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Published January 13, 2010 | Supplemental Material + Published
Journal Article Open

Regulation of Synaptic Pumilio Function by an Aggregation-Prone Domain


We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a "dominant-negative" effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.

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© 2010 Society for Neuroscience. For the first six months after publication SfN's license will be exclusive. Beginning six months after publication the Work will be made freely available to the public on SfN's website to copy, distribute, or display under a Creative Commons Attribution 4.0 International (CC BY 4.0) license (https://creativecommons.org/licenses/by/4.0/). Received May 31, 2009; revised Oct. 21, 2009; accepted Nov. 10, 2009. This work was supported by a McKnight Foundation Brain Disorders Award and by National Institutes of Health Grants RO1 NS28182, R01 NS62821, and RO1 NS43416 (K.Z.). We thank the following: Erich Schwarz (WormBase at the California Institute of Technology, Pasadena, CA) for conducting the computational search of the fly and worm proteomes for us; Dale Cameron, Lev Osherovich, and Jonathan Weissman for help with gamma integration; James Shorter, Johannes Graumann, Ashley Wright, Nina Sherwood, Alice Schmid, and Nicki Fox for helpful discussions; the Weissman (University of California, San Francisco, San Francisco, CA), Lindquist (Massachusetts Institute of Technology, Cambridge, MA), Deshaies (California Institute of Technology), and Wharton (Duke University, Durham, NC) groups for materials; and Elena Armand, Violana Nesterova, Lisa Nesterova, and Carlos Diaz-Balzac for technical assistance.Wethank Bill Tivol for assistance with EM, which was performed at the Broad CenterEMfacility. Confocal microscopy was performed at the Caltech Biological Imaging Facility. We thank Sean Brennan for assistance with graphics and good music.

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