Human Pat1b Connects Deadenylation with mRNA Decapping and Controls the Assembly of Processing Bodies

ABSTRACT In eukaryotic cells, degradation of many mRNAs is initiated by removal of the poly(A) tail followed by decapping and 5′-3′ exonucleolytic decay. Although the order of these events is well established, we are still lacking a mechanistic understanding of how deadenylation and decapping are linked. In this report we identify human Pat1b as a protein that is tightly associated with the Ccr4-Caf1-Not deadenylation complex as well as with the Dcp1-Dcp2 decapping complex. In addition, the RNA helicase Rck and Lsm1 proteins interact with human Pat1b. These interactions are mediated via at least three independent domains within Pat1b, suggesting that Pat1b serves as a scaffold protein. By tethering Pat1b to a reporter mRNA, we further provide evidence that Pat1b is also functionally linked to both deadenylation and decapping. Finally, we report that Pat1b strongly induces the formation of processing (P) bodies, cytoplasmic foci that contain most enzymes of the RNA decay machinery. An amino-terminal region within Pat1b serves as an aggregation-prone domain that nucleates P bodies, whereas an acidic domain controls the size of P bodies. Taken together, these findings provide evidence that human Pat1b is a central component of the RNA decay machinery by physically connecting deadenylation with decapping.

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IP6K1 upregulates the formation of processing bodies by promoting proteome remodeling on the mRNA cap

10.1101/2020.07.13.199828 ◽

2020 ◽

Cited By ~ 1

Author(s):

Akruti Shah ◽

Rashna Bhandari

Keyword(s):

Protein Interactions ◽

Inositol Phosphate ◽

Binding Protein ◽

Mrna Translation ◽

Rna Helicase ◽

Scaffolding Protein ◽

Processing Bodies ◽

Activator Proteins ◽

Mrna Decapping ◽

P Bodies

AbstractInositol hexakisphosphate kinases (IP6Ks) are ubiquitously expressed small molecule kinases that catalyze the conversion of the inositol phosphate IP6 to 5-IP7. IP6Ks have been reported to influence cellular functions by protein-protein interactions independent of their enzymatic activity. Here, we show that IP6K1 regulates the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that serve as sites for storage of translationally repressed mRNA. Cells with reduced levels of IP6K1 display a dramatic reduction in the number of P-bodies, which can be restored by the expression of active or catalytically inactive IP6K1. IP6K1 does not localize to P-bodies, but instead facilitates the formation of P-bodies by promoting translation suppression. We demonstrate that IP6K1 is present on ribosomes, where it interacts with proteins that constitute the mRNA decapping complex – the scaffold protein EDC4, activator proteins DCP1A/B, and the decapping enzyme DCP2. IP6K1 also interacts with components of the eIF4F translation initiation complex – the scaffolding protein eIF4G1, the RNA helicase eIF4A2, and the cap binding protein eIF4E. The RNA helicase DDX6 and the eIF4E binding protein 4E-T are known to promote translation suppression to facilitate P-body formation. We show that IP6K1 binds to DDX6 and promotes the interaction of DDX6 and 4E-T with the cap binding protein eIF4E, and also enhances the binding between DDX6 and EDC4, thus acting to suppress mRNA translation and promote mRNA decapping. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translation repression over initiation, and thus leading to the formation of P-bodies.

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Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis

The Journal of Cell Biology ◽

10.1083/jcb.200801183 ◽

2008 ◽

Vol 182 (3) ◽

pp. 543-557 ◽

Cited By ~ 77

Author(s):

Peter R. Boag ◽

Arzu Atalay ◽

Stacey Robida ◽

Valerie Reinke ◽

T. Keith Blackwell

Keyword(s):

Caenorhabditis Elegans ◽

Messenger Rnas ◽

Processing Bodies ◽

Body Protein ◽

Translation Inhibition ◽

Mrna Decapping ◽

P Bodies ◽

Somatic Tissues ◽

Translational Regulators ◽

Dependent Mechanism

During oogenesis, numerous messenger RNAs (mRNAs) are maintained in a translationally silenced state. In eukaryotic cells, various translation inhibition and mRNA degradation mechanisms congregate in cytoplasmic processing bodies (P bodies). The P body protein Dhh1 inhibits translation and promotes decapping-mediated mRNA decay together with Pat1 in yeast, and has been implicated in mRNA storage in metazoan oocytes. Here, we have investigated in Caenorhabditis elegans whether Dhh1 and Pat1 generally function together, and how they influence mRNA sequestration during oogenesis. We show that in somatic tissues, the Dhh1 orthologue (CGH-1) forms Pat1 (patr-1)-dependent P bodies that are involved in mRNA decapping. In contrast, during oogenesis, CGH-1 forms patr-1–independent mRNA storage bodies. CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded. Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1–dependent mechanism.

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In planta expression screens of candidate effector proteins from the wheat yellow rust fungus reveal processing bodies as a pathogen-targeted plant cell compartment

10.1101/032276 ◽

2015 ◽

Author(s):

Benjamin Petre ◽

Diane GO Saunders ◽

Jan Sklenar ◽

Cecile Lorrain ◽

Ksenia V Krasileva ◽

...

Keyword(s):

Plant Cell ◽

Molecular Mechanisms ◽

Puccinia Striiformis ◽

Yellow Rust ◽

In Planta ◽

Cell Compartment ◽

Processing Bodies ◽

Subcellular Compartment ◽

Mrna Decapping ◽

P Bodies

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. Our work identifies P-bodies as a novel plant cell compartment targeted by pathogen effectors.

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Analysis of P-Body Assembly in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0199 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2274-2287 ◽

Cited By ~ 156

Author(s):

Daniela Teixeira ◽

Roy Parker

Keyword(s):

Deletion Mutants ◽

Processing Bodies ◽

Mrna Decapping ◽

P Bodies ◽

Translation Repression ◽

Rate Limiting Step ◽

Limiting Step ◽

Body Component ◽

Rate Limiting ◽

Insight Into

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.

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A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay

The Journal of Cell Biology ◽

10.1083/jcb.200504039 ◽

2005 ◽

Vol 170 (6) ◽

pp. 913-924 ◽

Cited By ~ 146

Author(s):

Maria A. Ferraiuolo ◽

Sanjukta Basak ◽

Josee Dostie ◽

Elizabeth L. Murray ◽

Daniel R. Schoenberg ◽

...

Keyword(s):

Mrna Decay ◽

Binding Protein ◽

Initiation Factor ◽

Binding Motif ◽

Processing Bodies ◽

Mrna Decapping ◽

P Bodies ◽

Eukaryotic Initiation Factor 4E ◽

Messenger Ribonucleoprotein ◽

Bona Fide

4E-transporter (4E-T) is one of several proteins that bind the mRNA 5′cap-binding protein, eukaryotic initiation factor 4E (eIF4E), through a conserved binding motif. We previously showed that 4E-T is a nucleocytoplasmic shuttling protein, which mediates the import of eIF4E into the nucleus. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described processing bodies (P-bodies), which are believed to be sites of mRNA decay. In this paper, we demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies. Moreover, 4E-T controls mRNA half-life, because its depletion from cells using short interfering RNA increases mRNA stability. The 4E-T binding partner, eIF4E, also is localized in P-bodies. 4E-T interaction with eIF4E represses translation, which is believed to be a prerequisite for targeting of mRNAs to P-bodies. Collectively, these data suggest that 4E-T interaction with eIF4E is a priming event in inducing messenger ribonucleoprotein rearrangement and transition from translation to decay.

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IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap

Journal of Cell Science ◽

10.1242/jcs.259117 ◽

2021 ◽

Author(s):

Akruti Shah ◽

Rashna Bhandari

Keyword(s):

Protein Interactions ◽

Inositol Phosphate ◽

Translational Repression ◽

Protein Protein Interactions ◽

Processing Bodies ◽

Activator Proteins ◽

Mrna Decapping ◽

P Bodies ◽

Decapping Enzyme ◽

Translational Suppression

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independent of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localize to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2, and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5’ mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T and the cap binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly.

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Interaction Between Mutations in the suppressor of Hairy wing and modifier of mdg4 Genes of Drosophila melunogaster Affecting the Phenotype of gypsy-Induced Mutations

Genetics ◽

10.1093/genetics/142.2.425 ◽

1996 ◽

Vol 142 (2) ◽

pp. 425-436 ◽

Cited By ~ 5

Author(s):

Pavel Georgiev ◽

Marina Kozycina

Keyword(s):

Mutagenic Effect ◽

Induced Mutations ◽

Binding Region ◽

Direct Inhibition ◽

Amino Terminal ◽

Acidic Domain ◽

Complete Inactivation ◽

Insulation Effect ◽

Gypsy Retrotransposon ◽

Carboxy Terminal

Abstract The suppressor of Hairy-wing [su(Hw)] protein mediates the mutagenic effect of the gypsy retrotransposon by repressing the function of transcriptional enhancers located distally from the promoter with respect to the position of the su(Hw)-binding region. Mutations in a second gene, modifier of mdg4, also affect the gypsy-induced phenotype. Two major effects of the mod(mdg4)lul mutation can be distinguished: the interference with insulation by the su(Hw)-binding region and direct inhibition of gene expression that is not dependent on the su(Hw)-binding region position. The mod(mdg4)lul mutation partially suppresses ct6, scD1 and Hw1 mutations, possibly by interfering with the insulation effect of the su(Hw)-binding region. An example of the second effect of mod(mdg4)lul is a complete inactivation of yellow expression in combination with the y 2 allele. Phenotypic analyses of flies with combinations of mod(mdg4)lul and different su(Hw) mutations, or with constructions carrying deletions of the acidic domains of the su(Hw) protein, suggest that the carboxy-terminal acidic domain is important for direct inhibition of yellow transcription in bristles, while the amino-terminal acidic domain is more essential for insulation.

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RNA decay in processing bodies is indispensable for adipogenesis

Cell Death and Disease ◽

10.1038/s41419-021-03537-7 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Ryotaro Maeda ◽

Daisuke Kami ◽

Akira Shikuma ◽

Yosuke Suzuki ◽

Toshihiko Taya ◽

...

Keyword(s):

Lipid Droplets ◽

Adipogenic Differentiation ◽

Negative Regulator ◽

Translational Repression ◽

Rna Decay ◽

Significant Suppression ◽

Intracellular Lipid ◽

Processing Bodies ◽

Binding Partner ◽

The Stability

AbstractThe RNA decay pathway plays key regulatory roles in cell identities and differentiation processes. Although adipogenesis is transcriptionally and epigenetically regulated and has been thoroughly investigated, how RNA metabolism that contributes to the stability of phenotype-shaping transcriptomes participates in differentiation remains elusive. In this study, we investigated Ddx6, an essential component of processing bodies (PBs) that executes RNA decay and translational repression in the cytoplasm and participates in the cellular transition of reprogramming. Upon adipogenic induction, Ddx6 dynamically accumulated to form PBs with a binding partner, 4E-T, at the early phase prior to emergence of intracellular lipid droplets. In contrast, preadipocytes with Ddx6 knockout (KO) or 4E-T knockdown (KD) failed to generate PBs, resulting in significant suppression of adipogenesis. Transcription factors related to preadipocytes and negative regulators of adipogenesis that were not expressed under adipogenic stimulation were maintained in Ddx6-KO and 4E-T-KD preadipocytes under adipogenic induction. Elimination of Dlk1, a major negative regulator of adipogenesis, in 3T3L1 Ddx6-KO cells did not restore adipogenic differentiation capacity to any extent. Similar to murine cells, human primary mesenchymal stem cells, which can differentiate into adipocytes upon stimulation with adipogenic cocktails, required DDX6 to maturate into adipocytes. Therefore, RNA decay of the entire parental transcriptome, rather than removal of a strong negative regulator, could be indispensable for adipogenesis.

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Defects in the Secretory Pathway and High Ca2+ Induce Multiple P-bodies

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0099 ◽

2010 ◽

Vol 21 (15) ◽

pp. 2624-2638 ◽

Cited By ~ 34

Author(s):

Cornelia Kilchert ◽

Julie Weidner ◽

Cristina Prescianotto-Baschong ◽

Anne Spang

Keyword(s):

Osmotic Stress ◽

Secretory Pathway ◽

Small Gtpase ◽

Processing Bodies ◽

P Bodies ◽

Intracellular Signals ◽

Intracellular Ca ◽

Response To Stress ◽

Cellular Stresses ◽

Translational Block

mRNA is sequestered and turned over in cytoplasmic processing bodies (PBs), which are induced by various cellular stresses. Unexpectedly, in Saccharomyces cerevisiae, mutants of the small GTPase Arf1 and various secretory pathway mutants induced a significant increase in PB number, compared with PB induction by starvation or oxidative stress. Exposure of wild-type cells to osmotic stress or high extracellular Ca2+ mimicked this increase in PB number. Conversely, intracellular Ca2+-depletion strongly reduced PB formation in the secretory mutants. In contrast to PB induction through starvation or osmotic stress, PB formation in secretory mutants and by Ca2+ required the PB components Pat1 and Scd6, and calmodulin, indicating that different stressors act through distinct pathways. Consistent with this hypothesis, when stresses were combined, PB number did not correlate with the strength of the translational block, but rather with the type of stress encountered. Interestingly, independent of the stressor, PBs appear as spheres of ∼40–100 nm connected to the endoplasmic reticulum (ER), consistent with the idea that translation and silencing/degradation occur in a spatially coordinated manner at the ER. We propose that PB assembly in response to stress occurs at the ER and depends on intracellular signals that regulate PB number.

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