Dcp1-Bodies in Mouse Oocytes

Processing bodies (P-bodies) are cytoplasmic granules involved in the storage and degradation of mRNAs. In somatic cells, their formation involves miRNA-mediated mRNA silencing. Many P-body protein components are also found in germ cell granules, such as in mammalian spermatocytes. In fully grown mammalian oocytes, where changes in gene expression depend entirely on translational control, RNA granules have not as yet been characterized. Here we show the presence of P-body-like foci in mouse oocytes, as revealed by the presence of Dcp1a and the colocalization of RNA-associated protein 55 (RAP55) and the DEAD box RNA helicase Rck/p54, two proteins associated with P-bodies and translational control. These P-body-like structures have been called Dcp1-bodies and in meiotically arrested primary oocytes, two types can be distinguished based on their size. They also have different protein partners and sensitivities to the depletion of endogenous siRNA/miRNA and translational inhibitors. However, both type progressively disappear during in vitro meiotic maturation and are virtually absent in metaphase II–arrested secondary oocytes. Moreover, this disassembly of hDcp1a-bodies is concomitant with the posttranslational modification of EGFP-hDcp1a.

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The arrested state of processing bodies supports mRNA regulation in early development

10.1101/2021.03.16.435709 ◽

2021 ◽

Cited By ~ 1

Author(s):

M. Sankaranarayanan ◽

Ryan J. Emenecker ◽

Marcus Jahnel ◽

Irmela R. E. A. Trussina ◽

Matt Wayland ◽

...

Keyword(s):

Physical Properties ◽

Electrostatic Interactions ◽

Processing Bodies ◽

P Bodies ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Dead Box ◽

Premature Release ◽

Liquid Liquid Phase Separation

ABSTRACTBiomolecular condensates that form via liquid-liquid phase separation can exhibit diverse physical states. Despite considerable progress, the relevance of condensate physical states forin vivobiological function remains limited. Here, we investigated the physical properties ofin vivoprocessing bodies (P bodies) and their impact on mRNA storage in matureDrosophilaoocytes. We show that the conserved DEAD-box RNA helicase Me31B forms P body condensates which adopt a less dynamic, arrested physical state. We demonstrate that structurally distinct proteins and hydrophobic and electrostatic interactions, together with RNA and intrinsically disordered regions, regulate the physical properties of P bodies. Finally, using live imaging, we show that the arrested state of P bodies is required to prevent the premature release ofbicoid(bcd) mRNA, a body axis determinant, and that P body dissolution leads tobcdrelease. Together, this work establishes a role for arrested states of biomolecular condensates in regulating cellular function in a developing organism.

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RNA granules and cytoskeletal links

Biochemical Society Transactions ◽

10.1042/bst20140067 ◽

2014 ◽

Vol 42 (4) ◽

pp. 1206-1210 ◽

Cited By ~ 15

Author(s):

Dipen Rajgor ◽

Catherine M. Shanahan

Keyword(s):

Mammalian Cells ◽

Dynamic Properties ◽

Translational Repression ◽

Processing Bodies ◽

P Bodies ◽

Rna Granules ◽

Messenger Ribonucleoprotein ◽

Cytoskeletal Networks ◽

And Control ◽

Lower Eukaryote

In eukaryotic cells, non-translating mRNAs can accumulate into cytoplasmic mRNP (messenger ribonucleoprotein) granules such as P-bodies (processing bodies) and SGs (stress granules). P-bodies contain the mRNA decay and translational repression machineries and are ubiquitously expressed in mammalian cells and lower eukaryote species including Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. In contrast, SGs are only detected during cellular stress when translation is inhibited and form from aggregates of stalled pre-initiation complexes. SGs and P-bodies are related to NGs (neuronal granules), which are essential in the localization and control of mRNAs in neurons. Importantly, RNA granules are linked to the cytoskeleton, which plays an important role in mediating many of their dynamic properties. In the present review, we discuss how P-bodies, SGs and NGs are linked to cytoskeletal networks and the importance of these linkages in maintaining localization of their RNA cargoes.

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Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

10.1101/402404 ◽

2018 ◽

Author(s):

Ruchika Sachdev ◽

Maria Hondele ◽

Miriam Linsenmeier ◽

Pascal Vallotton ◽

Christopher F. Mugler ◽

...

Keyword(s):

Phase Separation ◽

Direct Interaction ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

The Dead ◽

Processing Body ◽

Liquid Liquid Phase Separation

AbstractProcessing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid-liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. We recently identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo [Mugler et al., 2016]. Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

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

Nucleic Acids Research ◽

10.1093/nar/gkz683 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9368-9385 ◽

Cited By ~ 5

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.

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NcPuf1 Is a Key Virulence Factor in Neospora caninum

Pathogens ◽

10.3390/pathogens9121019 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1019

Author(s):

Chenrong Wang ◽

Congshan Yang ◽

Jing Liu ◽

Qun Liu

Keyword(s):

Neospora Caninum ◽

Wide Distribution ◽

Key Factors ◽

Processing Bodies ◽

Puf Proteins ◽

P Bodies ◽

Intracellular Parasites ◽

Binding Factor ◽

Puf Protein

Background: Neospora caninum is an apicomplexan parasite that infects many mammals and particularly causes abortion in cattle. The key factors in its wide distribution are its virulence and ability to transform between tachyzoite and bradyzoite forms. However, the factors are not well understood. Although Puf protein (named after Pumilio from Drosophila melanogaster and fem-3 binding factor from Caenorhabditis elegans) have a functionally conserved role in promoting proliferation and inhibiting differentiation in many eukaryotes, the function of the Puf proteins in N. caninum is poorly understood. Methods: The CRISPR/CAS9 system was used to identify and study the function of the Puf protein in N. caninum. Results: We showed that N. caninum encodes a Puf protein, which was designated NcPuf1. NcPuf1 is found in the cytoplasm in intracellular parasites and in processing bodies (P-bodies), which are reported for the first time in N. caninum in extracellular parasites. NcPuf1 is not needed for the formation of P-bodies in N. caninum. The deletion of NcPuf1 (ΔNcPuf1) does not affect the differentiation in vitro and tissue cysts formation in the mouse brain. However, ΔNcPuf1 resulted in decreases in the proliferative capacity of N. caninum in vitro and virulence in mice. Conclusions: Altogether, the disruption of NcPuf1 does not affect bradyzoites differentiation, but seriously impairs tachyzoite proliferation in vitro and virulence in mice. These results can provide a theoretical basis for the development of attenuated vaccines to prevent the infection of N. caninum.

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

The Journal of Cell Biology ◽

10.1083/jcb.200704147 ◽

2007 ◽

Vol 179 (3) ◽

pp. 437-449 ◽

Cited By ~ 304

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.

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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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Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

eLife ◽

10.7554/elife.41415 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 20

Author(s):

Ruchika Sachdev ◽

Maria Hondele ◽

Miriam Linsenmeier ◽

Pascal Vallotton ◽

Christopher F Mugler ◽

...

Keyword(s):

Phase Separation ◽

Direct Interaction ◽

Liquid Droplets ◽

Processing Bodies ◽

Dead Box ◽

The Dead ◽

Processing Body ◽

Liquid Liquid Phase Separation

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.

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RGG-motif protein Sbp1 is required for Processing body (P-body) disassembly

10.1101/2021.02.23.432385 ◽

2021 ◽

Author(s):

Raju Roy ◽

Ishwarya Achappa Kuttanda ◽

Nupur Bhatter ◽

Purusharth I Rajyaguru

Keyword(s):

Mrna Decay ◽

Glucose Deprivation ◽

Low Complexity ◽

Wild Type ◽

Processing Bodies ◽

P Bodies ◽

Rna Granules ◽

Processing Body ◽

Mrna Fate

AbstractRNA granules are conserved mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. In this report, we identify RGG-motif translation repressor protein Sbp1 as a disassembly factor of P-bodies. Disassembly of Edc3 granules but not the Pab1 granules (a conserved stress granule marker) that arise upon sodium azide and glucose deprivation stress are defective in Δsbp1. Disassembly of other P-body proteins such as Dhh1 and Scd6 is also defective in Δsbp1. Complementation experiments suggest that the wild type Sbp1 but not an RGG-motif deletion mutant rescues the Edc3 granule disassembly defect in Δsbp1. We observe that purified Edc3 forms assemblies, which is promoted by the presence of RNA and NADH. Strikingly, addition of purified Sbp1 leads to significantly decreased Edc3 assemblies. Although low complexity sequences have been in general implicated in assembly, our results reveal the role of RGG-motif (a low-complexity sequence) in the disassembly of P-bodies.

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Abstract 20439: Novel Long Intergenic Noncoding RNAs Modulate Adipose Functions in Human

Circulation ◽

10.1161/circ.130.suppl_2.20439 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Xuan Zhang ◽

Rachel Ballantyne ◽

Chenyi Xue ◽

Jane F Ferguson ◽

Brian Gregory ◽

...

Keyword(s):

Single Molecule ◽

Noncoding Rnas ◽

Cellular Functions ◽

Protein Coding ◽

Processing Bodies ◽

P Bodies ◽

Genome Wide ◽

Potential Binding ◽

Competing Endogenous Rnas

Recently long intergenic noncoding RNAs (lincRNAs) have emerged as key mediators of cellular differentiation and functions in a variety of cell systems critical to cardiovascular and metabolic disorders. To identify and investigate novel functional lincRNAs in human adipose, we performed deep high-throughput RNAseq (>200 million reads/sample) in subcutaneous adiposes of 13 health volunteers. Of an integrated dataset of 54,944 human lincRNAs, 6,558 lincRNAs were detected. Here we report 2 cytoplasmic adipose lincRNAs, linc-DMRT2 and linc-NFE2L3-1, were detected in human adipocytes but not monocytes or macrophages. Linc-DMRT2, one of the most abundant adipose lincRNAs, was markedly induced during in vitro human adipocyte differentiation. Notably, single molecule RNA FISH (fluorescence in situ hybridization) demonstrated that linc-DMRT2 were exclusively present in adipocyte cytoplasma and co-localized with processing bodies (P-bodies) marker, GW182, suggesting its potential role in modulating turnover of certain RNA species. In addition, linc-NFE2L3-1, predominantly detected in adipose and skeleton muscle, is localized near an established GWAS locus associated with waist-hip ratio adjusted BMI. We identified 4 SNPs in linc-NFE2L3-1 reaching genome wide significance for BMI (lead SNP rs10267498, P=2.73х10 -8 ). Linkage disequilibrium analysis confirmed linc-NFE2L3-1 harbors stronger GWAS signals than protein-coding genes in the locus, suggesting lincRNA might be causal for GWAS association with BMI. Bioinformatic prediction algorithms identified potential binding sites in linc-DMRT2 and linc-NFE2L3-1 for multiple microRNAs that have been demonstrated to regulate adipogenesis (e.g. miR-15 a/b and let-7) or adipocyte functions (e.g. miR-320). In summary, our data suggest that cytoplasmic linc-DMRT2 and linc-NFE2L3-1 may play important roles in adipocyte biology by functioning as competing endogenous RNAs and binding specific microRNAs that mediate adipocyte cellular functions. Genetic variation in such human linRNAs may contribute to cardiometabolic traits.

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