteins typically share the C-terminal GG motif, which acts as a recognition motif for a protease that cleaves between the two glycines, initiating the process of conjugation. The occurrence of the GG motif in the structurally identical position in human and mouse DWNN domains (highlighted in pink in Figure ) suggests that the domain may be involved in a similar process of conjugation, which we may call "DWNNylation". As in the case of ubiquitin, the GG lies outside of the structured region, as can be clearly seen in Figure . The absence of the GG in lower organisms is more difficult to rationalise; however preliminary EST analysis suggests that organisms which do not contain the GG motif also do not contain the UBL form of the DWNN domain (unpublished data), so it is possible that the DWNN domain does not act as a covalent modifier in lower organisms. In the yeast protein Hub1, which has also been shown to be involved in pre-mRNA splicing [31
], the role of the di-glycine motif is taken by a YY motif [32
]. The structurally equivalent position in DWNN is taken by a highly conserved RR motif (see Figure ), which may therefore act as the activation signal.
Ubiquitin contains four conserved lysine residues which are the sites of attachment of additional ubiquitin moieties, leading to the formation of poly-ubiquitin chains in some contexts [33
-linked chains are recognised by the 26S proteosome, leading to degradation of the attached protein, whereas K6
-linked and K63
-linked chains are involved in a number of non-proteolytic processes, including stress response, DNA repair and endocytosis [34
-linked and K29
-linked chains may also target substrates to the proteosome. In addition, mono-ubiquitination of proteins has been shown to be associated with receptor endocytosis, as well as the sorting and trafficking of proteins [35
]. The DWNN domain contains no equivalent of K48
(see Figure ), although the equivalents of K6
in DWNN) are highly, although not absolutely, conserved.
A number of lines of evidence suggest a role for RBBP6 in both mRNA processing and ubiquitin-like protein modification. The close association between domains involved in RNA metabolism and ubiquitination has previously been pointed out in a number of proteins, including MDM2 [24
]. In yeast, the RBBP6 homologue Mpe1p has been shown to be a component of the CPF complex [11
]. Mammalian RBBP6 has been identified as an SR protein on the basis of an SR domain (residues 477–570) [2
], the CCHC RNA binding domain, its localisation within nuclear speckles [9
] and its associate with heterogeneous nuclear ribonucleoproteins (hnRNPs) [3
]. SR proteins are involved in splicing, whereas hnRNPs are thought to play a central role in organising the polyadenylation, splicing and export of mRNA transcripts [36
]. A number of SR proteins are known to interact directly with the C-terminal domain of the RNA Polymerase II complex. A role for RBBP6 in mRNA processing therefore seems highly probable. The presence of a RING finger domain in all eukaryotes, combined with the ubiquitin-like structure of the DWNN domain, makes it highly probable that RBBP6 also has ubiquitin-ligase activity, possibly involving modification of hnRNPs with a ubiquitin-like moiety. Several hnRNPs have recently been shown to be SUMOylated [37
], which resulted in a decreased affinity of the hnRNP for mRNA.
Furthermore, since p53 and Rb have both been shown to bind to mammalian RBBP6, it is possible that RBBP6 plays a role in the regulation of these two proteins similar to that played by MDM2 [38
], suggesting a possible model for the integration of the regulation of transcription, cell cycle control and apoptosis. Given the fact that the DWNN domain can be independently expressed in vertebrates, an interesting possibility is that the function of RBBP6 is to DWNNylate other proteins.