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Roles of Puf proteins in mRNA degradation and translation

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Abstract Puf proteins are regulators of diverse eukaryotic processes including stem cell maintenance, organelle biogenesis, oogenesis, neuron function, and memory formation. At the molecular level, Puf proteins promote translational repression and/or degradation of target mRNAs by first interacting with conserved cis‐elements in the 3′ untranslated region (UTR). Once bound to an mRNA, Puf proteins elicit RNA repression by complex interactions with protein cofactors and regulatory machinery involved in translation and degradation. Recent work has dramatically increased our understanding of the targets of Puf protein regulation, as well as the mechanisms by which Puf proteins recognize and regulate those mRNA targets. Crystal structure analysis of several Puf–RNA complexes has demonstrated that while Puf proteins are extremely conserved in their RNA‐binding domains, Pufs attain target specificity by utilizing different structural conformations to recognize 8–10 nt sequences. Puf proteins have also evolved modes of protein interactions that are organism and transcript‐specific, yet two common mechanisms of repression have emerged: inhibition of cap‐binding events to block translation initiation, and recruitment of the CCR4–POP2–NOT deadenylase complex for poly(A) tail removal. Finally, multiple schemes to regulate Puf protein activity have been identified, including post‐translational mechanisms that allow rapid changes in the repression of mRNA targets. WIREs RNA 2011 2 471–492 DOI: 10.1002/wrna.69 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition Translation > Translation Regulation RNA Turnover and Surveillance > Regulation of RNA Stability

Puf‐mediated regulation of mRNAs often involves interactions with other regulatory pathways, other Puf proteins, and recognition of specific classes of transcripts. (a) In some cases, one Puf protein is sufficient to regulate a single transcript.7,24,25,46,48,58,59,87 In other cases, the Puf protein may repress the transcript by working in conjunction with another regulatory pathway, such as the miRNA regulatory system.49,94 The Puf protein (green arc) binds conserved UGUR elements within the 3′ UTR of the mRNA target, while the miRNA (red nucleotides) binds to a complementary sequence in the 3′ UTR. The combinatorial functions of the Puf and the miRNA can result in both deadenylation and decay of the mRNA target, as well as translational repression. (b) Multiple Puf proteins can work together to promote repression and/or turnover of a single mRNA target.23,36,70,79,80,82 (c) A single Puf protein can regulate several mRNAs that belong to a specific class of transcripts.34 Different Puf proteins recognize and interact with distinct classes of mRNAs (represented as different colored lines).

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Regulation of Puf protein activity. (a) Puf proteins can autoregulate their own mRNAs. In this model of fbf mRNA post‐transcriptional regulation, FBF‐1 or FBF‐2 (green arc) may bind to the FBF recognition element (UGUR) in the 3′ UTR of fbf‐1 or fbf‐2 mRNA to promote deadenylation and inhibit translation.92 (b) PUF mRNAs can be regulated post‐transcriptionally by the miRNA regulatory system. A miRNA (red RNA bases) may bind to the 3′ UTR of PUF mRNA (green RNA bases), resulting in translational repression or degradation.123 (c) Puf protein activity can be negatively regulated post‐translationally. Phosphorylation of yeast Puf6p can turn off its activity.118 (d) Puf‐mediated translational repression can be inhibited by disrupting interactions with the mRNA target.124,125 In this model, Bam or GLD3 (yellow circle labeled X) interacts with the Puf protein (green arc) and prevents it from binding the PRE in the 3′ UTR of the targeted transcript. (e) Puf protein–mRNA interactions and activity are regulated by environmental stimuli. In Xenopus oocytes, progesterone disrupts Pum2 interactions with its mRNA target.72 Yeast Puf3p‐mediated mRNA decay occurs in the presence of dextrose, but its activity is inhibited in the presence of ethanol.22,87,126 (f) Puf proteins can form aggregates mediated by the Q‐rich domain (modeled as green barbells with Q‐rich and repeat domains),26 which potentially prevent or promote Puf‐mediated regulation of mRNA targets.48,72 These aggregates could be sequestered from the mRNA target, preventing binding interactions and alleviating RNA repression.

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Translational repression and decay mechanisms through direct interactions with Puf proteins. (a) XPum2 prevents translational activation of the RINGO/Spy mRNA by competing with eIF4E for interaction with the mRNA cap.117 (b) Yeast Puf6p represses the translation of ASH1 mRNA by either interacting with the translation factor Fun12p/eIF5B or competing with it for interactions with other translation initiation factors. Puf6p also prevents the formation of the 80S ribosome complex.118 (c) Yeast Pufs directly recruit the Pop2p subunit of the Ccr4p–Pop2p–Notp deadenylase complex, which in turn binds Ccr4p and presumably the rest of the NOT complex. The Puf protein also recruits Dcp1p, which cleaves the 5′ cap, and Dhh1, a regulator of mRNA degradation.79,80,82 After deadenylation and decapping of the transcript, the 5′ → 3′ exonuclease Xrn1p rapidly degrades the mRNA.119

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Puf‐mediated repression of mRNA targets requires Nanos, Brat, CPEB and/or other protein partners on a transcript‐specific basis. (a) Model of Drosophila hunchback translational repression. For simplicity, only one NRE is shown. Repression of hunchback mRNA requires the formation of a quaternary complex including hunchback mRNA, Pumilio (green arc), Nanos (gold rectangle), and Brat (blue triangle) proteins.58 Brat interacts with the cap binding protein d4EHP, which in turn may disrupt eIF4E‐cap interactions and prevent translation initiation.113 (b) Model of Drosophila cyclin B translational repression. Repression of cyclin B mRNA requires the formation of a ternary complex including cyclin B mRNA, Pumilio (green arc), and Nanos (gold rectangle) proteins.58 Nanos interacts with NOT4,25 while Pumilio associates with POP2, and these interactions likely recruit the entire CCR4–POP2–NOT deadenylase complex to the mRNA.25 (c) Model of C. elegans fem‐3 translational repression. Repression of fem‐3 mRNA requires the formation of a ternary complex including fem‐3 mRNA, FBF (green arc), and NANOS‐3 (gold rectangle) proteins.10,69 FBF interacts with the POP2 homolog CCF‐1 in vitro to stimulate deadenylation of gld‐1 mRNA,45 and this deadenylation mechanism is likely to occur with fem‐3 mRNA as well. (d) Model of Xenopus cyclin B1 translational repression. For simplicity, only one cytoplasmic polyadenylation element (CPE) is represented. Repression of cyclin B1 mRNA requires the formation of a large complex including hunchback mRNA, Pumilio1 (green arc), Xcat‐2 (gold rectangle), and CPEB (purple) proteins.16,43,71 Small light blue circles represent poly(A)‐binding proteins to which eIF4G binds. Pumilio1 binds UGUR elements and interacts with CPEB to stabilize its interactions with the transcript via CPE sequences. CPEB interacts with Maskin (tan ellipse), which prevents eIF4E–eIF4G interactions, thus inhibiting translation initiation.114 Arrowheads in models denote stimulation of deadenylation.

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Puf proteins bind recognition elements in either a one base to one repeat modular‐manner or by inclusion of spacer/flipped bases. (a) Cocrystal structure of the canonical human PUM1‐RD bound to a Nanos Response Element (NRE) from Drosophila hunchback mRNA. (Reprinted with permission from Ref 104. Copyright 2002 Elsevier.) (b) Cocrystal structure of C. elegans FBF‐RD bound to a Puf Recognition Element (PRE) in gld‐1. Arrow denotes flipped RNA base that does not interact with FBF amino acids. (Reprinted with permission from Ref 106. Copyright 2009 National Academy of Sciences.) (c) Cocrystal structure of yeast Puf4RDp bound to the HO PRE. Arrow denotes flipped RNA base that does not interact with Puf4p amino acids. (Reprinted with permission from Ref 107. Copyright 2008 Nature Publishing Group.) (d) Cocrystal structure of yeast Puf3RDp bound to a PRE in COX17. Arrow denotes novel interaction between an upstream cytosine base and amino acids of Puf repeat 8′. (Reprinted with permission from Ref 100. Copyright 2009 Library of Sciences.) (e) Binding interactions between RNA bases and amino acids of PufRDs. Conserved interactions are represented in black. Amino acid–RNA base interactions and spacer/flipped bases that are unique to each Puf protein are indicated by color: C. elegans FBF (red), human PUM1 (gold), yeast Puf4p (green), and yeast Puf3p (orange). (Reprinted with permission from Ref 106. Copyright 2009 National Academy of Sciences.)

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Consensus sequence weight matrices were developed based on sequences in the 3′ UTRs of mRNAs associated with human PUM1 and PUM2, Drosophila Pumilio, and S. cerevisiae Puf3p, Puf4p, and Puf5p. The height of the nucleotide represents the probability that it will occur at that position. Positions that are conserved across organisms and proteins are highlighted in yellow. (Reprinted with permission from Ref 94. Copyright 2008 Public Library of Science.)

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