A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 11/2009; 106(48):20192-7. DOI: 10.1073/pnas.0812079106
Source: PubMed


A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

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Available from: Juno M Krahn, Sep 03, 2014
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    • "Moreover, Puf3p was able to bind a target mRNA comparably in both glucose and glucose depletion conditions as assayed by RIP, suggesting that its phosphorylation does not significantly impact RNA binding (Figure S3D). A previous study suggested that mutation of one of these sites (S866A) near the end of the PUF domain reduces its binding affinity for RNA (Zhu et al., 2009). However, a single S866A mutant in the context of the full-length protein was still able to bind RNA and grew at identical rates compared to WT following glucose depletion (Figure S3A, S3B, and S3E). "
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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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    • "To model for RBP-mRNA binding occupancy, we used a biophysical model similar to that presented by Foat et al. (2006). We assume that the free protein concentration is low relative to the dissociation constant for protein-RNA interaction (Ghaemmaghami et al. 2003; Miller et al. 2008; Zhu et al. 2009). The occupancy (N) of sequence (S) of mRNA of gene (g) by an RBP (ϕ) is given as follows: "
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    ABSTRACT: Understanding how genomic variation influences phenotypic variation through the molecular networks of the cell is one of the central challenges of biology. Transcriptional regulation has received much attention, but equally important is the post-transcriptional regulation of mRNA stability. Here we applied a systems genetics approach to dissect post-transcriptional regulatory networks in the budding yeast Saccharomyces cerevisiae. Quantitative sequence-to-affinity models were built from high-throughput in vivo RNA Binding Protein (RBP) binding data for 15 yeast RBPs. Integration of these models with genomewide mRNA expression data allowed us to estimate protein-level RBP regulatory activity for individual segregants from a genetic cross between two yeast strains. Treating these activities as a quantitative trait, we mapped trans-acting loci ("aQTLs") that act via post-transcriptional regulation of transcript stability. We predicted and experimentally confirmed that a coding polymorphism at the IRA2 locus modulates Puf4p activity. Our results also indicate that Puf3p activity is modulated by distinct loci, depending on whether it acts via the 5' or the 3' untranslated region of its target mRNAs. Together, our results validate a general strategy for dissecting the connectivity between post-translational regulators and their upstream signaling pathways.
    Full-text · Article · Jun 2014 · G3-Genes Genomes Genetics
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    • "PUF proteins were named after the two founding members: Pumilio in Drosophila melanogaster and FBF in Caenorhabditis elegans. They contain up to 8 copies of a nucleotide-binding repeat in their RNA binding ‘PUF’ domain [1] which recognises sequences that include the motif UGUR [2], [3], [4], [5]. PUF proteins play various roles in RNA metabolism. "
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    ABSTRACT: PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the Trypanosoma brucei genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of PUF5 did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.
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