title: Pumilio binds para mRNA and requires Nanos and Brat to regulate sodium current in Drosophila motoneurons.
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 (I(Na)) and excitability in Drosophila motoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3' end of the para open 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 of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I(Na) in 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 of nanos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling between I(Na) (para) and I(K) (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as 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.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom. nara.muraro@manchester.ac.uk
authors.forename: Nara I
authors.initials: NI
authors.lastname: Muraro
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Weston
authors.forename: Andre P
authors.initials: AP
authors.lastname: Gerber
authors.forename: Stefan
authors.initials: S
authors.lastname: Luschnig
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Moffat
authors.forename: Richard A
authors.initials: RA
authors.lastname: Baines
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004330:Drosophila; D029721:Drosophila Proteins / Q000502:physiology*; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D007814:Larva; D008564:Membrane Potentials / Q000235:genetics; D009046:Motor Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000502:physiology*; D015222:Sodium Channels / Q000502:physiology*
chemical_list: D029721:Drosophila Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015222:Sodium Channels; C074333:pum protein, Drosophila; C070574:nos protein, Drosophila