title: Human Pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs.
abstract: PUF proteins are a conserved family of eukaryotic RNA-binding proteins that regulate specific mRNAs: they control many processes including stem cell proliferation, fertility, and memory formation. PUFs repress protein expression from their target mRNAs but the mechanism by which they do so remains unclear, especially for humans. Humans possess two PUF proteins, PUM1 and PUM2, which exhibit similar RNA binding specificities. Here we report new insights into their regulatory activities and mechanisms of action. We developed functional assays to measure sequence-specific repression by PUM1 and PUM2. Both robustly inhibit translation and promote mRNA degradation. Purified PUM complexes were found to contain subunits of the CCR4-NOT (CNOT) complex, which contains multiple enzymes that catalyze mRNA deadenylation. PUMs interact with the CNOT deadenylase subunits in vitro. We used three approaches to determine the importance of deadenylases for PUM repression. First, dominant-negative mutants of CNOT7 and CNOT8 reduced PUM repression. Second, RNA interference depletion of the deadenylases alleviated PUM repression. Third, the poly(A) tail was necessary for maximal PUM repression. These findings demonstrate a conserved mechanism of PUF-mediated repression via direct recruitment of the CCR4-POP2-NOT deadenylase leading to translational inhibition and mRNA degradation. A second, deadenylation independent mechanism was revealed by the finding that PUMs repress an mRNA that lacks a poly(A) tail. Thus, human PUMs are repressors capable of deadenylation-dependent and -independent modes of repression.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0600, USA.
authors.forename: Jamie
authors.initials: J
authors.lastname: Van Etten
authors.forename: Trista L
authors.initials: TL
authors.lastname: Schagat
authors.forename: Joel
authors.initials: J
authors.lastname: Hrit
authors.forename: Chase A
authors.initials: CA
authors.lastname: Weidmann
authors.forename: Justin
authors.initials: J
authors.lastname: Brumbaugh
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Coon
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Goldstrohm
mesh_terms: D001681:Biological Assay; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D011061:Poly A / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000502:physiology*; D017434:Protein Structure, Tertiary; D020871:RNA Stability / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: C489780:CNOT8 protein, human; D009097:Multienzyme Complexes; C471242:PUM1 protein, human; C471243:PUM2 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D011061:Poly A; C545829:CNOT7 protein, human; D005095:Exoribonucleases