scholarly journals Poly(A) binding protein is required for mRNP remodeling to form P-bodies in mammalian cells

2021 ◽  
Author(s):  
Jingwei Xie ◽  
Yu Chen ◽  
Xiaoyu Wei ◽  
Guennadi Kozlov
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Stress Granules ◽  
Body Formation ◽  
P Bodies ◽  
Rna Granules ◽  
Cellular Processes ◽  
Early Stages ◽  
Summary Statement

AbstractCompartmentalization of mRNA through formation of RNA granules is involved in many cellular processes, yet it is not well understood. mRNP complexes undergo dramatic changes in protein compositions, reflected by markers of P-bodies and stress granules. Here, we show that PABPC1, albeit absent in P-bodies, plays important role in P-body formation. Depletion of PABPC1 decreases P-body population in unstressed cells. Upon stress in PABPC1 depleted cells, individual P-bodies fail to form and instead P-body proteins assemble on PABPC1-containing stress granules. We hypothesize that mRNP recruit proteins via PABPC1 to assemble P-bodies, before PABPC1 is displaced from mRNP. Further, we demonstrate that GW182 can mediate P-body assembly. These findings help us understand the early stages of mRNP remodeling and P-body formation.Summary statementA novel role of poly(A) binding protein is reported in P-body formation

scholarly journals P bodies promote stress granule assembly in Saccharomyces cerevisiae

2008 ◽  
Vol 183 (3) ◽  
pp. 441-455 ◽  
Author(s):  
J. Ross Buchan ◽  
Denise Muhlrad ◽  
Roy Parker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Protein Composition ◽  
Stress Granules ◽  
Glucose Deprivation ◽  
Stress Granule ◽  
Granule Formation ◽  
P Bodies ◽  
Rna Granules ◽  
Kinetics Of

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

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.

scholarly journals RNA Binding Protein TAF15 Suppresses Toxicity in a Yeast Model of FUS Proteinopathy

2021 ◽  
Author(s):  
Elliott Hayden ◽  
Aicha Kebe ◽  
Shuzhen Chen ◽  
Abagail Chumley ◽  
Chenyi Xia ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stress Granules ◽  
Rna Recognition Motif ◽  
Body Formation ◽  
P Bodies ◽  
Associated Proteins ◽  
Rnp Granules ◽  
Yeast Model

Abstract Mutations in Fused in Sarcoma (FUS), an RNA binding protein that functions in multiple steps in gene expression regulation and RNA processing, are known to cause familial amyotrophic lateral sclerosis (ALS). Since this discovery, mutations in several other RNA binding proteins (RBPs) have also been linked to ALS. Some of these ALS-associated RBPs have been shown to colocalize with ribonucleoprotein (RNP) granules such as stress granules and processing bodies (p-bodies). Characterization of ALS-associated proteins, their mis-localization, aggregation and toxicity in cellular and animal models have provided critical insights in disease. More and more evidence has emerged supporting a hypothesis that impaired clearance, inappropriate assembly, and dysregulation of RNP granules play a role in ALS. Through genome-scale overexpression screening of a yeast model of FUS toxicity, we found that TAF15, a human RBP with a similar protein domain structure and belonging to the same FET protein family as FUS, suppresses FUS toxicity. The suppressor effect of TAF15 is specific to FUS and not found in other yeast models of neurodegenerative disease-associated proteins. We showed that the RNA recognition motif (RRM) of TAF15 is required for its rescue of FUS toxicity. Furthermore, FUS and TAF15 physically interact, and the C-terminus of TAF15 is required for both the physical protein-protein interaction and its protection against FUS toxicity. Finally, while FUS induces and colocalizes with both stress granules and p-bodies, TAF15 only induces and colocalizes with p-bodies. Importantly, co-expression of FUS and TAF15 induces more p-bodies than individually expressing each gene alone, and FUS toxicity is exacerbated in yeast that is deficient in p-body formation. Overall, our findings suggest a role of p-body formation in the suppression of FUS toxicity by TAF15.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

scholarly journals Regulation of circular dorsal ruffles, macropinocytosis, and cell migration by RhoG and its exchange factor, Trio

2017 ◽  
Vol 28 (13) ◽  
pp. 1768-1781 ◽  
Author(s):  
Alejandra Valdivia ◽  
Silvia M. Goicoechea ◽  
Sahezeel Awadia ◽  
Ashtyn Zinn ◽  
Rafael Garcia-Mata
Keyword(s):  
Cell Migration ◽  
Mammalian Cells ◽  
Dorsal Surface ◽  
Receptor Internalization ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Unique Type ◽  
Exchange Factor ◽  
Cellular Processes

Circular dorsal ruffles (CDRs) are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation. CDRs represent a unique type of structure that forms transiently and only once upon stimulation. The formation of CDRs involves a drastic rearrangement of the cytoskeleton, which is regulated by the Rho family of GTPases. So far, only Rac1 has been consistently associated with CDR formation, whereas the role of other GTPases in this process is either lacking or inconclusive. Here we show that RhoG and its exchange factor, Trio, play a role in the regulation of CDR dynamics, particularly by modulating their size. RhoG is activated by Trio downstream of PDGF in a PI3K- and Src-dependent manner. Silencing RhoG expression decreases the number of cells that form CDRs, as well as the area of the CDRs. The regulation of CDR area by RhoG is independent of Rac1 function. In addition, our results show the RhoG plays a role in the cellular functions associated with CDR formation, including macropinocytosis, receptor internalization, and cell migration. Taken together, our results reveal a novel role for RhoG in the regulation of CDRs and the cellular processes associated with their formation.

RNA granules and cytoskeletal links

2014 ◽  
Vol 42 (4) ◽  
pp. 1206-1210 ◽  
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.

scholarly journals Telomeric DNA Mediates De Novo PML Body Formation

2009 ◽  
Vol 20 (22) ◽  
pp. 4804-4815 ◽  
Author(s):  
Anneke K. Brouwer ◽  
Joost Schimmel ◽  
Joop C.A.G. Wiegant ◽  
Alfred C.O. Vertegaal ◽  
Hans J. Tanke ◽  
...  
Keyword(s):  
De Novo ◽  
Cell Types ◽  
Promyelocytic Leukemia ◽  
Telomeric Dna ◽  
Body Formation ◽  
Cellular Processes ◽  
Sumo Ligase ◽  
Pml Bodies ◽  
Nuclear Processes

The cell nucleus harbors a variety of different bodies that vary in number, composition, and size. Although these bodies coordinate important nuclear processes, little is known about how they are formed. Among the most intensively studied bodies in recent years is the PML body. These bodies have been implicated in gene regulation and other cellular processes and are disrupted in cells from patients suffering from acute promyelocytic leukemia. Using live cell imaging microscopy and immunofluorescence, we show in several cell types that PML bodies are formed at telomeric DNA during interphase. Recent studies revealed that both SUMO modification sites and SUMO interaction motifs in the promyelocytic leukemia (PML) protein are required for PML body formation. We show that SMC5, a component of the SUMO ligase MMS21-containing SMC5/6 complex, localizes temporarily at telomeric DNA during PML body formation, suggesting a possible role for SUMO in the formation of PML bodies at telomeric DNA. Our data identify a novel role of telomeric DNA during PML body formation.

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.

scholarly journals Blocking the Bromodomains Function Contributes to Disturbances in Alga Chara vulgaris Spermatids Differentiation

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1352
Author(s):  
Agnieszka Wojtczak
Keyword(s):  
Endoplasmic Reticulum ◽  
Essential Role ◽  
Developmental Stages ◽  
Chromatin Condensation ◽  
Ultrastructural Level ◽  
Prolonged Treatment ◽  
Chara Vulgaris ◽  
Cellular Processes ◽  
Early Stages

Bromodomain containing (BRD) proteins play an essential role in many cellular processes. The aim of this study was to estimate activity of bromodomains during alga Chara vulgaris spermatids differentiation. The effect of a bromodomain inhibitor, JQ1 (100 μM), on the distribution of individual stages of spermatids and their ultrastructure was studied. The material was Feulgen stained and analysed in an electron microscope. JQ1 caused shortening of the early stages of spermiogenesis and a reverse reaction at the later stages. Additionally, in the same antheridium, spermatids at distant developmental stages were present. On the ultrastructural level, chromatin fibril system disorders and significantly distended endoplasmic reticulum (ER) cisternae already at the early stages were observed. Many autolytic vacuoles were also visible. The ultrastructural disturbances intensified after prolonged treatment with JQ1. The obtained data show that JQ1 treatment led to changes in the spermatid number and disturbances in chromatin condensation and to cytoplasm reduction. The current studies show some similarities between C. vulgaris and mammals spermiogenesis. Taken together, these results suggest that JQ1 interferes with the spermatid differentiation on many interdependent levels and seems to induce ER stress, which leads to spermatid degeneration. Studies on the role of bromodomains in algae spermiogenesis have not been conducted so far.

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.

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