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

2020 ◽  
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.

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

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.

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

2005 ◽  
Vol 170 (6) ◽  
pp. 913-924 ◽  
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.

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

2010 ◽  
Vol 30 (17) ◽  
pp. 4308-4323 ◽  
Author(s):  
Sevim Ozgur ◽  
Marina Chekulaeva ◽  
Georg Stoecklin
Keyword(s):  
Rna Helicase ◽  
Rna Decay ◽  
Eukaryotic Cells ◽  
Central Component ◽  
Processing Bodies ◽  
Mrna Decapping ◽  
P Bodies ◽  
Amino Terminal ◽  
Acidic Domain ◽  
Reporter Mrna

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.

scholarly journals Spatial engineering of E. coli with addressable phase-separated RNAs

2020 ◽  
Author(s):  
Haotian Guo ◽  
Joseph C. Ryan ◽  
Adeline Mallet ◽  
Xiaohu Song ◽  
Victor Pabst ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Fluorescent Proteins ◽  
Mrna Translation ◽  
Cag Repeats ◽  
Scaffolding Protein ◽  
Branch Points ◽  
Multilayered Structures ◽  
E Coli ◽  
Versatile Tool

AbstractBiochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within the Escherichia coli. TEARS are assembled from RNA-binding protein recruitment domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures and a non-equilibrium mechanism controlling their composition and reaction robustness. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. TEARS are a simple and versatile tool for spatially controlling E. coli biochemistry.

scholarly journals KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182

2019 ◽  
Vol 47 (17) ◽  
pp. 9368-9385 ◽  
Author(s):  
Nishi R Sharma ◽  
Vladimir Majerciak ◽  
Michael J Kruhlak ◽  
Lulu Yu ◽  
Jeong Gu Kang ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Time Lapse ◽  
Processing Bodies ◽  
P Bodies ◽  
Rna Granules ◽  
Inhibitory Function ◽  
Invasion Mechanisms ◽  
Initial Stage

Abstract Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.

Translation of IGBP1 mRNA Contributes to the Regulation of Expansion and Differentiation of Erythroid Progenitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 72-72
Author(s):  
Godfrey Grech ◽  
Montserrat Blazquez-Domingo ◽  
Andrea Kolbus ◽  
Hartmut Beug ◽  
Bob Lowenberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathways ◽  
Binding Protein ◽  
Expression Profiles ◽  
Constitutive Expression ◽  
Mrna Translation ◽  
Erythroid Differentiation ◽  
Scaffolding Protein ◽  
Initiation Factor ◽  
Erythroid Progenitors

Abstract Erythroid progenitors can be expanded in vitro in the presence of erythropoietin (Epo), stem cell factor (SCF) and dexamethasone, while they differentiate to enucleated erythrocytes in presence of Epo only. Our study aims to identify (i) signaling pathways that control expansion of erythroid progenitors and (i) genes regulated by these signaling pathways. Since (i) SCF strongly activates phosphotidylinositol 3 kinase (PI3K) and (ii) inhibition of PI3K with LY294002 induces terminal differentiation of erythroid progenitors under Epo and SCF stimulation, SCF seems to enhance renewal divisions of erythroid progenitors via a PI3K-dependent mechanism. An important PI3K-dependent process in cell proliferation is regulation of mRNA translation. PI3K controls the activity of mTOR (mammalian target of rapamycin), whose activation results in phosphorylation of eIF4E (eukaryote Initiation Factor 4E)-binding protein (4E-BP). Fully phosphorylated 4E-BP releases eIF4E, which can subsequently bind eIF4G, the scaffolding protein of the eIF4F cap-binding and scanning complex. In particular mRNAs with a structured UTR (untranslated region) require optimal availability of eIF4E to be translated. SCF, but not Epo can induce full phosphorylation of 4E-BP and efficient formation of the eIF4F cap-binding complex. Overexpression of eIF4E inhibited erythroid differentiation as if SCF were present, indicating that SCF-induced release of eIF4E from 4E-BP is an important mechanism regulating the balance between progenitor expansion and differentiation. A major step in mRNA translation controlled by eIF4F is polysome recruitment. To identify genes whose expression is regulated by signaling-induced polysome recruitment, we compared total and polysome-bound mRNA from factor deprived and Epo-, SCF- or Epo plus SCF restimulated progenitors on gene-expression micro-arrays (Affymetrix). The profiling was performed with 4 biological replicates and candidate genes were selected using ANOVA. In subsequent cluster analysis we combined these data with (polysomal) expression profiles of differentiating erythroid cells. Thus we identified a cluster containing genes, upregulated in part or completely at the level of mRNA polysome recruitment and downregulated during erythroid differentiation. Targets involved in signal transduction and gene expression were analyzed in more detail. Polysome recruitment of 15/17 targets tested so far appeared to be dependent on PI3K activation and eIF4E expression. Constitutive expression of these targets in erythroid progenitors revealed that one target in particular was able to inhibit erythroid differentiation comparable to overexpression of eIF4E. This target was IGBP1 (Immunoglobulin binding protein 1). IGBP1 binds to and modulates the activity of the catalytic subunit of PP2A, a major cellular serine/threonine phosphatase, which also dephosphorylates 4E-BP. Overexpression of IGBP1 does not inhibit 4E-BP dephosphorylation in absence of factor, but enhances phosphorylation of 4E-BP in presence of Epo. Nevertheless, constitutive expression of IGBP1 does not increase polysome association of structured mRNAs. The multiple functions of PP2A suggest that the potent inhibition of erythroid differentiation by IGBP1 may be due to deregulation of several cellular mechanisms.

scholarly journals Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

2007 ◽  
Vol 179 (3) ◽  
pp. 437-449 ◽  
Author(s):  
Carolyn J. Decker ◽  
Daniela Teixeira ◽  
Roy Parker
Keyword(s):  
Protein Interactions ◽  
Messenger Rna ◽  
Mrna Decay ◽  
Processing Bodies ◽  
P Bodies ◽  
Rna Granules ◽  
Rich Domain ◽  
Cytoplasmic Rna ◽  
Processing Body ◽  
The Individual

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the “prionlike” domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA–protein complex that then aggregates through multiple specific mechanisms.

scholarly journals The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

2011 ◽  
Vol 192 (4) ◽  
pp. 583-598 ◽  
Author(s):  
Cornelia Kurischko ◽  
Hong Kyung Kim ◽  
Venkata K. Kuravi ◽  
Juliane Pratzka ◽  
Francis C. Luca
Keyword(s):  
Binding Protein ◽  
Cellular Stress ◽  
Stress Granules ◽  
Polarized Growth ◽  
Cell Integrity ◽  
Processing Bodies ◽  
P Bodies ◽  
Mrna Binding Protein ◽  
Cortical Localization ◽  
Mrna Binding

The mRNA-binding protein Ssd1 is a substrate for the Saccharomyces cerevisiae LATS/NDR orthologue Cbk1, which controls polarized growth, cell separation, and cell integrity. We discovered that most Ssd1 localizes diffusely within the cytoplasm, but some transiently accumulates at sites of polarized growth. Cbk1 inhibition and cellular stress cause Ssd1 to redistribute to mRNA processing bodies (P-bodies) and stress granules, which are known to repress translation. Ssd1 recruitment to P-bodies is independent of mRNA binding and is promoted by the removal of Cbk1 phosphorylation sites. SSD1 deletion severely impairs the asymmetric localization of the Ssd1-associated mRNA, SRL1. Expression of phosphomimetic Ssd1 promotes polarized localization of SRL1 mRNA, whereas phosphorylation-deficient Ssd1 causes constitutive localization of SRL1 mRNA to P-bodies and causes cellular lysis. These data support the model that Cbk1-mediated phosphorylation of Ssd1 promotes the cortical localization of Ssd1–mRNA complexes, whereas Cbk1 inhibition, cellular stress, and Ssd1 dephosphorylation promote Ssd1–mRNA interactions with P-bodies and stress granules, leading to translational repression.

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

2008 ◽  
Vol 182 (3) ◽  
pp. 543-557 ◽  
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.

scholarly journals Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing

2013 ◽  
Vol 42 (5) ◽  
pp. 3298-3313 ◽  
Author(s):  
Anastasiia Kamenska ◽  
Wei-Ting Lu ◽  
Dorota Kubacka ◽  
Helen Broomhead ◽  
Nicola Minshall ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Mrna Translation ◽  
Developmental Model ◽  
Model Systems ◽  
P Bodies ◽  
Specific Mrnas

Abstract A key player in translation initiation is eIF4E, the mRNA 5′ cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4Lϕ site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4Lϕ, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

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