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1.  Pumilio-2 Function in the Mouse Nervous System 
PLoS ONE  2011;6(10):e25932.
Coordinated mRNA translation at the synapse is increasingly recognized as a critical mechanism for neuronal regulation. Pumilio, a translational regulator, is known to be involved in neuronal homeostasis and memory formation in Drosophila. Most recently, the mammalian Pumilio homolog Pumilio-2 (Pum2) has been found to play a role in the mammalian nervous system, in particular in regulating morphology, arborization and excitability of neuronal dendrites, in vitro. However, the role of Pum2 in vivo remains unclear. Here, we report our investigation of the functional and molecular consequences of Pum2 disruption in vivo using an array of neurophysiology, behavioral and gene expression profiling techniques. We used Pum2-deficient mice to monitor in vivo brain activity using EEG and to study behavior traits, including memory, locomotor activity and nesting capacities. Because of the suspected role of Pum2 in neuronal excitability, we also examined the susceptibility to seizure induction. Finally, we used a quantitative gene expression profiling assay to identify key molecular partners of Pum2. We found that Pum2-deficient mice have abnormal behavioral strategies in spatial and object memory test. Additionally, Pum2 deficiency is associated with increased locomotor activity and decreased body weight. We also observed environmentally-induced impairment in nesting behavior. Most importantly, Pum2-deficient mice showed spontaneous EEG abnormalities and had lower seizure thresholds using a convulsing dosage of pentylenetetrazole. Finally, some genes, including neuronal ion channels, were differentially expressed in the hippocampus of Pum2-deficient mice. These findings demonstrate that Pum2 serves key functions in the adult mammalian central nervous system encompassing neuronal excitability and behavioral response to environmental challenges.
doi:10.1371/journal.pone.0025932
PMCID: PMC3189250  PMID: 22016787
2.  The translational repressors Nanos and Pumilio have divergent effects on presynaptic terminal growth and postsynaptic glutamate receptor subunit composition 
Pumilio (Pum) is a translational repressor that binds selectively to target mRNAs and recruits Nanos (Nos) as a corepressor. In the larval neuromuscular system, Pum represses expression of the translation factor eIF-4E and the glutamate receptor subunit GluRIIA. Here we show that Nos, like Pum, is expressed at the neuromuscular junction (NMJ) and in neuronal cell bodies. Surprisingly, however, Nos and Pum have divergent functions on both the pre- and postsynaptic sides of the NMJ. In nos mutant and nos RNAi larvae, the number of NMJ boutons is increased, while loss of Pum reduces bouton number. On the postsynaptic side, Nos acts in opposition to Pum in regulating the subunit composition of the glutamate receptor. NMJ active zones are associated with GluRIIA- and GluRIIB-containing receptor clusters. Loss of Nos causes downregulation of GluRIIA and increases the levels of GluRIIB. Consistent with this finding, the electrophysiological properties of NMJs lacking postsynaptic Nos suggest that they employ primarily GluRIIB-containing receptors. Nos can regulate GluRIIB in the absence of GluRIIA, suggesting that the effects of Nos on GluRIIB levels are at least partially independent of synaptic competition between GluRIIA and GluRIIB. Nos is a target for Pum repression, and Pum binds selectively to the 3' UTRs of the nos and GluRIIA mRNAs. Our results suggest a model in which regulatory interplay among Pum, Nos, GluRIIA, and GluRIIB could cause a small change in Pum activity to be amplified into a large shift in the balance between GluRIIA and GluRIIB synapses.
doi:10.1523/JNEUROSCI.0520-09.2009
PMCID: PMC2750846  PMID: 19403823
translational repression; glutamate receptor; neuromuscular junction; Drosophila; mRNA binding; synaptic bouton
3.  Pumilio Binds para mRNA and Requires Nanos and Brat to Regulate Sodium Current in Drosophila Motoneurons 
Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodelling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (INa) and excitability in Drosophila motoneurons. In this current study we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3′ end of the para open reading frame (ORF). Characterisation of the mechanism of action of Pum, using whole-cell patch clamp and real time RT-PCR, reveals that the full length protein is required for translational repression of para mRNA. Additionally, the co-factor Nanos (Nos) is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell-type specific. Thus, Pum-dependent regulation of INa in motoneurons requires both Nos and Brat, whilst regulation in other neuronal types seemingly requires only Nos but not Brat. We also show that Pum is able to reduce the level of nos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from over-activity of Pum. Finally, we show coupling between INa (para) and IK (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as a direct target of Pum represents the first ion channel to be translationally-regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3′ untranslated region of target transcripts.
doi:10.1523/JNEUROSCI.5092-07.2008
PMCID: PMC2323674  PMID: 18305244
Pumilio; Nanos; Brat; paralytic; aCC; RP2
4.  Negative Regulation of EGFR/MAPK Pathway by Pumilio in Drosophila melanogaster 
PLoS ONE  2012;7(4):e34016.
In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)–like sequences in their 3’UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila.
doi:10.1371/journal.pone.0034016
PMCID: PMC3326002  PMID: 22514614
5.  Regulation of synaptic Pumilio function by an aggregation-prone domain 
We identified Pumilio (Pum), a Drosophila translational repressor, in a computational search for metazoan proteins whose activities might be regulated by assembly into ordered aggregates. The search algorithm was based on evolutionary sequence conservation patterns observed for yeast prion proteins, which contain aggregation-prone glutamine/asparagine (Q/N)-rich domains attached to functional domains of normal amino acid composition. We examined aggregation of Pum and its nematode ortholog PUF-9 by expression in yeast. A domain of Pum containing the Q/N-rich sequence, denoted as NQ1, the entire Pum N-terminus, and the complete PUF-9 protein localize to macroscopic aggregates (foci) in yeast. NQ1 and PUF-9 can generate the yeast Pin+ trait, which is transmitted by a heritable aggregate. NQ1 also assembles into amyloid fibrils in vitro. In Drosophila, Pum regulates postsynaptic translation at neuromuscular junctions (NMJs). To assess whether NQ1 affects synaptic Pum activity in vivo, we expressed it in muscles. We found that it negatively regulates endogenous Pum, producing gene dosage-dependent pum loss-of-function NMJ phenotypes. NQ1 coexpression also suppresses lethality and NMJ phenotypes caused by overexpression of Pum in muscles. The Q/N block of NQ1 is required for these phenotypic effects. Negative regulation of Pum by NQ1 might be explained by formation of inactive aggregates, but we have been unable to demonstrate that NQ1 aggregates in Drosophila. NQ1 could also regulate Pum by a “dominant-negative” effect, in which it would block Q/N-mediated interactions of Pum with itself or with cofactors required for translational repression.
doi:10.1523/JNEUROSCI.2523-09.2010
PMCID: PMC2847508  PMID: 20071514
translational repression; aggregate; neuromuscular junction; amyloid; Drosophila; prion; RNA-binding protein; stress granule; synaptic bouton
6.  Ribonomic Analysis of Human Pum1 Reveals cis-trans Conservation across Species despite Evolution of Diverse mRNA Target Sets▿ † 
Molecular and Cellular Biology  2008;28(12):4093-4103.
PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes.
doi:10.1128/MCB.00155-08
PMCID: PMC2423135  PMID: 18411299
7.  Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System 
PLoS ONE  2008;3(9):e3164.
Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3′-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3′-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.
doi:10.1371/journal.pone.0003164
PMCID: PMC2522278  PMID: 18776931
8.  The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization 
BMC Plant Biology  2010;10:44.
Background
Puf proteins have important roles in controlling gene expression at the post-transcriptional level by promoting RNA decay and repressing translation. The Pumilio homology domain (PUM-HD) is a conserved region within Puf proteins that binds to RNA with sequence specificity. Although Puf proteins have been well characterized in animal and fungal systems, little is known about the structural and functional characteristics of Puf-like proteins in plants.
Results
The Arabidopsis and rice genomes code for 26 and 19 Puf-like proteins, respectively, each possessing eight or fewer Puf repeats in their PUM-HD. Key amino acids in the PUM-HD of several of these proteins are conserved with those of animal and fungal homologs, whereas other plant Puf proteins demonstrate extensive variability in these amino acids. Three-dimensional modeling revealed that the predicted structure of this domain in plant Puf proteins provides a suitable surface for binding RNA. Electrophoretic gel mobility shift experiments showed that the Arabidopsis AtPum2 PUM-HD binds with high affinity to BoxB of the Drosophila Nanos Response Element I (NRE1) RNA, whereas a point mutation in the core of the NRE1 resulted in a significant reduction in binding affinity. Transient expression of several of the Arabidopsis Puf proteins as fluorescent protein fusions revealed a dynamic, punctate cytoplasmic pattern of localization for most of these proteins. The presence of predicted nuclear export signals and accumulation of AtPuf proteins in the nucleus after treatment of cells with leptomycin B demonstrated that shuttling of these proteins between the cytosol and nucleus is common among these proteins. In addition to the cytoplasmically enriched AtPum proteins, two AtPum proteins showed nuclear targeting with enrichment in the nucleolus.
Conclusions
The Puf family of RNA-binding proteins in plants consists of a greater number of members than any other model species studied to date. This, along with the amino acid variability observed within their PUM-HDs, suggests that these proteins may be involved in a wide range of post-transcriptional regulatory events that are important in providing plants with the ability to respond rapidly to changes in environmental conditions and throughout development.
doi:10.1186/1471-2229-10-44
PMCID: PMC2848763  PMID: 20214804
9.  Alternate Modes of Cognate RNA Recognition by Human PUMILIO Proteins 
Summary
Human PUMILIO1 (PUM1) and PUMILIO2 (PUM2) are members of the PUMILIO/FBF (PUF) family that regulate specific target mRNAs posttranscriptionally. Recent studies have identified mRNA targets associated with human PUM1 and PUM2. Here we explore the structural basis of natural target RNA recognition by human PUF proteins through crystal structures of the RNA-binding domains of PUM1 and PUM2 in complex with four cognate RNA sequences including sequences from p38α and erk2 MAP kinase mRNAs. We observe three distinct modes of RNA binding around the 5th RNA base, two of which are different from the prototypical 1 repeat:1 RNA base binding mode previously identified with model RNA sequences. RNA-binding affinities of PUM1 and PUM2 are not affected dramatically by the different binding modes in vitro. However, these modes of binding create structurally variable recognition surfaces that suggest a mechanism in vivo for recruitment of downstream effector proteins defined by the PUF:RNA complex.
doi:10.1016/j.str.2010.12.019
PMCID: PMC3063405  PMID: 21397187
10.  Pumilio-2 regulates translation of Nav1.6 to mediate homeostasis of membrane excitability 
The ability to regulate intrinsic membrane excitability, in order to maintain consistency of action potential firing, is critical for stable neural circuit activity. Without such mechanisms, Hebbian-based synaptic plasticity could push circuits toward activity-saturation or, alternatively, quiescence. Although now well documented, the underlying molecular components of these homeostatic mechanisms remain poorly understood. Recent work in the fruit fly, Drosophila melanogaster, has identified Pumilio (Pum), a translational repressor, as an essential component of one such mechanism. In response to changing synaptic excitation, Pum regulates the translation of the voltage-gated sodium conductance leading to a concomitant adjustment in action potential firing. While similar homeostatic mechanisms are operational in mammalian neurons, it is unknown whether Pum is similarly involved.
In this study, we report that Pum2 is indeed central to the homeostatic mechanism regulating membrane excitability in rat visual cortical pyramidal neurons. Using RNA interference, we observed that loss of Pum2 leads to increased sodium current (INa) and action potential firing, mimicking the response by these neurons to being deprived of synaptic depolarisation. By contrast, increased synaptic depolarisation results in increased Pum2 expression and subsequent reduction in INa and membrane excitability. We further show that Pum2 is able to directly bind the predominant voltage-gated sodium channel transcript (NaV1.6) expressed in these neurons and, through doing so, regulates translation of this key determinant of membrane excitability. Taken together, our results show that Pum2 forms part of a homeostatic mechanism that matches membrane excitability to synaptic depolarization in mammalian neurons.
doi:10.1523/JNEUROSCI.0921-13.2013
PMCID: PMC3678506  PMID: 23739961
action potential; pumilio; sodium channel; homeostasis; translational repression
11.  The CCR4 Deadenylase Acts with Nanos and Pumilio in the Fine-Tuning of Mei-P26 Expression to Promote Germline Stem Cell Self-Renewal 
Stem Cell Reports  2013;1(5):411-424.
Summary
Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation.
Highlights
•The CCR4 deadenylase is required for female germline stem cell self-renewal•Nos/Pum recruit CCR4-NOT for translational repression in germline stem cells•mei-P26 mRNA is a major target of translational repression by Nos/Pum/CCR4•Fine-tuning of mei-P26 by CCR4 is required for germline stem cell self-renewal
doi:10.1016/j.stemcr.2013.09.007
PMCID: PMC3841267  PMID: 24286029
12.  Computational Assessment of the Cooperativity between RNA Binding Proteins and MicroRNAs in Transcript Decay 
PLoS Computational Biology  2013;9(5):e1003075.
Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3′UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3′UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.
Author Summary
Transcript degradation represents an important mechanism of regulation used in diverse biological processes, including during development to eliminate maternally inherited transcripts, in adult tissues to define cell lineages, and as part of signaling pathways to down-regulate unneeded transcripts. RNA binding proteins (RBPs) and microRNAs are two major classes of molecules utilized to degrade transcripts. Using computational methods, we analyzed the genomewide cooperativity between microRNA and RBP recognition sites. We observed cooperativity between Pumilio (PUM) and specific microRNAs that impacts transcript decay. Our analysis suggests that approximately seven mammalian microRNAs preferentially co-localize with PUM binding sites, and these microRNAs have recognition motifs that are reverse complements to the PUM recognition motif. Their binding sites are more likely to form RNA hairpin structures with proximal PUM recognition sites that would limit microRNA efficiency, but would be more accessible to microRNAs in response to the binding of PUM. These results indicate that rescuing microRNA recognition sites from hairpin structures may be an important role for PUM.
doi:10.1371/journal.pcbi.1003075
PMCID: PMC3667768  PMID: 23737738
13.  Brat Promotes Stem Cell Differentiation via Control of a Bistable Switch that Restricts BMP Signaling 
Developmental Cell  2011;20(1):72-83.
Summary
Drosophila ovarian germline stem cells (GSCs) are maintained by Dpp signaling and the Pumilio (Pum) and Nanos (Nos) translational repressors. Upon division, Dpp signaling is extinguished, and Nos is downregulated in one daughter cell, causing it to switch to a differentiating cystoblast (CB). However, downstream effectors of Pum-Nos remain unknown, and how CBs lose their responsiveness to Dpp is unclear. Here, we identify Brain Tumor (Brat) as a potent differentiation factor and target of Pum-Nos regulation. Brat is excluded from GSCs by Pum-Nos but functions with Pum in CBs to translationally repress distinct targets, including the Mad and dMyc mRNAs. Regulation of both targets simultaneously lowers cellular responsiveness to Dpp signaling, forcing the cell to become refractory to the self-renewal signal. Mathematical modeling elucidates bistability of cell fate in the Brat-mediated system, revealing how autoregulation of GSC number can arise from Brat coupling extracellular Dpp regulation to intracellular interpretation.
Highlights
► Pumilio and Nanos translationally repress brat mRNA in germline stem cells ► Brat promotes differentiation by limiting Dpp signaling and cellular growth ► Brat acts with Pumilio to repress translation of the Mad and dMyc mRNAs ► Modeling shows Brat creates bistability and provides robust cell-fate control
doi:10.1016/j.devcel.2010.11.019
PMCID: PMC3178012  PMID: 21238926
14.  Drosophila Pumilio Protein Contains Multiple Autonomous Repression Domains That Regulate mRNAs Independently of Nanos and Brain Tumor 
Molecular and Cellular Biology  2012;32(2):527-540.
Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.
doi:10.1128/MCB.06052-11
PMCID: PMC3255780  PMID: 22064486
15.  A Novel Function of Human Pumilio Proteins in Cytoplasmic Sensing of Viral Infection 
PLoS Pathogens  2014;10(10):e1004417.
RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.
Author Summary
Mammals utilize innate immune system to counteract viral infections. The host pattern-recognition receptors, such as RIG-I-like receptors (RLRs), sense invading pathogens and initiate innate immune responses. RLRs are composed of three RNA helicases, RIG-I, MDA5 and LGP2, and detect a series of RNA viruses, such as influenza or hepatitis C virus, in the cytoplasm. Upon RNA virus infection, RLRs transmit signals through mitochondrial adaptor protein, IPS-1, to activate transcription factor IRF-3/7, resulting in the production of type I interferon (IFN). Type I IFN plays a crucial role in innate immune system by inducing a hundreds of interferon-stimulated genes and its induction is tightly controlled at transcriptional and translational steps. Pumilio proteins are originally identified as translational repressor through direct binding to specific sequence motifs in the 3′ untranslated regions of specific mRNA, and regulate critical biological processes, such as development and differentiation. In this report, we identified human Pumilio proteins, PUM1 and PUM2, as candidate regulators of IFN signaling. Our results demonstrated an unknown function of Pumilio in viral recognition by LGP2.
doi:10.1371/journal.ppat.1004417
PMCID: PMC4207803  PMID: 25340845
16.  Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins 
PLoS Genetics  2007;3(12):e233.
Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.
Author Summary
The mitogen-activated protein (MAP) kinase (MAPK) enzyme is crucial for regulation of both stem cell maintenance and tumorigenesis. Two conserved controls of MAPK include its activation by RAS signaling and a kinase cascade as well as its inactivation by MAPK phosphatases (MKPs). We identify a third mode of conserved MAPK regulation. We demonstrate that PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins repress mRNAs encoding MAPK enzymes in both the Caenorhabditis elegans germline and human embryonic stem cells. PUF proteins have emerged as conserved regulators of germline stem cells in C. elegans, Drosophila, and probably vertebrates. Their molecular mode of action relies on binding to sequence elements in the 3′ untranslated region of target mRNAs. We report that PUF proteins bind and repress mRNAs encoding C. elegans MPK-1 as well as human ERK2 and p38α. We also report that PUF repression and MKP inactivation function redundantly in the C. elegans germline to restrict MPK-1/MAPK activity and prevent germ cell apoptosis. We suggest that this dual regulation of MAPK activity by PUF and MKP proteins may be a conserved mechanism for the control of growth and differentiation during animal development and tissue homeostasis.
doi:10.1371/journal.pgen.0030233
PMCID: PMC2323325  PMID: 18166083
17.  PUMILIO-2 Is Involved in the Positive Regulation of Cellular Proliferation in Human Adipose-Derived Stem Cells 
Stem Cells and Development  2011;21(2):217-227.
Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.
doi:10.1089/scd.2011.0143
PMCID: PMC3258435  PMID: 21649561
18.  A structural-based statistical approach suggests a cooperative activity of PUM1 and miR-410 in human 3'-untranslated regions 
Silence  2010;1:17.
Background
Micro (mi)RNAs comprise a large family of small non-coding RNAs that are thought to regulate a large fraction of protein-coding genes. Generally, miRNAs downregulate messenger (m)RNA expression by binding to the 3' untranslated regions (UTRs) of the RNA molecules. An important factor for binding specificity is the matching in the seed region. In addition, target site accessibility is thought to be crucial for efficient repression of miRNA targets. Several recent studies indicated that miRNA repression can be facilitated by RNA-binding proteins. In this study, we examine the conjecture that RNA-binding proteins are involved in ushering miRNAs to bind targets that are initially less accessible.
Results
We analyzed human 3'-UTR sequences containing potential binding sites of 153 conserved miRNA families, and ranked sequences around the sites according to their miRNA accessibility. By applying a rank-based motif search tool to these miRNA targets, we found motifs that are enriched among less accessible targets. As expected from our ranking method, most of the significant motifs were GC-rich. However, one AU-rich motif was found to be enriched among miR-410 less accessible targets. This motif resembles the Pumilio homolog 1 (PUM1) consensus binding site. We observed a stronger enrichment of the PUM1 motif in conserved targets than in non-conserved targets; moreover, the enrichment of this motif was found to be conserved in a subset of placental mammals. Further, we analyzed publicly available gene expression data, and found that the mutual expression of PUM1 and miR-410 has a greater negative influence on the expression of low accessibility targets than on other targets, an effect that was stronger than when considering both miR-410 and PUM1 separately.
Conclusions
Taken together, our findings suggest a cooperative relationship between miR-410 and PUM1 in regulating human highly structured 3'-UTRs. This kind of cooperation can allow a second level of regulation of such targets. Considering cases in which miRNAs bind low accessibility targets may help to improve current miRNA prediction tools and to obtain a better understanding of the mechanisms underlying miRNA regulation activity.
doi:10.1186/1758-907X-1-17
PMCID: PMC2955683  PMID: 20860814
19.  MREdictor: a two-step dynamic interaction model that accounts for mRNA accessibility and Pumilio binding accurately predicts microRNA targets 
Nucleic Acids Research  2013;41(18):8421-8433.
The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3′-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods.
doi:10.1093/nar/gkt629
PMCID: PMC3794589  PMID: 23863844
20.  Morphotype Transition and Sexual Reproduction Are Genetically Associated in a Ubiquitous Environmental Pathogen 
PLoS Pathogens  2014;10(6):e1004185.
Sexual reproduction in an environmental pathogen helps maximize its lineage fitness to changing environment and the host. For the fungal pathogen Cryptococcus neoformans, sexual reproduction is proposed to have yielded hyper virulent and drug resistant variants. The life cycle of this pathogen commences with mating, followed by the yeast-hypha transition and hyphal growth, and it concludes with fruiting body differentiation and sporulation. How these sequential differentiation events are orchestrated to ensure developmental continuality is enigmatic. Here we revealed the genetic network of the yeast-to-hypha transition in Cryptococcus by analyzing transcriptomes of populations with a homogeneous morphotype generated by an engineered strain. Among this network, we found that a Pumilio-family protein Pum1 and the matricellular signal Cfl1 represent two major parallel circuits directing the yeast-hypha transition. Interestingly, only Pum1 coordinates the sequential morphogenesis events during a-α bisexual and α unisexual reproduction. Pum1 initiates the yeast-to-hypha transition, partially through a novel filament-specific secretory protein Fas1; Pum1 is also required to sustain hyphal growth after the morphological switch. Furthermore, Pum1 directs subsequent differentiation of aerial hyphae into fruiting bodies in both laboratory and clinical isolates. Pum1 exerts its control on sexual reproduction partly through regulating the temporal expression of Dmc1, the meiosis-specific recombinase. Therefore, Pum1 serves a pivotal role in bridging post-mating morphological differentiation events with sexual reproduction in Cryptococcus. Our findings in Cryptococcus illustrate how an environmental pathogen can ensure the completion of its life cycle to safeguard its long-term lineage success.
Author Summary
Sex, despite its cost, is an important means to maximize species fitness in coping with unpredictable environmental challenges. In the human fungal pathogen Cryptococcus neoformans, sexual reproduction has yielded hyper virulent and drug resistant variants, and produces airborne infectious spores. Developmentally, sexual spores are generated from fruiting bodies that are differentiated from aerial hyphae. Cryptococcus cells typically grow as yeast cells with a subpopulation that respond to mating stimulation and switch to hyphal growth after mating. However, mechanisms that connect sexual reproduction and multiple differentiation events to ensure the developmental continuality are unknown. Here we revealed a network of yeast-to-hypha transition in Cryptococcus. From this network we identified a Pumilio-family RNA binding protein Pum1 that acts in concert with the matricellular signal Cfl1 in regulating the yeast-to-hyphal transition following mating. Interestingly, Pum1 is also important in sustaining hyphal growth and in directing the progression from aerial hyphal morphogenesis to the formation of fruiting bodies. Intriguingly, mutations of Pum1 affect the spatiotemporal expression pattern of the filament- and meiosis-specific proteins Fas1 and Dmc1. Our study opens a new avenue to investigate how a microbe controls development continuity while maintaining population heterogeneity.
doi:10.1371/journal.ppat.1004185
PMCID: PMC4047104  PMID: 24901238
21.  Regulation of membrane excitability: a convergence on voltage-gated sodium conductance 
Molecular Neurobiology  2014;51:57-67.
The voltage-gated sodium channel (Nav) plays a key role in regulation of neuronal excitability. Aberrant regulation of Nav expression and/or function can result in an imbalance in neuronal activity which can progress to epilepsy. Regulation of Nav activity is achieved by coordination of a multitude of mechanisms including RNA alternative splicing and translational repression. Understanding of these regulatory mechanisms is complicated by extensive genetic redundancy: the mammalian genome encodes ten Navs. By contrast, the genome of the fruitfly, Drosophila melanogaster, contains just one Nav homologue, encoded by paralytic (DmNav). Analysis of splicing in DmNav shows variants exhibit distinct gating properties including varying magnitudes of persistent sodium current (INaP). Splicing by Pasilla, an identified RNA splicing factor, alters INaP magnitude as part of an activity-dependent mechanism. Enhanced INaP promotes membrane hyperexcitability that is associated with seizure-like behaviour in Drosophila. Nova-2, a mammalian Pasilla homologue, has also been linked to splicing of Navs and, moreover, mouse gene knockouts display seizure-like behaviour.
Expression level of Navs is also regulated through a mechanism of translational repression in both flies and mammals. The translational repressor Pumilio (Pum) can bind to Nav transcripts and repress the normal process of translation, thus regulating sodium current (INa) density in neurons. Pum2-deficient mice exhibit spontaneous EEG abnormalities. Taken together, aberrant regulation of Nav function and/or expression is often epileptogenic. As such, a better understanding of regulation of membrane excitability through RNA alternative splicing and translational repression of Navs should provide new leads to treat epilepsy.
doi:10.1007/s12035-014-8674-0
PMCID: PMC4309913  PMID: 24677068
Excitability; Drosophila; Epilepsy; Paralytic; Splicing; Translational repression
22.  A conserved PUF/Ago/eEF1A complex attenuates translation elongation 
SUMMARY
PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar yet distinct mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode and mammalian PUF/Ago/eEF1A complexes were identified, using co-immunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promotes repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1/CSR-1 heterodimer inhibits EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2/Ago/eEF1A inhibits translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurs after translation initiation and leads to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerges from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF/Ago/eEF1A complex attenuates translation elongation.
doi:10.1038/nsmb.2214
PMCID: PMC3293257  PMID: 22231398
23.  Cap-Dependent Translational Inhibition Establishes Two Opposing Morphogen Gradients in Drosophila Embryos 
Current biology : CB  2006;16(20):2035-2041.
In the early Drosophila embryo, asymmetric distribution of transcription factors, established as a consequence of translational control of their maternally-derived mRNAs, initiates pattern formation1-4. For instance, translation of the uniformly distributed maternal hunchback (hb) mRNA is inhibited at the posterior to form an anterior-to-posterior protein concentration gradient along the longitudinal axis5, 6. Inhibition of hb mRNA translation requires an mRNP complex (the NRE-complex) that consists of Nanos (Nos), Pumilio (Pum) and Brain tumor (Brat) proteins, and the Nos responsive element (NRE) present in the 3’ UTR of hb mRNA7-9. The identity of the mRNA 5’ effector protein that is responsible for this translational inhibition remained elusive. Here we show that d4EHP, a cap-binding protein which represses caudal (cad) mRNA translation10, also inhibits hb mRNA translation by interacting simultaneously with the mRNA 5’ cap structure (m7GpppN, where N is any nucleotide)11 and Brat. Thus, by regulating Cad and Hb expression, d4EHP plays a key role in establishing anterior-posterior axis polarity in the Drosophila embryo.
doi:10.1016/j.cub.2006.08.093
PMCID: PMC2238800  PMID: 17055983
24.  A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A 
PLoS ONE  2011;6(5):e19718.
Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/CCdh1. Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/CCdh1-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis.
doi:10.1371/journal.pone.0019718
PMCID: PMC3092770  PMID: 21589936
25.  Identification of Human Proteins That Modify Misfolding and Proteotoxicity of Pathogenic Ataxin-1 
PLoS Genetics  2012;8(8):e1002897.
Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.
Author Summary
Spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative diseases with around 30 subtypes, which are characterized by a progressive loss of cerebellar neurons. Neuronal death has been linked to the aggregation of mutated disease-causing proteins, such as ataxin-1 (ATXN1). Pathogenic ATXN1 contains an elongated glutamine stretch, which triggers spontaneous misfolding and self-assembly of the protein into aggregates. Earlier studies in lower organisms have discovered many non-human proteins that alter aggregation and/or toxicity of mutant ATXN1. Here, we combine an experimental screening approach with bioinformatics to find human proteins that modulate aggregation and toxicity of ATXN1. We identified 21 proteins affecting mutant ATXN1 in mammalian cells. Further characterization revealed that enhancers of ATXN1-mediated toxicity contain α-helical coiled-coil domains as structural motifs, while suppressors do not. Detailed studies with the ATXN1 interacting proteins MED15 and Pum1 finally demonstrated that coiled-coil domains are indeed critical for the aggregation and toxicity promoting effects of human proteins. Our study contributes to a deeper understanding of ATXN1 aggregation and SCA1 pathogenesis and highlights potential therapeutic targets for further investigations.
doi:10.1371/journal.pgen.1002897
PMCID: PMC3420947  PMID: 22916034

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