Abstract
Nova proteins are neuron-specific RNA binding proteins targeted by autoantibodies in a disorder manifest by failure of motor inhibition, and they regulate splicing and alternative 3′ processing. Nova regulates splicing of RNAs encoding synaptic proteins, including the inhibitory glycine receptor α2 subunit (GlyRα2), and binds to others, including the GIRK2 channel. We found that Nova harbors functional NES and NLS elements, shuttles between the nucleus and cytoplasm, and that 50% of the protein localizes to the soma-dendritic compartment. Immunofluoresence and EM analysis of spinal cord motor neurons demonstrated that Nova co-localizes beneath synaptic contacts in dendrites with the same RNA, GlyRα2, whose splicing it regulates in the nucleus. HITS-CLIP identified intronic and 3′ UTR sites where Nova binds to GlyRα2 and GIRK2 transcripts in the brain. This led directly to the identification of a 3′ UTR localization element that mediates Nova-dependent localization of GIRK2 in primary neurons. These data demonstrate that HITS-CLIP can identify functional RNA localization elements, and they suggest new links between the regulation of nuclear RNA processing and mRNA localization.
Highlights
Posttranscriptional regulation is thought to play a crucial role in generating diversity in the postsynaptic dendrite
Nova1 can be seen in the cytoplasm in cells transfected with the WT but not in cells transfected with the ΔNES construct; in contrast the ΔNLS construct is largely excluded from nuclei
Since Nova DKO mice die at birth (Ule et al, 2006; Ruggiu et al, 2009), Nova1 null animals die in the first 7–10 days postnatally (Jensen et al, 2000) and Nova2 null animals usually die in the second postnatal week, we examined mRNA localization of a different Nova target
Summary
Posttranscriptional regulation is thought to play a crucial role in generating diversity in the postsynaptic dendrite. This includes links between RNA splicing, RNA localization, RNA translation and synaptic formation, long term potentiation and synaptic plasticity (Schuman et al, 2006; Sutton and Schuman, 2006; Lin and Holt, 2007; Richter and Klann, 2009). Neurons have been found to generate preferentially long 3′ UTRs (Wang et al, 2008) in a manner regulated by neuronal RNABPs (Licatalosi et al, 2008), and these are potential sites for controlling RNA turnover or regulation by miRNAs in the synapse, as suggested by studies of Arc (Bramham et al, 2008) or CaMKII synaptic RNAs (Ashraf et al, 2006). One means of exploring this question is to ask whether the regulatory RNABPs regulating alternative RNA processing of specific transcripts participate in their synaptic regulation
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