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Journal of Molecular Biology Mar 2023Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E...
Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.
Topics: Animals; Male; Drosophila melanogaster; Drosophila Proteins; Eukaryotic Initiation Factor-4E; Protein Binding; Protein Biosynthesis; Protein Isoforms; Protein Interaction Domains and Motifs
PubMed: 36638908
DOI: 10.1016/j.jmb.2023.167949 -
Journal of Virology Sep 2021Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation...
Several viruses have been proven to inhibit the formation of RNA processing bodies (P-bodies); however, knowledge regarding whether enterovirus blocks P-body formation remains unclear, and the detailed molecular mechanisms and functions of picornavirus regulation of P-bodies are limited. Here, we show the crucial role of 2A protease in inhibiting P-bodies to promote viral replication during enterovirus 71 infection. Moreover, we found that the activity of 2A protease is essential to inhibit P-body formation, which was proven by the result that infection with EV71-2A, a 2A protease activity-inactivated recombinant virus, failed to block the formation of P-bodies. Furthermore, we show that DDX6, a scaffolding protein of P-bodies, interacted with viral RNA to facilitate viral replication rather than viral translation, by using a luciferase mRNA reporter system and nascent RNA capture assay. Altogether, our data first demonstrate that the 2A protease of enterovirus inhibits P-body formation to facilitate viral RNA synthesis by recruiting the P-body components to viral RNA. Processing bodies (P-bodies) are constitutively present in eukaryotic cells and play an important role in the mRNA cycle, including regulation of gene expression and mRNA degradation. The P-body is the structure that viruses manipulate to facilitate their survival. Here, we show that the 2A protease alone was efficient to block P-body formation during enterovirus 71 infection, and its activity is essential. When the assembly of P-bodies was blocked by 2A protease, DDX6 and 4E-T, which were required for P-body formation, bound to viral RNA to facilitate viral RNA synthesis. We propose a model revealing that EV71 manipulates P-body formation to generate an environment that is conducive to viral replication by facilitating viral RNA synthesis: 2A protease blocked P-body assembly to make it possible for virus to take advantage of P-body components.
Topics: Cell Line, Tumor; Cytoplasmic Granules; DEAD-box RNA Helicases; Enterovirus A, Human; HeLa Cells; Humans; Nucleocytoplasmic Transport Proteins; Peptide Hydrolases; Proto-Oncogene Proteins; RNA, Viral; Ribonucleoproteins; Virus Replication
PubMed: 34287048
DOI: 10.1128/JVI.00922-21 -
Frontiers in Bioscience (Landmark... Jun 2015Extensive research has been carried out in the past two decades to provide insights into the molecular mechanisms by which the Nucleophosmin-Anaplastic Lymphoma Kinase... (Review)
Review
Extensive research has been carried out in the past two decades to provide insights into the molecular mechanisms by which the Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK) exerts its oncogenic effects. These studies led to the concept that NPM-ALK acts at the transcriptional level through the activation of several transcription factors downstream of many different signaling pathways including JAK3/STAT3, PI3K/AKT and RAS/ERK. Nevertheless, the discovery of several RNA-binding proteins (RBPs) within ALK interactome suggested an additional and complementary role of this oncogenic kinase at the post-transcriptional level. This review gives emerging views in ALK-mediated post-transcriptional regulation with a focus on RBPs that are associated with ALK. We will summarize the capacity of NPM-ALK in modulating the biological properties of RBPs and then discuss the role of cytoplasmic aggregates, called AGs for "ALK granules", which are observed in anaplastic large cell lymphoma (ALCL) expressing the ALK kinase. AGs contain polyadenylated mRNAs and numerous RBPs but are distinct from processing bodies (PBs) and stress granules (SGs), two well-known discrete cytoplasmic sites involved in mRNA fate.
Topics: Anaplastic Lymphoma Kinase; Gene Expression Regulation; Models, Genetic; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; Receptor Protein-Tyrosine Kinases; Ribonucleoproteins
PubMed: 25961555
DOI: 10.2741/4369 -
Arabidopsis TAF15b Localizes to RNA Processing Bodies and Contributes to snc1-Mediated Autoimmunity.Molecular Plant-microbe Interactions :... Apr 2016In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the...
In both animals and plants, messenger (m)RNA export has been shown to contribute to immune response regulation. The Arabidopsis nuclear protein MOS11, along with the nucleoporins MOS3/Nup96/SAR3 and Nup160/SAR1 are components of the mRNA export machinery and contribute to immunity mediated by nucleotide binding leucine-rich repeat immune receptors (NLR). The human MOS11 ortholog CIP29 is part of a small protein complex with three additional members: the RNA helicase DDX39, ALY, and TAF15b. We systematically assessed the biological roles of the Arabidopsis homologs of these proteins in toll interleukin 1 receptor-type NLR (TNL)-mediated immunity using reverse genetics. Although mutations in ALY and DDX39 did not result in obvious defects, taf15b mutation partially suppressed the autoimmune phenotypes of a gain-of-function TNL mutant, snc1. An additive effect on snc1 suppression was observed in mos11-1 taf15b snc1 triple mutant plants, suggesting that MOS11 and TAF15b have independent functions. TAF15b-GFP fusion protein, which fully complemented taf15b mutant phenotypes, localized to nuclei similarly to MOS11. However, it was also targeted to cytosolic granules identified as processing bodies. In addition, we observed no change in SNC1 mRNA levels, whereas less SNC1 protein accumulated in taf15b mutant, suggesting that TAF15b contributes to SNC1 homeostasis through posttranscriptional mechanisms. In summary, this study highlights the importance of posttranscriptional RNA processing mediated by TAF15b in the regulation of TNL-mediated immunity.
Topics: Active Transport, Cell Nucleus; Arabidopsis; Arabidopsis Proteins; Biological Transport; Gene Expression Regulation, Plant; Genes, Reporter; Multiprotein Complexes; Mutation; Nuclear Pore Complex Proteins; Phenotype; Plants, Genetically Modified; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Plant; RNA-Binding Proteins; Seedlings
PubMed: 26713351
DOI: 10.1094/MPMI-11-15-0246-R -
PloS One 2015Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are...
Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.
Topics: Cell Line; Cytoplasmic Granules; Endoribonucleases; Gene Expression Regulation; Humans; Interleukin-6; Macrophage Activation; Macrophages; Protein Biosynthesis; Proteins; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Trans-Activators; Tumor Necrosis Factor-alpha
PubMed: 25970328
DOI: 10.1371/journal.pone.0123223 -
Autophagy Jul 2020Classical macroautophagy/autophagy functions to maintain cell health during stressful conditions by targeting cytosolic components for degradation and recycling through...
UNLABELLED
Classical macroautophagy/autophagy functions to maintain cell health during stressful conditions by targeting cytosolic components for degradation and recycling through the lysosomal pathway. In contrast, nondegradative secretory autophagy functions as an alternative autophagy mechanism to mediate extracellular secretion. A recent study published in from the laboratory of Jayanta Debnath has identified components of the LC3-conjugation machinery as mediators in the selection of cargo for nonclassical secretion. Termed LC3-dependent extracellular vesicle loading and secretion (LDELS), this mechanism provides an additional link between secretory autophagy and the release of extracellular vesicles.
ABBREVIATIONS
ATG, autophagy-related; BioID, proximity-dependent biotinylation; CM, conditioned medium; EV, extracellular vesicle; HNRNPK, heterogeneous nuclear ribonuclear protein K; ILVs, intralumenal vesicles; LDELS, LC3-dependent EV loading and secretion; LIR, LC3-interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MS, mass spectrometry; MVBs, multivesicular bodies; ncRNA, non-coding RNA; NSMAF/FAN, neutral sphingomyelinase activation associated factor; P-bodies, processing bodies; PE, phosphatidylethanolamine; RB1CC1/FIP200, RB1 inducible coiled-coil 1; RBP, RNA-binding protein; RNA-seq, RNA sequencing; SAFB, scaffold-attachment factor B; SILAC, stable isotope labeling of amino acids in cell culture; SMPD3/nSMase2, sphingomyelin phosphodiesterase 3; TEM, transmission electron microscopy; TMT, tandem mass tagging.
Topics: Amino Acid Motifs; Animals; Autophagy; Endosomes; Extracellular Vesicles; Humans; Microtubule-Associated Proteins; Mutation; RNA-Binding Proteins; rab5 GTP-Binding Proteins
PubMed: 32401566
DOI: 10.1080/15548627.2020.1760057 -
The Journal of Cell Biology Jun 2024Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress...
Stress granules and P-bodies are ribonucleoprotein (RNP) granules that accumulate during the stress response due to the condensation of untranslating mRNPs. Stress granules form in part by intermolecular RNA-RNA interactions and can be limited by components of the RNA chaperone network, which inhibits RNA-driven aggregation. Herein, we demonstrate that the DEAD-box helicase DDX6, a P-body component, can also limit the formation of stress granules, independent of the formation of P-bodies. In an ATPase, RNA-binding dependent manner, DDX6 limits the partitioning of itself and other RNPs into stress granules. When P-bodies are limited, proteins that normally partition between stress granules and P-bodies show increased accumulation within stress granules. Moreover, we show that loss of DDX6, 4E-T, and DCP1A increases P-body docking with stress granules, which depends on CNOT1 and PAT1B. Taken together, these observations identify a new role for DDX6 in limiting stress granules and demonstrate that P-body components can influence stress granule composition and docking with P-bodies.
Topics: Adenosine Triphosphatases; Processing Bodies; RNA; Stress Granules; Humans; Cell Line, Tumor; DEAD-box RNA Helicases
PubMed: 38536035
DOI: 10.1083/jcb.202306022 -
International Review of Cytology 2002As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to... (Review)
Review
As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleolus; Coiled Bodies; Eukaryotic Cells; Humans; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Small Nucleolar; Ribosomes; Signal Transduction
PubMed: 12211630
DOI: 10.1016/s0074-7696(02)19014-0 -
Nucleic Acids Research Jul 2022Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to...
Processing bodies (P-bodies) are ribonucleoprotein granules that contain mRNAs, RNA-binding proteins and effectors of mRNA turnover. While P-bodies have been reported to contain translationally repressed mRNAs, a causative role for P-bodies in regulating mRNA decay has yet to be established. Enhancer of decapping protein 4 (EDC4) is a core P-body component that interacts with multiple mRNA decay factors, including the mRNA decapping (DCP2) and decay (XRN1) enzymes. EDC4 also associates with the RNA endonuclease MARF1, an interaction that antagonizes the decay of MARF1-targeted mRNAs. How EDC4 interacts with MARF1 and how it represses MARF1 activity is unclear. In this study, we show that human MARF1 and XRN1 interact with EDC4 using analogous conserved short linear motifs in a mutually exclusive manner. While the EDC4-MARF1 interaction is required for EDC4 to inhibit MARF1 activity, our data indicate that the interaction with EDC4 alone is not sufficient. Importantly, we show that P-body architecture plays a critical role in antagonizing MARF1-mediated mRNA decay. Taken together, our study suggests that P-bodies can directly regulate mRNA turnover by sequestering an mRNA decay enzyme and preventing it from interfacing with and degrading targeted mRNAs.
Topics: Cell Cycle Proteins; Endoribonucleases; Exoribonucleases; Humans; Microtubule-Associated Proteins; Proteins; RNA Stability; RNA, Messenger; RNA-Binding Proteins
PubMed: 35801873
DOI: 10.1093/nar/gkac557 -
Trends in Cell Biology Aug 2010The germline originates from primordial embryonic germ cells which give rise to sperm and egg cells and consequently, to the next generation. Germ cells of many... (Review)
Review
The germline originates from primordial embryonic germ cells which give rise to sperm and egg cells and consequently, to the next generation. Germ cells of many organisms contain electron-dense granules that comprise RNA and proteins indispensable for germline development. Here we review recent reports that provide important insights into the structure and function of crucial RNA and protein components of the granules, including DEAD-box helicases, Tudor domain proteins, Piwi/Argonaute proteins and piRNA. Collectively, these components function in translational control, remodeling of ribonucleoprotein complexes and transposon silencing. Furthermore, they interact with each other by means of conserved structural modules and post-translationally modified amino acids. These data suggest a widespread use of several protein motifs in germline development and further our understanding of other ribonucleoprotein structures, for example, processing bodies and neuronal granules.
Topics: Animals; Cytoplasmic Granules; DEAD-box RNA Helicases; Germ Cells; Humans; Membrane Transport Proteins; RNA, Small Interfering; Ribonucleoproteins
PubMed: 20541937
DOI: 10.1016/j.tcb.2010.05.004