Phospho-dependent phase separation of FMRP and CAPRIN1 recapitulates regulation of translation and deadenylation

Science ◽  
2019 ◽  
Vol 365 (6455) ◽  
pp. 825-829 ◽  
Author(s):  
Tae Hun Kim ◽  
Brian Tsang ◽  
Robert M. Vernon ◽  
Nahum Sonenberg ◽  
Lewis E. Kay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Processing ◽  
Intrinsically Disordered Protein ◽  
Resonance Spectroscopy ◽  
Specific Interactions ◽  
Intrinsically Disordered ◽  
Functional Consequences ◽  
Translational Regulators

Membraneless organelles involved in RNA processing are biomolecular condensates assembled by phase separation. Despite the important role of intrinsically disordered protein regions (IDRs), the specific interactions underlying IDR phase separation and its functional consequences remain elusive. To address these questions, we used minimal condensates formed from the C-terminal disordered regions of two interacting translational regulators, FMRP and CAPRIN1. Nuclear magnetic resonance spectroscopy of FMRP-CAPRIN1 condensates revealed interactions involving arginine-rich and aromatic-rich regions. We found that different FMRP serine/threonine and CAPRIN1 tyrosine phosphorylation patterns control phase separation propensity with RNA, including subcompartmentalization, and tune deadenylation and translation rates in vitro. The resulting evidence for residue-specific interactions underlying co–phase separation, phosphorylation-modulated condensate architecture, and enzymatic activity within condensates has implications for how the integration of signaling pathways controls RNA processing and translation.

scholarly journals Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jarrett Smith ◽  
Deepika Calidas ◽  
Helen Schmidt ◽  
Tu Lu ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Intrinsically Disordered Protein ◽  
General Mechanism ◽  
P Granules ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Disordered Protein

RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time.

scholarly journals Characterization of AMBN I and II Isoforms and Study of Their Ca2+-Binding Properties

2020 ◽  
Vol 21 (23) ◽  
pp. 9293
Author(s):  
Veronika Vetyskova ◽  
Monika Zouharova ◽  
Lucie Bednarova ◽  
Ondřej Vaněk ◽  
Petra Sázelová ◽  
...  
Keyword(s):  
Intrinsically Disordered Protein ◽  
Significant Feature ◽  
Amino Acid Residues ◽  
Binding Properties ◽  
Specific Sequence ◽  
Intrinsically Disordered ◽  
Disordered Protein

Ameloblastin (Ambn) as an intrinsically disordered protein (IDP) stands for an important role in the formation of enamel—the hardest biomineralized tissue commonly formed in vertebrates. The human ameloblastin (AMBN) is expressed in two isoforms: full-length isoform I (AMBN ISO I) and isoform II (AMBN ISO II), which is about 15 amino acid residues shorter than AMBN ISO I. The significant feature of AMBN—its oligomerization ability—is enabled due to a specific sequence encoded by exon 5 present at the N-terminal part in both known isoforms. In this study, we characterized AMBN ISO I and AMBN ISO II by biochemical and biophysical methods to determine their common features and differences. We confirmed that both AMBN ISO I and AMBN ISO II form oligomers in in vitro conditions. Due to an important role of AMBN in biomineralization, we further addressed the calcium (Ca2+)-binding properties of AMBN ISO I and ISO II. The binding properties of AMBN to Ca2+ may explain the role of AMBN in biomineralization and more generally in Ca2+ homeostasis processes.

scholarly journals Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

2016 ◽  
Author(s):  
Jarrett Smith ◽  
Deepika Calidas ◽  
Helen Schmidt ◽  
Tu Lu ◽  
Dominique Rasoloson ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Binding ◽  
Intrinsically Disordered Protein ◽  
P Granules ◽  
C Elegans ◽  
Rna Granules ◽  
Intrinsically Disordered ◽  
Disordered Protein

ABSTRACTRNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings support a model whereby MEX-5 functions as an mRNA sink to locally suppress MEG-3 phase separation and drive P granule asymmetry.HIGHLIGHTS- The intrinsically-disordered protein MEG-3 is essential for localized assembly of P granules in C. elegans zygotes.- MEG-3 binds RNA and RNA stimulates MEG-3 phase separation.- The RNA-binding protein MEX-5 inhibits MEG-3 granule assembly in the anterior cytoplasm by sequestering RNA.

scholarly journals Membrane-associated phase separation: organization and function emerge from a two-dimensional milieu

2021 ◽  
Author(s):  
Jonathon A Ditlev
Keyword(s):  
Phase Separation ◽  
Cell Biology ◽  
Cellular Environment ◽  
Condensate Formation ◽  
Associated Proteins ◽  
Available Technology ◽  
And Function ◽  
Surrounding Membrane

Abstract Liquid‒liquid phase separation (LLPS) of biomolecules has emerged as an important mechanism that contributes to cellular organization. Phase separated biomolecular condensates, or membrane-less organelles, are compartments composed of specific biomolecules without a surrounding membrane in the nucleus and cytoplasm. LLPS also occurs at membranes, where both lipids and membrane-associated proteins can de-mix to form phase separated compartments. Investigation of these membrane-associated condensates using in vitro biochemical reconstitution and cell biology has provided key insights into the role of phase separation in membrane domain formation and function. However, these studies have generally been limited by available technology to study LLPS on model membranes and the complex cellular environment that regulates condensate formation, composition, and function. Here, I briefly review our current understanding of membrane-associated condensates, establish why LLPS can be advantageous for certain membrane-associated condensates, and offer a perspective for how these condensates may be studied in the future.

scholarly journals Backbone and nearly complete side-chain chemical shift assignments reveal the human uncharacterized protein CXorf51A as intrinsically disordered

2021 ◽  
Vol 15 (2) ◽  
pp. 441-448
Author(s):  
Christoph Wiedemann ◽  
Kingsley Benjamin Obika ◽  
Sandra Liebscher ◽  
Jan Jirschitzka ◽  
Oliver Ohlenschlãger ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Intrinsically Disordered Protein ◽  
Genome Project ◽  
Side Chain ◽  
Resonance Spectroscopy ◽  
Chemical Shift Assignments ◽  
Uncharacterized Protein ◽  
Protein Coding ◽  
Intrinsically Disordered ◽  
Side Chain Chemical Shift

AbstractEven though the human genome project showed that our DNA contains a mere 20,000 to 25,000 protein coding genes, an unexpectedly large number of these proteins remain functionally uncharacterized. A structural characterization of these “unknown” proteins may help to identify possible cellular tasks. We therefore used a combination of bioinformatics and nuclear magnetic resonance spectroscopy to structurally de-orphanize one of these gene products, the 108 amino acid human uncharacterized protein CXorf51A. Both our bioinformatics analysis as well as the $$^1$$ 1 H, $$^{13}$$ 13 C, $$^{15}$$ 15 N backbone and near-complete side-chain chemical shift assignments indicate that it is an intrinsically disordered protein.

scholarly journals G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

2021 ◽  
Author(s):  
Jun Gao ◽  
Zhaofeng Gao ◽  
Andrea A. Putnam ◽  
Alicia K. Byrd ◽  
Sarah L. Venus ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Intrinsically Disordered ◽  
Dead Box ◽  
G4 Dna ◽  
G Quadruplex ◽  
The Dead ◽  
Liquid Liquid Phase Separation

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

scholarly journals Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

2018 ◽  
Vol 294 (5) ◽  
pp. 1451-1463 ◽  
Author(s):  
Roubina Tatavosian ◽  
Samantha Kent ◽  
Kyle Brown ◽  
Tingting Yao ◽  
Huy Nguyen Duc ◽  
...  
Keyword(s):  
Phase Separation ◽  
Polycomb Group ◽  
Site Directed Mutagenesis ◽  
Developmental Defects ◽  
Heterochromatin Formation ◽  
Intrinsically Disordered ◽  
Polycomb Protein ◽  
Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

scholarly journals Distinct regions of the intrinsically disordered protein MUT-16 mediate assembly of a small RNA amplification complex and promote phase separation of Mutator foci

PLoS Genetics ◽  
2018 ◽  
Vol 14 (7) ◽  
pp. e1007542 ◽  
Author(s):  
Celja J. Uebel ◽  
Dorian C. Anderson ◽  
Lisa M. Mandarino ◽  
Kevin I. Manage ◽  
Stephan Aynaszyan ◽  
...  
Keyword(s):  
Phase Separation ◽  
Small Rna ◽  
Intrinsically Disordered Protein ◽  
Rna Amplification ◽  
Intrinsically Disordered ◽  
Disordered Protein

scholarly journals Nitric oxide interactions with cobalamins: biochemical and functional consequences

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1857-1864 ◽  
Author(s):  
M Brouwer ◽  
W Chamulitrat ◽  
G Ferruzzi ◽  
DL Sauls ◽  
JB Weinberg
Keyword(s):  
Nitric Oxide ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
No Formation ◽  
Iron Nitrosyl ◽  
Wide Range ◽  
Functional Consequences

Abstract Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

scholarly journals Phase transition of RNA−protein complexes into ordered hollow condensates

2020 ◽  
Vol 117 (27) ◽  
pp. 15650-15658 ◽  
Author(s):  
Ibraheem Alshareedah ◽  
Mahdi Muhammad Moosa ◽  
Muralikrishna Raju ◽  
Davit A. Potoyan ◽  
Priya R. Banerjee
Keyword(s):  
Phase Separation ◽  
Protein Complexes ◽  
Intrinsically Disordered Protein ◽  
Liquid Droplets ◽  
Isotropic Liquid ◽  
Size Dependent ◽  
Intrinsically Disordered ◽  
Condensate Phase ◽  
Rna Complexes ◽  
Rna Protein Complexes

Liquid−liquid phase separation of multivalent intrinsically disordered protein−RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA−protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA−protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA−protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein−RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

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