Human Pumilio Proteins Recruit Multiple Deadenylases to Efficiently Repress Messenger RNAs

From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0600.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2012; 287(43):36370-83. DOI: 10.1074/jbc.M112.373522
Source: PubMed


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.

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    • "It remains unclear precisely how PUF family proteins regulate their target transcripts. Most studies to date suggest PUF proteins repress translation either through decapping or deadenylation of mRNA (Houshmandi and Olivas, 2005; Kershner and Kimble, 2010; Merritt and Seydoux, 2010; Van Etten et al., 2012). However, other studies suggest their function is to promote mRNA translation, perhaps by controlling mRNA localization for the purpose of local translation (Archer et al., 2009; Deng et al., 2008; Gadir et al., 2011; Kaye et al., 2009). "
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    ABSTRACT: PUF proteins are post-transcriptional regulators that bind to the 3' UTRs of mRNA transcripts. Herein, we show how a yeast PUF protein, Puf3p, responds to glucose availability to switch the fate of its bound transcripts that encode proteins required for mitochondrial biogenesis. Upon glucose depletion, Puf3p becomes heavily phosphorylated within its N-terminal region of low complexity, associates with polysomes, and promotes translation of its target mRNAs. Such nutrient-responsive phosphorylation toggles the activity of Puf3p to promote either degradation or translation of these mRNAs according to the needs of the cell. Moreover, activation of translation of pre-existing mRNAs might enable rapid adjustment to environmental changes without the need for de novo transcription. Strikingly, a Puf3p phosphomutant no longer promotes translation but becomes trapped in intracellular foci in an mRNA-dependent manner. Our findings suggest that the inability to properly resolve Puf3p-containing RNA-protein granules via a phosphorylation-based mechanism might be toxic to a cell. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Jun 2015 · Cell Reports
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    • "Cytoplasmic polyadenylation element-binding protein (CPEB) binds specific mRNAs, to which Tob, as an adapter protein, mediates the recruitment of the CCR4–NOT deadenylase (Hosoda et al., 2011; Ogami et al., 2014). Other RNA-binding proteins, such as Nanos, Pumilio and fem-3 binding factor (PUF) proteins, Smaug, tristetrapolin (TTP), and Bicaudal-C (Bic-C), were reported to interact with the components of CCR4–NOT and cause the suppression of target mRNAs (Semotok et al., 2005; Goldstrohm et al., 2006, 2007; Chicoine et al., 2007; Suzuki et al., 2010, 2012; Sandler et al., 2011; Van Etten et al., 2012; Fabian et al., 2013; Joly et al., 2013; Bhandari et al., 2014). In addition, CCR4–NOT is involved in the microRNA (miRNA)-mediated deadenylation of mRNAs through interaction with the GW182 proteins (Behm-Ansmant et al., 2006; Braun et al., 2011; Chekulaeva et al., 2011; Fabian et al., 2011). "
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    ABSTRACT: The carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) complex serves as one of the major deadenylases of eukaryotes. Although it was originally identified and characterized in yeast, recent studies have revealed that the CCR4-NOT complex also exerts important functions in mammals, -including humans. However, there are some differences in the composition and functions of the CCR4-NOT complex between mammals and yeast. It is noteworthy that each subunit of the CCR4-NOT complex has unique, multifunctional roles and is responsible for various physiological phenomena. This heterogeneity and versatility of the CCR4-NOT complex makes an overall understanding of this complex difficult. Here, we describe the functions of each subunit of the mammalian CCR4-NOT complex and discuss the molecular mechanisms by which it regulates homeostasis in mammals. Furthermore, a possible link between the disruption of the CCR4-NOT complex and various diseases will be discussed. Finally, we propose that the analysis of mice with each CCR4-NOT subunit knocked out is an effective strategy for clarifying its complicated functions and networks in mammals.
    Full-text · Article · Aug 2014 · Frontiers in Genetics
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    • "We attempted to test the effect of Pumilio knockdown on CNOT6 accumulation; however, these embryos were developmentally arrested before blastula stage, probably pointing to pleiotropic effects (data not shown). Indeed, Nanosindependent roles for Pumilio have been identified (Van Etten et al., 2012; Weidmann and Goldstrohm, 2012). Indicative of its diverse functions, Pumilio protein is detectable in granules in all cells of the Fig. 2 "
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    ABSTRACT: A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo.
    Full-text · Article · Aug 2014 · Development
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