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

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 3Ј 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 3Ј-untranslated region of target transcripts.

A Caenorhabditis elegans PUF protein family with distinct RNA binding specificity

“…Cold Spring Harbor Laboratory Press on May 7, 2018 -Published by rnajournal.cshlp.org Downloaded from para mRNA, which encodes a voltage-gated sodium channel (Muraro et al 2008). Our preliminary data suggest that PUF-6 is also expressed in neurons (D. Byrd, C.R.…”

Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells

“…Additionally, several negative regulators of PUF activity have been documented, including a kinase that inhibits PUF activity by phosphorylation, as well as proteins that inhibit PUF protein-mRNA interactions (Miller and Olivas, 2011). Most known regulatory interactions involve the conserved PUF RNA-binding domain, despite the fact that the regions flanking the RNA-binding domain are required for full PUF activity (Muraro et al, 2008;Weidmann and Goldstrohm, 2012). There are no reports of PUF protein interactors that promote PUF subcellular localization or binding to the target mRNA.…”

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation

“…For example, Brat and Pum repress myc and mad translation to promote differentiation during oogenesis (Harris et al 2011). Furthermore, Brat, Pum, and Nos inhibit the paralytic mRNA to control excitability in motorneurons (Muraro et al 2008), and, finally, in neuromuscular synapses, repression of mad mRNA by Brat regulates synaptic size (Shi et al 2013).…”