scholarly journals Quantifying mRNA targeting to P-bodies in living human cells reveals their dual role in mRNA decay and storage

2014 ◽  
Vol 127 (20) ◽  
pp. 4443-4456 ◽  
Author(s):  
Adva Aizer ◽  
Alon Kalo ◽  
Pinhas Kafri ◽  
Amit Shraga ◽  
Rakefet Ben-Yishay ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Dual Role ◽  
Human Cells ◽  
P Bodies ◽  
And Storage ◽  
Mrna Targeting

scholarly journals Direct and Indirect Effects on Viral Translation and RNA Replication Are Required for AUF1 Restriction of Enterovirus Infections in Human Cells

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Wendy Ullmer ◽  
Bert L. Semler
Keyword(s):  
Mrna Decay ◽  
Coxsackievirus B3 ◽  
Human Cells ◽  
Human Rhinovirus ◽  
Host Cells ◽  
Paralytic Poliomyelitis ◽  
Common Mechanism ◽  
Restriction Factor ◽  
Nucleocytoplasmic Trafficking ◽  
Viral Translation

ABSTRACTThe cellular mRNA decay protein AUF1 acts as a restriction factor during infection by picornaviruses, including poliovirus, coxsackievirus, and human rhinovirus. AUF1 relocalizes from the nucleus to the cytoplasm during infection by these viruses due to the disruption of nucleocytoplasmic trafficking by viral proteinases. Previous studies have demonstrated that AUF1 binds to poliovirus and coxsackievirus B3 (CVB3) RNA during infection, with binding shown to occur within the internal ribosome entry site (IRES) of the 5′ noncoding region (NCR) or the 3′ NCR, respectively. Binding to different sites within the viral RNA suggests that AUF1 may negatively regulate infection by these viruses using different mechanisms. The work presented here addresses the mechanism of AUF1 inhibition of the replication of poliovirus and CVB3. We demonstrate that AUF1 knockdown in human cells results in increased viral translation, RNA synthesis, and virus production. AUF1 is shown to negatively regulate translation of a poliovirus and CVB3 IRES reporter RNA during infection but not in uninfected cells. We found that this inhibitory activity is not mediated through destabilization of viral genomic RNA; however, it does require virus-induced relocalization of AUF1 from the nucleus to the cytoplasm during the early phases of infection. Our findings suggest that AUF1 restriction of poliovirus and CVB3 replication uses a common mechanism through the viral IRES, which is distinct from the canonical role that AUF1 plays in regulated mRNA decay in uninfected host cells.IMPORTANCEPicornaviruses primarily infect the gastrointestinal or upper respiratory tracts of humans and animals and may disseminate to tissues of the central nervous system, heart, skin, liver, or pancreas. Many common human pathogens belong to thePicornaviridaefamily, which includes viruses known to cause paralytic poliomyelitis (poliovirus); myocarditis (coxsackievirus B3 [CVB3]); the common cold (human rhinovirus [HRV]); and hand, foot, and mouth disease (enterovirus 71 [EV71]), among other illnesses. There are no specific treatments for infection, and vaccines exist for only two picornaviruses: poliovirus and hepatitis A virus. Given the worldwide distribution and prevalence of picornaviruses, it is important to gain insight into the host mechanisms used to restrict infection. Other than proteins involved in the innate immune response, few host factors have been identified that restrict picornavirus replication. The work presented here seeks to define the mechanism of action for the host restriction factor AUF1 during infection by poliovirus and CVB3.

scholarly journals Cytoplasmic foci are sites of mRNA decay in human cells

2004 ◽  
Vol 165 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Nicolas Cougot ◽  
Sylvie Babajko ◽  
Bertrand Séraphin
Keyword(s):  
Mrna Decay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cells ◽  
Eukaryotic Cells ◽  
Mrna Turnover ◽  
Expression Control ◽  
Gene Expression Control ◽  
Cytoplasmic Structures

Understanding gene expression control requires defining the molecular and cellular basis of mRNA turnover. We have previously shown that the human decapping factors hDcp2 and hDcp1a are concentrated in specific cytoplasmic structures. Here, we show that hCcr4, hDcp1b, hLsm, and rck/p54 proteins related to 5′–3′ mRNA decay also localize to these structures, whereas DcpS, which is involved in cap nucleotide catabolism, is nuclear. Functional analysis using fluorescence resonance energy transfer revealed that hDcp1a and hDcp2 interact in vivo in these structures that were shown to differ from the previously described stress granules. Our data indicate that these new structures are dynamic, as they disappear when mRNA breakdown is abolished by treatment with inhibitors. Accumulation of poly(A)+ RNA in these structures, after RNAi-mediated inactivation of the Xrn1 exonuclease, demonstrates that they represent active mRNA decay sites. The occurrence of 5′–3′ mRNA decay in specific subcellular locations in human cells suggests that the cytoplasm of eukaryotic cells may be more organized than previously anticipated.

On track with P-bodies

2010 ◽  
Vol 38 (1) ◽  
pp. 242-251 ◽  
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.

scholarly journals dTIS11 Protein-dependent Polysomal Deadenylation Is the Key Step in AU-rich Element-mediated mRNA Decay in Drosophila Cells

2012 ◽  
Vol 287 (42) ◽  
pp. 35527-35538 ◽  
Author(s):  
Caroline Vindry ◽  
Aurélien Lauwers ◽  
David Hutin ◽  
Romuald Soin ◽  
Corinne Wauquier ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Molecular Mechanism ◽  
Mrna Decay ◽  
Degradation Process ◽  
Mrna Degradation ◽  
Decay Process ◽  
Human Protein ◽  
P Bodies ◽  
Drosophila Cells ◽  
Degradation Intermediates

The destabilization of AU-rich element (ARE)-containing mRNAs mediated by proteins of the TIS11 family is conserved among eukaryotes including Drosophila. Previous studies have demonstrated that Tristetraprolin, a human protein of the TIS11 family, induces the degradation of ARE-containing mRNAs through a large variety of mechanisms including deadenylation, decapping, and P-body targeting. We have previously shown that the degradation of the mRNA encoding the antimicrobial peptide Cecropin A1 (CecA1) is controlled by the TIS11 protein (dTIS11) in Drosophila cells. In this study, we used CecA1 mRNA as a model to investigate the molecular mechanism of dTIS11-mediated mRNA decay. We observed that during the biphasic deadenylation and decay process of this mRNA, dTIS11 enhances deadenylation performed by the CCR4-CAF-NOT complex while the mRNA is still associated with ribosomes. Sequencing of mRNA degradation intermediates revealed that the complete deadenylation of the mRNA triggers its decapping and decay in both the 5′-3′ and the 3′-5′ directions. Contrary to the observations made for its mammalian homologs, overexpression of dTIS11 does not promote the localization of ARE-containing mRNAs in P-bodies but rather decreases the accumulation of CecA1 mRNA in these structures by enhancing the degradation process. Therefore, our results suggest that proteins of the TIS11 family may have acquired additional functions in the course of evolution from invertebrates to mammals.

scholarly journals The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms

2007 ◽  
Vol 178 (7) ◽  
pp. 1133-1143 ◽  
Author(s):  
Rona Lotan ◽  
Vicky Goler-Baron ◽  
Lea Duek ◽  
Gal Haimovich ◽  
Mordechai Choder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Component ◽  
Mrna Decay ◽  
State Level ◽  
Mrna Levels ◽  
Exonuclease Activity ◽  
Steady State Level ◽  
Pol Ii ◽  
P Bodies

The steady-state level of mRNAs is determined by the balance between their synthesis by RNA polymerase II (Pol II) and their decay. In the cytoplasm, mRNAs are degraded by two major pathways; one requires decapping and 5′ to 3′ exonuclease activity and the other involves 3′ to 5′ degradation. Rpb7p is a Pol II subunit that shuttles between the nucleus and the cytoplasm. Here, we show that Rpb7p is involved in the two mRNA decay pathways and possibly couples them. Rpb7p stimulates the deadenylation stage required for execution of both pathways. Additionally, Rpb7p is both an active component of the P bodies, where decapping and 5′ to 3′ degradation occur, and is capable of affecting the P bodies function. Moreover, Rpb7p interacts with the decapping regulator Pat1p in a manner important for the mRNA decay machinery. Rpb7p is also involved in the second pathway, as it stimulates 3′ to 5′ degradation. Our genetic analyses suggest that Rpb7p plays two distinct roles in mRNA decay, which can both be uncoupled from Rpb7p's role in transcription. Thus, Rpb7p plays pivotal roles in determining mRNA levels.

scholarly journals A non-canonical role for the EDC4 decapping factor in regulating MARF1-mediated mRNA decay

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
William R Brothers ◽  
Steven Hebert ◽  
Claudia L Kleinman ◽  
Marc R Fabian
Keyword(s):  
Essential Role ◽  
Mrna Decay ◽  
Rna Binding ◽  
Mrna Turnover ◽  
Core Component ◽  
P Bodies ◽  
Target Mrnas ◽  
Binding Domains ◽  
Bona Fide ◽  
Decapping Enzyme

EDC4 is a core component of processing (P)-bodies that binds the DCP2 decapping enzyme and stimulates mRNA decay. EDC4 also interacts with mammalian MARF1, a recently identified endoribonuclease that promotes oogenesis and contains a number of RNA binding domains, including two RRMs and multiple LOTUS domains. How EDC4 regulates MARF1 action and the identity of MARF1 target mRNAs is not known. Our transcriptome-wide analysis identifies bona fide MARF1 target mRNAs and indicates that MARF1 predominantly binds their 3’ UTRs via its LOTUS domains to promote their decay. We also show that a MARF1 RRM plays an essential role in enhancing its endonuclease activity. Importantly, we establish that EDC4 impairs MARF1 activity by preventing its LOTUS domains from binding target mRNAs. Thus, EDC4 not only serves as an enhancer of mRNA turnover that binds DCP2, but also as a repressor that binds MARF1 to prevent the decay of MARF1 target mRNAs.

scholarly journals UPF3A and UPF3B are redundant and modular activators of nonsense-mediated mRNA decay in human cells

2021 ◽  
Author(s):  
Damaris Wallmeroth ◽  
Volker Boehm ◽  
Jan-Wilm Lackmann ◽  
Janine Altmueller ◽  
Christoph Dieterich ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Mrna Decay ◽  
Fault Tolerant ◽  
Human Cells ◽  
Interaction Sites ◽  
Exon Junction ◽  
Human Proteins ◽  
Nonsense Mediated Mrna Decay ◽  
Experimental Approaches ◽  
Redundant Functions

The paralogous human proteins UPF3A and UPF3B are involved in recognizing mRNAs targeted by nonsense-mediated mRNA decay (NMD). While UPF3B has been demonstrated to support NMD, contradicting reports describe UPF3A either as an NMD activator or inhibitor. Here, we present a comprehensive functional analysis of UPF3A and UPF3B in human cells using combinatory experimental approaches. Overexpression or knockout of UPF3A as well as knockout of UPF3B did not detectably change global NMD activity. In contrast, the co-depletion of UPF3A and UPF3B resulted in a marked NMD inhibition and a transcriptome-wide upregulation of NMD substrates, demonstrating a functional redundancy between both NMD factors. Although current models assume that UPF3 bridges NMD-activating exon-junction complexes (EJC) to the NMD factor UPF2, UPF3B exhibited normal NMD activity in rescue experiments when UPF2 or EJC binding was impaired. Further rescue experiments revealed partially redundant functions of UPF3B domains in supporting NMD, involving both UPF2 and EJC interaction sites and the central region of UPF3. Collectively, UPF3A and UPF3B serve as fault-tolerant NMD activators in human cells.

Endoplasmic reticulum contact sites regulate the dynamics of membraneless organelles

Science ◽  
2020 ◽  
Vol 367 (6477) ◽  
pp. eaay7108 ◽  
Author(s):  
Jason E. Lee ◽  
Peter I. Cathey ◽  
Haoxi Wu ◽  
Roy Parker ◽  
Gia K. Voeltz
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Granules ◽  
Human Cells ◽  
Stress Granule ◽  
Processing Bodies ◽  
P Bodies ◽  
Contact Sites ◽  
Membrane Bound ◽  
Efficient Transfer ◽  
Er Membrane

Tethered interactions between the endoplasmic reticulum (ER) and other membrane-bound organelles allow for efficient transfer of ions and/or macromolecules and provide a platform for organelle fission. Here, we describe an unconventional interface between membraneless ribonucleoprotein granules, such as processing bodies (P-bodies, or PBs) and stress granules, and the ER membrane. We found that PBs are tethered at molecular distances to the ER in human cells in a tunable fashion. ER-PB contact and PB biogenesis were modulated by altering PB composition, ER shape, or ER translational capacity. Furthermore, ER contact sites defined the position where PB and stress granule fission occurs. We thus suggest that the ER plays a fundamental role in regulating the assembly and disassembly of membraneless organelles.

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

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.

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.

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