While there is growing evidence that distinct protein isoforms resulting from alternative splicing play roles in learning and memory, cell recognition and ion channel function, little is known about how this splicing is regulated in the nervous system. We have cloned the
unc-75 locus and find that it encodes a predicted RNA-binding protein related to the Bruno/Elav/Etr family (C17D12.2). Members of this family are involved in diverse aspects of RNA regulation including translational control, mRNA transport and splicing.
unc-75 is expressed broadly in the nervous system as well as in the excretory gland. We have visualized UNC-75 protein in living animals using rescuing GFP fusion constructs and find that it localizes to subnuclear puncta. The puncta are quite dynamic and reminiscent of splicing speckles previously observed in cell culture. Furthermore, we have shown that this subnuclear localization is essential for rescue of
unc-75 mutant phenotypes.
unc-75 animals are uncoordinated, are resistant to cholinergic agonists, lay eggs constitutively, and are severely compromised for defecation. Mutants also have subtle neuroanatomical defects that parallel those seen in synaptic activity mutants. We speculate that
unc-75 has a modulatory role in neurotransmission. Consistent with this non-developmental role, we can supply mutants with a dose of
unc-75 as adults and rescue their locomotory defects. Furthermore, we find that the aldicarb resistance phenotype of
unc-75 can be rescued at elevated temperatures. Hypomorphs in presynaptic machinery components such as
unc-64 and
unc-13 show a similar temperature-dependent response to aldicarb while those involved in neurotransmitter synthesis/transport (
unc-17) do not. Taken together, our data suggest that
unc-75 is a neural-specific splicing factor required for the fine-tuning of forward neurotransmission, possibly at the level of synaptic vesicle fusion.Interestingly, we have also found that the three closest human orthologs of
unc-75 (CELF3, hBRUNOL4 and hBRUNOL5) are expressed nearly exclusively in brain. Each has been shown to modify mRNA splicing in vitro. When expressed in C.elegans, hBRUNOL4 is localized to nuclear speckles and can rescue
unc-75 defects. Therefore, it is likely that information obtained on
unc-75 function in C.elegans will be directly translatable to studies of the human orthologs. We are currently designing experiments to identify
unc-75 targets in C.elegans.