Human pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs

Jamie Van Etten, Trista L. Schagat, Joel Hrit, Chase A. Weidmann, Justin Brumbaugh, Joshua J. Coon, Aaron C. Goldstrohm

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)36370-36383
Number of pages14
JournalJournal of Biological Chemistry
Volume287
Issue number43
DOIs
StatePublished - Oct 19 2012

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Messenger RNA
RNA Stability
Proteins
RNA-Binding Proteins
RNA
RNA Interference
Degradation
Fertility
human pumilio protein
Cell proliferation
Stem Cells
Stem cells
Cell Proliferation
Assays
Enzymes
Data storage equipment

Cite this

Human pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs. / Van Etten, Jamie; Schagat, Trista L.; Hrit, Joel; Weidmann, Chase A.; Brumbaugh, Justin; Coon, Joshua J.; Goldstrohm, Aaron C.

In: Journal of Biological Chemistry, Vol. 287, No. 43, 19.10.2012, p. 36370-36383.

Research output: Contribution to journalArticle

Van Etten, J, Schagat, TL, Hrit, J, Weidmann, CA, Brumbaugh, J, Coon, JJ & Goldstrohm, AC 2012, 'Human pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs', Journal of Biological Chemistry, vol. 287, no. 43, pp. 36370-36383. https://doi.org/10.1074/jbc.M112.373522
Van Etten J, Schagat TL, Hrit J, Weidmann CA, Brumbaugh J, Coon JJ et al. Human pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs. Journal of Biological Chemistry. 2012 Oct 19;287(43):36370-36383. https://doi.org/10.1074/jbc.M112.373522
Van Etten, Jamie ; Schagat, Trista L. ; Hrit, Joel ; Weidmann, Chase A. ; Brumbaugh, Justin ; Coon, Joshua J. ; Goldstrohm, Aaron C. / Human pumilio proteins recruit multiple deadenylases to efficiently repress messenger RNAs. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 43. pp. 36370-36383.
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