Pumilio BindsparamRNA and Requires Nanos and Brat to Regulate Sodium Current inDrosophilaMotoneurons

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Abstract
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (INa) and excitability inDrosophilamotoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding theDrosophilavoltage-gated sodium channelparalytic(para). We identify a putative binding site for Pum in the 3′ end of theparaopen reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression ofparamRNA. Additionally, the cofactor Nanos is essential for Pum-dependentpararepression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation ofINain motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. We also show that Pum is able to reduce the level ofnanosmRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling betweenINa(para) andIK(Shal) such that Pum-mediated change inpararesults in a compensatory change inShal. The identification ofparaas a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3′-untranslated region of target transcripts.