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

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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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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The C-Terminal RGG Domain of Human Lsm4 Promotes Processing Body Formation Stimulated by Arginine Dimethylation

Molecular and Cellular Biology ◽

10.1128/mcb.01102-15 ◽

2016 ◽

Vol 36 (17) ◽

pp. 2226-2235 ◽

Cited By ~ 23

Author(s):

Marcos Arribas-Layton ◽

Jaclyn Dennis ◽

Eric J. Bennett ◽

Christian K. Damgaard ◽

Jens Lykke-Andersen

Keyword(s):

Posttranslational Modifications ◽

Mrna Decay ◽

Low Complexity ◽

Translational Repression ◽

Processing Bodies ◽

Content Type ◽

Rich Domain ◽

Pb Accumulation ◽

Processing Body ◽

Protein Components

Processing bodies (PBs) are conserved cytoplasmic aggregations of translationally repressed mRNAs assembled with mRNA decay factors. The aggregation of mRNA-protein (mRNP) complexes into PBs involves interactions between low-complexity regions of protein components of the mRNPs. InSaccharomyces cerevisiae, the carboxy (C)-terminal Q/N-rich domain of the Lsm4 subunit of the Lsm1-7 complex plays an important role in PB formation, but the C-terminal domain of Lsm4 in most eukaryotes is an RGG domain rather than Q/N rich. Here we show that the Lsm4 RGG domain promotes PB accumulation in human cells and that symmetric dimethylation of arginines within the RGG domain stimulates this process. A mutant Lsm4 protein lacking the RGG domain failed to rescue PB formation in cells depleted of endogenous Lsm4, although this mutant protein retained the ability to assemble with Lsm1-7, associate with decapping factors, and promote mRNA decay and translational repression. Mutation of the symmetrically dimethylated arginines within the RGG domain impaired the ability of Lsm4 to promote PB accumulation. Depletion of PRMT5, the primary protein arginine methyltransferase responsible for symmetric arginine dimethylation, including Lsm4, resulted in loss of PBs. We also uncovered the histone acetyltransferase 1 (HAT1)-RBBP7 lysine acetylase complex as an interaction partner of the Lsm4 RGG domain but found no evidence of a role for this complex in PB metabolism. Together, our findings suggest a stimulatory role for posttranslational modifications in PB accumulation and raise the possibility that mRNP dynamics are posttranslationally regulated.

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RNA granules

The Journal of Cell Biology ◽

10.1083/jcb.200512082 ◽

2006 ◽

Vol 172 (6) ◽

pp. 803-808 ◽

Cited By ~ 709

Author(s):

Paul Anderson ◽

Nancy Kedersha

Keyword(s):

Messenger Rna ◽

Posttranscriptional Regulation ◽

Spatial Organization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulation Of Gene Expression ◽

Processing Bodies ◽

Rna Granules ◽

Cytoplasmic Rna ◽

Messenger Rna Translation

Cytoplasmic RNA granules in germ cells (polar and germinal granules), somatic cells (stress granules and processing bodies), and neurons (neuronal granules) have emerged as important players in the posttranscriptional regulation of gene expression. RNA granules contain various ribosomal subunits, translation factors, decay enzymes, helicases, scaffold proteins, and RNA-binding proteins, and they control the localization, stability, and translation of their RNA cargo. We review the relationship between different classes of these granules and discuss how spatial organization regulates messenger RNA translation/decay.

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Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

The Journal of Cell Biology ◽

10.1083/jcb.200707010 ◽

2007 ◽

Vol 179 (1) ◽

pp. 65-74 ◽

Cited By ~ 161

Author(s):

Nathaniel P. Hoyle ◽

Lydia M. Castelli ◽

Susan G. Campbell ◽

Leah E.A. Holmes ◽

Mark P. Ashe

Keyword(s):

Messenger Rna ◽

Complex Dynamics ◽

Kinetic Studies ◽

P Bodies ◽

Potential Control ◽

Rna Granules ◽

Cytoplasmic Rna ◽

Mrna Pool ◽

Stress Dependent ◽

Mrna Fate

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

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Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.200912019 ◽

2010 ◽

Vol 189 (5) ◽

pp. 813-827 ◽

Cited By ~ 60

Author(s):

Je-Hyun Yoon ◽

Eui-Ju Choi ◽

Roy Parker

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Interactions ◽

Messenger Rna ◽

Mrna Decay ◽

Control Point ◽

Stress Granules ◽

Mrna Degradation ◽

Stress Granule ◽

Granule Formation ◽

P Bodies

Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.

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RNA helicase DDX6 in P-bodies is essential for the assembly of stress granules

10.1101/2021.09.24.461736 ◽

2021 ◽

Author(s):

Vladimir Majerciak ◽

Tongqing Zhou ◽

Zhi-Ming Zheng

Keyword(s):

Rna Processing ◽

Stress Granules ◽

The Other ◽

Helicase Activity ◽

Dual Roles ◽

Processing Bodies ◽

P Bodies ◽

Rna Granules ◽

Cytoplasmic Rna ◽

Conceptual Advance

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate storage of translationally arrested mRNAs and are intimately related. In this study, we found the dependence of SG formation on PB in the cells under arsenite (ARS) stress, but not the other way around. GW182, 4E-T and DDX6 essential for PB formation differentially affect SG formation in the cells under ARS stress, with DDX6 being the most prominent. The cells with DDX6 deficiency display irregular shape of SG which could be rescued by ectopic wt DDX6, but not its helicase mutant E247A DDX6, which induces SG in the cells without stress, indicating that DDX6 helicase activity is essential for PB, but suppressive for SG. DDX6's dual roles are independent of DDX6 interactors EDC3, CNOT1, and PAT1B. This study provides a conceptual advance of how DDX6 involves in the biogenesis of PB and SG.

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Focusing on mRNA Granules and Stalled Polysomes Amidst Diverse Mechanisms Underlying mRNA Transport, mRNA Storage, and Local Translation

The Oxford Handbook of Neuronal Protein Synthesis ◽

10.1093/oxfordhb/9780190686307.013.20 ◽

2020 ◽

Author(s):

Mina N. Anadolu ◽

Wayne S. Sossin

Keyword(s):

Protein Synthesis ◽

Liquid Phase ◽

Mrna Translation ◽

Rna Transport ◽

P Bodies ◽

Rna Granules ◽

Mrna Granules ◽

Transport Particle ◽

As Stress ◽

The Individual

In neurons, mRNAs are transported to distal sites to allow for localized protein synthesis. There are many diverse mechanisms underlying this transport. For example, an individual mRNA can be transported in an RNA transport particle that is tailored to the individual mRNA and its associated binding proteins. In contrast, some mRNAs are transported in liquid-liquid phase separated structures called neuronal RNA granules that are made up of multiple stalled polysomes, allowing for rapid initiation-independent production of proteins required for synaptic plasticity. Moreover, neurons have additional types of liquid-liquid phase–separated structures containing mRNA, such as stress granules and P bodies. This chapter discusses the relationships between all of these structures, what proteins distinguish them, and the possible roles they play in the complex control of mRNA translation at distal sites that allow neurons to use protein synthesis to refine their local proteome in many different ways.

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On track with P-bodies

Biochemical Society Transactions ◽

10.1042/bst0380242 ◽

2010 ◽

Vol 38 (1) ◽

pp. 242-251 ◽

Cited By ~ 131

Author(s):

Meeta Kulkarni ◽

Sevim Ozgur ◽

Georg Stoecklin

Keyword(s):

Light Microscopy ◽

Mrna Decay ◽

Molecular Level ◽

Somatic Cells ◽

Present Review ◽

Dual Function ◽

Processing Bodies ◽

P Bodies ◽

Specific Mrnas ◽

Transient Interactions

P-bodies (processing bodies) are cytoplasmic foci visible by light microscopy in somatic cells of vertebrate and invertebrate origin as well as in yeast, plants and trypanosomes. At the molecular level, P-bodies are dynamic aggregates of specific mRNAs and proteins that serve a dual function: first, they harbour mRNAs that are translationally silenced, and such mRNA can exit again from P-bodies to re-engage in translation. Secondly, P-bodies recruit mRNAs that are targeted for deadenylation and degradation by the decapping/Xrn1 pathway. Whereas certain proteins are core constituents of P-bodies, others involved in recognizing short-lived mRNAs can only be trapped in P-bodies when mRNA decay is attenuated. This reflects the very transient interactions by which many proteins associate with P-bodies. In the present review, we summarize recent findings on the function, assembly and motility of P-bodies. An updated list of proteins and RNAs that localize to P-bodies will help in keeping track of this fast-growing field.

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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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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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