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RNA decay in processing bodies is indispensable for adipogenesis.

ABSTRACT: The RNA decay pathway plays key regulatory roles in cell identities and differentiation processes. Although adipogenesis is transcriptionally and epigenetically regulated and has been thoroughly investigated, how RNA metabolism that contributes to the stability of phenotype-shaping transcriptomes participates in differentiation remains elusive. In this study, we investigated Ddx6, an essential component of processing bodies (PBs) that executes RNA decay and translational repression in the cytoplasm and participates in the cellular transition of reprogramming. Upon adipogenic induction, Ddx6 dynamically accumulated to form PBs with a binding partner, 4E-T, at the early phase prior to emergence of intracellular lipid droplets. In contrast, preadipocytes with Ddx6 knockout (KO) or 4E-T knockdown (KD) failed to generate PBs, resulting in significant suppression of adipogenesis. Transcription factors related to preadipocytes and negative regulators of adipogenesis that were not expressed under adipogenic stimulation were maintained in Ddx6-KO and 4E-T-KD preadipocytes under adipogenic induction. Elimination of Dlk1, a major negative regulator of adipogenesis, in 3T3L1 Ddx6-KO cells did not restore adipogenic differentiation capacity to any extent. Similar to murine cells, human primary mesenchymal stem cells, which can differentiate into adipocytes upon stimulation with adipogenic cocktails, required DDX6 to maturate into adipocytes. Therefore, RNA decay of the entire parental transcriptome, rather than removal of a strong negative regulator, could be indispensable for adipogenesis.

SUBMITTER: Maeda R

PROVIDER: S-EPMC7969960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RNA Decay in Processing Bodies is Indispensable for Adipogenesis

Project description:The RNA decay pathway plays key regulatory roles in cell identities and differentiation processes. Although adipogenesis is transcriptionally and epigenetically regulated and has been thoroughly investigated, how RNA metabolism that contributes to the stability of phenotype-shaping transcriptomes participates in differentiation remains elusive. In this study, we investigated Ddx6, an essential component of processing bodies (PBs) that executes RNA decay and translational repression in the cytoplasm and participates in the cellular transition of reprogramming. Upon adipogenic induction, Ddx6 dynamically accumulated to form PBs with a binding partner, 4E-T, at the early phase prior to emergence of intracellular lipid droplets. In contrast, preadipocytes with Ddx6 knockout (KO) or 4E-T knockdown (KD) failed to generate PBs, resulting in significant suppression of adipogenesis. Transcription factors related to preadipocytes and negative regulators of adipogenesis that were not expressed under adipogenic stimulation were maintained in Ddx6-KO and 4E-T-KD preadipocytes under adipogenic induction. Elimination of Dlk1, a major negative regulator of adipogenesis, in 3T3L1 Ddx6-KO cells did not restore adipogenic differentiation capacity to any extent. Similar to murine cells, human primary mesenchymal stem cells, which can differentiate into adipocytes upon stimulation with adipogenic cocktails, required DDX6 to maturate into adipocytes. Therefore, RNA decay of the entire parental transcriptome, rather than removal of a strong negative regulator, could be indispensable for adipogenesis.

2021-02-10 | GSE166450 | GEO

RNA Decay in Processing Bodies is Indispensable for Adipogenesis

Project description:RNA Decay in Processing Bodies is Indispensable for Adipogenesis

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Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.

Project description:A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.

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Quantitative reconstitution of yeast RNA processing bodies.

Project description:Many biomolecular condensates appear to form through liquid-liquid phase separation (LLPS). Individual condensate components can often undergo LLPS in vitro, capturing some features of the native structures. However, natural condensates contain dozens of components with different concentrations, dynamics, and contributions to compartment formation. Most biochemical reconstitutions of condensates have not benefited from quantitative knowledge of these cellular features nor attempted to capture natural complexity. Here, we build on prior quantitative cellular studies to reconstitute yeast RNA processing bodies (P bodies) from purified components. Individually, five of the seven highly concentrated P-body proteins form homotypic condensates at cellular protein and salt concentrations, using both structured domains and intrinsically disordered regions. Combining the seven proteins together at their cellular concentrations with RNA yields phase-separated droplets with partition coefficients and dynamics of most proteins in reasonable agreement with cellular values. RNA delays the maturation of proteins within and promotes the reversibility of, P bodies. Our ability to quantitatively recapitulate the composition and dynamics of a condensate from its most concentrated components suggests that simple interactions between these components carry much of the information that defines the physical properties of the cellular structure.

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“Turbo–RNA Immunoprecipitation” (T–RIP) of Processing Bodies in Arabidopsis

Project description: Not available

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Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes.

Project description:Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5' to 3' exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.

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Dynamic Recruitment of Single RNAs to Processing Bodies Depends on RNA Functionality.

Project description:Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.

| S-EPMC6499680 | biostudies-literature

Human coronaviruses disassemble processing bodies.

Project description:A dysregulated proinflammatory cytokine response is characteristic of severe coronavirus infections caused by SARS-CoV-2, yet our understanding of the underlying mechanism responsible for this imbalanced immune response remains incomplete. Processing bodies (PBs) are cytoplasmic membraneless ribonucleoprotein granules that control innate immune responses by mediating the constitutive decay or suppression of mRNA transcripts, including many that encode proinflammatory cytokines. PB formation promotes turnover or suppression of cytokine RNAs, whereas PB disassembly corresponds with the increased stability and/or translation of these cytokine RNAs. Many viruses cause PB disassembly, an event that can be viewed as a switch that rapidly relieves cytokine RNA repression and permits the infected cell to respond to viral infection. Prior to this submission, no information was known about how human coronaviruses (CoVs) impacted PBs. Here, we show SARS-CoV-2 and the common cold CoVs, OC43 and 229E, induced PB loss. We screened a SARS-CoV-2 gene library and identified that expression of the viral nucleocapsid (N) protein from SARS-CoV-2 was sufficient to mediate PB disassembly. RNA fluorescent in situ hybridization revealed that transcripts encoding TNF and IL-6 localized to PBs in control cells. PB loss correlated with the increased cytoplasmic localization of these transcripts in SARS-CoV-2 N protein-expressing cells. Ectopic expression of the N proteins from five other human coronaviruses (OC43, MERS, 229E, NL63 and SARS-CoV) did not cause significant PB disassembly, suggesting that this feature is unique to SARS-CoV-2 N protein. These data suggest that SARS-CoV-2-mediated PB disassembly contributes to the dysregulation of proinflammatory cytokine production observed during severe SARS-CoV-2 infection.

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Project description:Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.

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BR-Bodies Provide Selectively Permeable Condensates that Stimulate mRNA Decay and Prevent Release of Decay Intermediates.

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