We describe here 2 muscle-specific alternative splice forms of the ubiquitin ligase, UFD2a. The smaller, UFD2a-7 contains the previously identified exon 7, while UFD2a-7/7a incorporates a novel exon 7a in addition to exon 7. Both of these alternatively spliced exons are located just downstream of the previously described MPAC regulatory domain and within the N-terminal extension of UFD2a, which is unique to vertebrate orthologs 
. The GenBank database includes expressed sequence tag entries containing exon 7 spliced to exon 8 in human primary fibroblasts and thalamus tissue 
, and a full-length cDNA sequence of UFD2a-7
cloned from a mouse fetal brain library. However, we did not detect UFD2a-7 protein in human or mouse fibroblast or brain tissues. Two recent genome-wide exon-junction and whole transcript microarrays, which examined the expression of alternative pre-mRNA splice forms across a total of 92 tissues and cell lines, both found that exon 7 was not present in UFD2a cDNA from human fetal brain tissue. In fact, consistent with the data presented here, exon 7 was exclusively expressed in adult human skeletal muscle, heart, tongue (therefore, predominantly in striated muscle cells) and to a lower extent peripheral blood leukocytes. Of the nine fetal tissues tested, only fetal heart expressed exon 7 (fetal skeletal muscle was not included). The tissue specificity of exon 7a was not examined since its presence in UFD2a
had not yet been reported.
Our RT-PCR and Western blot data suggest that UFD2a-7 is uniquely expressed in a transient manner during myogenic development and myoblast differentiation. The timing of UFD2a-7 expression during differentiation and after injury appeared to correlate with that of the myogenic regulatory factors Myf5 and MyoD. Interestingly, ubiquitin-dependent degradation regulates most of these myogenic regulatory factors during myogenesis. The degradation of Myf5, in particular, is required for myoblast fusion 
which occurs at the time of UFD2a-7 expression. In developing skeletal muscle, it coincided with the greatest expression of embryonic MHC (data not shown).
In cell culture models of differentiation and in vivo models of regeneration, UFD2a and UFD2a-7 protein persisted, most likely because differentiation was not complete. However, UFD2a-7 was not present in adult striated muscle tissue in vivo, and though small amounts of the ubiquitous UFD2a isoform were detected in tissue lysates, only UFD2a-7/7a was seen on Western blots of isolated EDL muscle fibers (data not shown). Therefore, UFD2a-7 expression is transient during muscle differentiation, a phenomenon which is mirrored during development, where UFD2a-7 was detected in both embryonic tissue from mice and zebrafish but disappeared by 7 days after birth.
The newly identified UFD2a-7/7a isoform was expressed with slightly delayed kinetics during differentiation in vitro
and during regeneration and development in vivo
, where its levels increased with time while UFD2a-7 decreased. Therefore, in both in vitro
and in vivo
models of myogenesis, the UFD2a alternatively spliced transcripts are expressed sequentially. The ubiquitous short isoform is likely expressed in dividing myoblasts due to its role in mitosis, while the transient expression of UFD2a-7 may be important for transitory events during differentiation such as myoblast fusion. Since expression of UFD2a-7/7a occurs late, it may have a unique function in terminal differentiation. Identification of the alternative splice form of Integrin α7β1, which appears late in differentiation 
, provides evidence that splicing can influence processes required for differentiation since α7β1 isoform specific protein-protein interactions regulate myotube formation and cell adherence in culture 
. The fact that UFD2a-7/7a did not bind to VCP/p97 suggests that UFD2a alternative splice forms also have different binding specificities, strongly suggesting a unique function for UFD2a-7 and -7/7a in differentiated muscle cells.
Although several other muscle-specific ubiquitin E3 ligases have been identified, including F-box (MAFbx)/atrogin-1 and the muscle-specific RING finger (MURF) family, and their importance during development and muscle atrophy are well described 
, UFD2a is unique in that it represents a ubiquitously expressed ligase that has distinct alternative-splice forms in striated muscle. The central section of the exon 7a sequence is unusually rich in acidic amino acids. Although the length of this acidic sequence is variable among species, its presence is conserved. This finding suggests that the exon contains a unique binding domain that mediates interaction with a positively charged molecule. Interestingly, the N-terminal domain of UFD2a contains an RRRL motif, which mediates its interaction with VCP/p97. In fact, the yeast 2-hybrid data presented here showed that UFD2a-7/7a fails to bind VCP/p97, suggesting that the acidic stretch of exon 7a may disrupt UFD2a folding, affecting this interaction.
One striated muscle protein whose proteasome-dependent degradation depends on UFD2a and VCP/p97 is UNC-45b 
. C. elegans
contains only one unc-45
gene, but vertebrates express UNC-45a
(also referred to as the General Cell (GC) form) in most tissues and UNC45b
exclusively in striated muscle tissue 
. UNC-45 proteins have myosin chaparonin activity which under certain conditions may involve forming a co-chaperone complex with HSP-90 
, however recent studies in C. elegans
suggest that UNC-45 function in muscle is inhibited by HSP-90 binding 
UNC-45a appears to be essential to basic cellular processes such as cytokinesis, myoblast proliferation, and early fusion events, and UNC-45b is required for proper assembly of sarcomeric myosin filaments 
. Interestingly, the expression of UNC-45 isoforms mirrors those of UFD2a, where UNC-45a is expressed early during differentiation, and later its levels decrease while those of UNC-45b increase 
. The degradation of human UNC-45b has been shown to be dependent upon human UFD2a and VCP/p97 
, but the mechanism of UNC-45a degradation has not been studied. Potentially, UFD2a may not target UNC-45a for degradation, thereby allowing levels of this isoform to remain high during myoblast proliferation. However, at this time and during early stages of differentiation, UFD2a may ubiquitinate UNC-45b and target its degradation in a manner dependent on its interaction with VCP/p97. The importance of the regulation of UNC-45b levels is illustrated by the detrimental effects of UNC-45b overexpression. Increased Unc-45b in zebrafish results in disorganized myosin filaments 
, and overexpression of Unc-45 in worms leads to paralysis 
. Later in differentiation, levels of UFD2a decrease concomitant with an increase in UFD2a-7 and -7/7a, the latter of which may not bind to VCP-p97, promoting UNC-45b degradation and subsequent accumulation of this vital chaperone. Moreover, UFD2a-7 may specifically target UNC-45a after the early myoblast fusion events have completed.
Like UFD2a, VCP/p97 has been implicated in the progression of cells through mitosis, a process that would be restricted to proliferating myoblasts, which express UFD2a-7. When VCP/p97 is bound to the UFD1-Npl4 cofactor complex, it extracts ubiquitinated Aurora B kinase from mitotic chromosomes, a process that is essential for decondensation, nuclear reformation, and spindle disassembly 
. If VCP/p97 were to interact with the UFD1-Nlp4 complex and UFD2a in a mutually exclusive fashion, UFD2a binding may inhibit premature extraction of Aurora B. The fact that both UFD2a and VCP/p97 are required for mitotic progression suggests that their interaction may be important only during myoblast proliferation. Later in differentiation, when myocytes have exited the cell cycle, the switching of UFD2a isoform expression to UFD2a-7/7a may promote complete differentiation by simply enabling VCP/p97 to bind other cofactors.
The differential binding of UFD2a and UFD2a-7/7a to VCP/p97 is also of interest given that mutations in this AAA-type ATPase are present in patients with inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia and cause complete disease pathology in transgenic mice 
. In vitro
studies have suggested that VCP/p97 is crucial for homeostasis in mature muscle and during differentiation. Indeed, expression of disease-associated mutant VCP/p97 leads to an accumulation of ubiquitin-loaded autophagosomes and decreased myoblast fusion 
In summary, the data reported here show that 2 alternatively spliced isoforms of the E3/4 ubiquitin ligase UFD2a, resulting from the inclusion of 2 exons highly conserved across vertebrate species, are specifically expressed in differentiating or mature striated muscle cells. Intron sequences immediately downstream of these muscle-specific exons contain recently described sequence motifs thought to convey muscle-specific alternative splicing. Given the sequential nature of isoform expression during zebrafish development and mammalian skeletal muscle differentiation and regeneration, we hypothesize that UFD2a-7 participates in key events of early myoblast differentiation, including the first cell-fusion event, and UFD2a-7/7a maintains the postmitotic state in mature muscle. We postulate that the differential binding capacity of these UFD2a isoforms to the ubiquitin-binding chaperone VCP/p97 is important to the regulation of UFD2a function during this process. These data are the first to suggest that UFD2a has divergent functions in striated muscle tissue and that elucidation of these functions during myocyte differentiation and physiology may provide further insight into the mechanisms of muscle regeneration and response to injury and disease.