title: The RNA binding domain of Pumilio antagonizes poly-adenosine binding protein and accelerates deadenylation.
abstract: PUF proteins are potent repressors that serve important roles in stem cell maintenance, neurological processes, and embryonic development. These functions are driven by PUF protein recognition of specific binding sites within the 3' untranslated regions of target mRNAs. In this study, we investigated mechanisms of repression by the founding PUF, Drosophila Pumilio, and its human orthologs. Here, we evaluated a previously proposed model wherein the Pumilio RNA binding domain (RBD) binds Argonaute, which in turn blocks the translational activity of the eukaryotic elongation factor 1A. Surprisingly, we found that Argonautes are not necessary for repression elicited by Drosophila and human PUFs in vivo. A second model proposed that the RBD of Pumilio represses by recruiting deadenylases to shorten the mRNA's polyadenosine tail. Indeed, the RBD binds to the Pop2 deadenylase and accelerates deadenylation; however, this activity is not crucial for regulation. Rather, we determined that the poly(A) is necessary for repression by the RBD. Our results reveal that poly(A)-dependent repression by the RBD requires the poly(A) binding protein, pAbp. Furthermore, we show that repression by the human PUM2 RBD requires the pAbp ortholog, PABPC1. Pumilio associates with pAbp but does not disrupt binding of pAbp to the mRNA. Taken together, our data support a model wherein the Pumilio RBD antagonizes the ability of pAbp to promote translation. Thus, the conserved function of the PUF RBD is to bind specific mRNAs, antagonize pAbp function, and promote deadenylation.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA Genetics Training Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Chase A
authors.initials: CA
authors.lastname: Weidmann
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA Genetics Training Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Raynard
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Blewett
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: Van Etten
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA Genetics Training Program, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Goldstrohm
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D029721:Drosophila Proteins / Q000378:metabolism; D005095:Exoribonucleases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D026723:Polyadenylation / Q000502:physiology*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D064570:Ubiquitin-Specific Proteases / Q000378:metabolism
chemical_list: D060565:Argonaute Proteins; D029721:Drosophila Proteins; D005095:Exoribonucleases; D020648:Peptide Elongation Factor 1; D011061:Poly A; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D064570:Ubiquitin-Specific Proteases; C586202:Puf protein, Drosophila
keywords: PUF; Pumilio; deadenylation; pAbp; poly(A) tail