Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

AbstractLong Interspersed Nuclear Element-1 (LINE-1 or L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as mRNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation while some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.Statement of significanceOver half of the human genome is comprised of repetitive sequences. The Long Interspersed Nuclear Element-1 (L1) is an autonomous mobile DNA element that can alter its genomic location, resulting in genomic instability and DNA damage. L1 encodes two proteins that are required for this function: the ORF1 RNA chaperone and the enzymatic ORF2. Here, we demonstrate that ORF1 forms liquid-liquid phase separated states in vitro, which is mediated by electrostatic interactions between the conserved, disordered N-terminus and coiled-coil domain. This work provides a framework to explore how L1 phase separation may enhance the ability of the retrotransposable element to colonize the genome by preventing degradation of the L1 transcript and evasion of host immune responses.

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Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression

10.1101/671230 ◽

2019 ◽

Cited By ~ 4

Author(s):

Tiantian Wu ◽

Yi Lu ◽

Orit Gutman ◽

Huasong Lu ◽

Qiang Zhou ◽

...

Keyword(s):

Gene Expression ◽

Phase Separation ◽

Transcriptional Activation ◽

Target Gene ◽

Coiled Coil ◽

Separation Mechanism ◽

Hippo Signaling ◽

Transcriptional Responses ◽

Coiled Coil Domain

AbstractTAZ promotes cell proliferation, development, and tumorigenesis by regulating target gene transcription. However, how TAZ orchestrates the transcriptional responses remains poorly defined. Here we demonstrate that TAZ forms nuclear condensates via liquid-liquid phase separation to compartmentalize its DNA binding co-factor TEAD4, the transcription co-activators BRD4 and MED1 and the transcription elongation factor CDK9 for activation of gene expression. TAZ, but not its paralog YAP, forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signaling via LATS-mediated phosphorylation and mediated by the coiled-coil domain. Deletion of the TAZ coiled-coil domain or substitution with the YAP coiled-coil domain does not affect the interaction of TAZ with its partners, but prevents its phase separation and more importantly, its ability to induce target gene expression. Thus, our study identifies a novel mechanism for the transcriptional activation by TAZ and demonstrates for the first time that pathway-specific transcription factors also engage the phase separation mechanism for efficient transcription activation.

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Cep57, a multidomain protein with unique microtubule and centrosomal localization domains

Biochemical Journal ◽

10.1042/bj20071501 ◽

2008 ◽

Vol 412 (2) ◽

pp. 265-273 ◽

Cited By ~ 28

Author(s):

Ko Momotani ◽

Alexander S. Khromov ◽

Tsuyoshi Miyake ◽

P. Todd Stukenberg ◽

Avril V. Somlyo

Keyword(s):

Ectopic Expression ◽

Coiled Coil ◽

Full Length ◽

Centrosomal Protein ◽

Multidomain Protein ◽

Coiled Coil Domain ◽

C Terminus ◽

N Terminus

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to γ-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

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Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

Biophysical Journal ◽

10.1016/j.bpj.2021.03.028 ◽

2021 ◽

Author(s):

Jocelyn C. Newton ◽

Mandar T. Naik ◽

Grace Y. Li ◽

Eileen L. Murphy ◽

Nicolas L. Fawzi ◽

...

Keyword(s):

Phase Separation ◽

Coiled Coil ◽

Orf1 Protein ◽

Coiled Coil Domain ◽

N Terminus

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Eukaryotic clamp loaders and unloaders in the maintenance of genome stability

Experimental & Molecular Medicine ◽

10.1038/s12276-020-00533-3 ◽

2020 ◽

Vol 52 (12) ◽

pp. 1948-1958

Author(s):

Kyoo-young Lee ◽

Su Hyung Park

Keyword(s):

Dna Damage ◽

Genomic Instability ◽

Genome Stability ◽

Critical Role ◽

Nuclear Antigen ◽

Checkpoint Activation ◽

Sliding Clamp ◽

Clamp Loaders ◽

Factor C

AbstractEukaryotic sliding clamp proliferating cell nuclear antigen (PCNA) plays a critical role as a processivity factor for DNA polymerases and as a binding and acting platform for many proteins. The ring-shaped PCNA homotrimer and the DNA damage checkpoint clamp 9-1-1 are loaded onto DNA by clamp loaders. PCNA can be loaded by the pentameric replication factor C (RFC) complex and the CTF18-RFC-like complex (RLC) in vitro. In cells, each complex loads PCNA for different purposes; RFC-loaded PCNA is essential for DNA replication, while CTF18-RLC-loaded PCNA participates in cohesion establishment and checkpoint activation. After completing its tasks, PCNA is unloaded by ATAD5 (Elg1 in yeast)-RLC. The 9-1-1 clamp is loaded at DNA damage sites by RAD17 (Rad24 in yeast)-RLC. All five RFC complex components, but none of the three large subunits of RLC, CTF18, ATAD5, or RAD17, are essential for cell survival; however, deficiency of the three RLC proteins leads to genomic instability. In this review, we describe recent findings that contribute to the understanding of the basic roles of the RFC complex and RLCs and how genomic instability due to deficiency of the three RLCs is linked to the molecular and cellular activity of RLC, particularly focusing on ATAD5 (Elg1).

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The Caenorhabditis elegans unc-64 Locus Encodes a Syntaxin That Interacts Genetically with Synaptobrevin

Molecular Biology of the Cell ◽

10.1091/mbc.9.6.1235 ◽

1998 ◽

Vol 9 (6) ◽

pp. 1235-1252 ◽

Cited By ~ 151

Author(s):

Owais Saifee ◽

Liping Wei ◽

Michael L. Nonet

Keyword(s):

Caenorhabditis Elegans ◽

Synaptic Vesicle ◽

Acetylcholinesterase Inhibitor ◽

Coiled Coil ◽

Vesicle Fusion ◽

Loss Of Function ◽

Hydrophobic Membrane ◽

Coiled Coil Domain ◽

Synaptic Vesicle Fusion

We describe the molecular cloning and characterization of theunc-64 locus of Caenorhabditis elegans. unc-64 expresses three transcripts, each encoding a molecule with 63–64% identity to human syntaxin 1A, a membrane- anchored protein involved in synaptic vesicle fusion. Interestingly, the alternative forms of syntaxin differ only in their C-terminal hydrophobic membrane anchors. The forms are differentially expressed in neuronal and secretory tissues; genetic evidence suggests that these forms are not functionally equivalent. A complete loss-of-function mutation in unc-64 results in a worm that completes embryogenesis, but arrests development shortly thereafter as a paralyzed L1 larva, presumably as a consequence of neuronal dysfunction. The severity of the neuronal phenotypes of C. elegans syntaxin mutants appears comparable to those ofDrosophila syntaxin mutants. However, nematode syntaxin appears not to be required for embryonic development, for secretion of cuticle from the hypodermis, or for the function of muscle, in contrast to Drosophila syntaxin, which appears to be required in all cells. Less severe viable unc-64 mutants exhibit a variety of behavioral defects and show strong resistance to the acetylcholinesterase inhibitor aldicarb. Extracellular physiological recordings from pharyngeal muscle of hypomorphic mutants show alterations in the kinetics of transmitter release. The lesions in the hypomorphic alleles map to the hydrophobic face of the H3 coiled-coil domain of syntaxin, a domain that in vitro mediates physical interactions with similar coiled-coil domains in SNAP-25 and synaptobrevin. Furthermore, the unc-64 syntaxin mutants exhibit allele-specific genetic interactions with mutants carrying lesions in the coiled-coil domain of synaptobrevin, providing in vivo evidence for the significance of these domains in regulating synaptic vesicle fusion.

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Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

The Journal of Cell Biology ◽

10.1083/jcb.200206113 ◽

2002 ◽

Vol 159 (6) ◽

pp. 993-1004 ◽

Cited By ~ 126

Author(s):

Christine L. Humphries ◽

Heath I. Balcer ◽

Jessica L. D'Agostino ◽

Barbara Winsor ◽

David G. Drubin ◽

...

Keyword(s):

Binding Protein ◽

Coiled Coil ◽

Actin Binding ◽

Complex Activity ◽

Actin Binding Protein ◽

Coiled Coil Domain ◽

Allele Specific ◽

And Function

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.

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Multimerization and tubulin binding are required for the SPIRAL2 protein to localize to and stabilize microtubule minus ends

10.1101/2021.10.05.463238 ◽

2021 ◽

Author(s):

YUANWEI FAN ◽

Natasha Bilkey ◽

Ram Dixit

Keyword(s):

Coiled Coil ◽

Spatial Arrangement ◽

In Planta ◽

Structural Determinants ◽

Coiled Coil Domain ◽

Tubulin Binding ◽

And Function ◽

Necessary And Sufficient ◽

Basic Region

Accruing evidence points to the control of microtubule minus-end dynamics as being crucial for the spatial arrangement and function of the microtubule cytoskeleton. In plants, the SPIRAL2 (SPR2) protein has emerged as a microtubule minus-end regulator that is structurally distinct from the animal minus-end regulators. Previously, SPR2 was shown to autonomously localize to microtubule minus ends and decrease their depolymerization rate. Here, we used in vitro and in planta experiments to identify the structural determinants required for SPR2 to recognize and stabilize microtubule minus ends. We show that SPR2 contains a single N-terminal TOG domain that binds to soluble tubulin. The TOG domain, a basic region, and coiled-coil domain are necessary and sufficient to target and stabilize microtubule minus ends. We demonstrate that the coiled-coil domain mediates multimerization of SPR2 that provides avidity for microtubule binding and is essential for binding to soluble tubulin. While TOG domain-containing proteins are traditionally thought to function as microtubule plus-end regulators, our results reveal that nature has repurposed the TOG domain of SPR2 to regulate microtubule minus ends.

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Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

Molecular Biology of the Cell ◽

10.1091/mbc.e10-03-0256 ◽

2011 ◽

Vol 22 (2) ◽

pp. 189-201 ◽

Cited By ~ 40

Author(s):

Roman Gorelik ◽

Changsong Yang ◽

Vasumathi Kameswaran ◽

Roberto Dominguez ◽

Tatyana Svitkina

Keyword(s):

Plasma Membrane ◽

Electrostatic Interactions ◽

Coiled Coil ◽

Small Gtpases ◽

Coincidence Detection ◽

Membrane Targeting ◽

Amino Terminal ◽

N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

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Regulation of c-Fes Tyrosine Kinase and Biological Activities by N-Terminal Coiled-Coil Oligomerization Domains

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8335 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8335-8343 ◽

Cited By ~ 26

Author(s):

Haiyun Cheng ◽

Jim A. Rogers ◽

Nancy A. Dunham ◽

Thomas E. Smithgall

Keyword(s):

Tyrosine Kinase ◽

Mammalian Cells ◽

Protein Tyrosine Kinase ◽

Biological Activities ◽

Coiled Coil ◽

Protein Tyrosine ◽

Coiled Coil Domain ◽

Fibroblast Transformation

ABSTRACT The cytoplasmic protein-tyrosine kinase Fes has been implicated in cytokine signal transduction, hematopoiesis, and embryonic development. Previous work from our laboratory has shown that active Fes exists as a large oligomeric complex in vitro. However, when Fes is expressed in mammalian cells, its kinase activity is tightly repressed. The Fes unique N-terminal sequence has two regions with strong homology to coiled-coil-forming domains often found in oligomeric proteins. Here we show that disruption or deletion of the first coiled-coil domain upregulates Fes tyrosine kinase and transforming activities in Rat-2 fibroblasts and enhances Fes differentiation-inducing activity in myeloid leukemia cells. Conversely, expression of a Fes truncation mutant consisting only of the unique N-terminal domain interfered with Rat-2 fibroblast transformation by an activated Fes mutant, suggesting that oligomerization is essential for Fes activation in vivo. Coexpression with the Fes N-terminal region did not affect the transforming activity of v-Src in Rat-2 cells, arguing against a nonspecific suppressive effect. Taken together, these findings suggest a model in which Fes activation may involve coiled-coil-mediated interconversion of monomeric and oligomeric forms of the kinase. Mutation of the first coiled-coil domain may activate Fes by disturbing intramolecular coiled-coil interaction, allowing for oligomerization via the second coiled-coil domain. Deletion of the second coiled-coil domain blocks fibroblast transformation by an activated form of c-Fes, consistent with this model. These results provide the first evidence for regulation of a nonreceptor protein-tyrosine kinase by coiled-coil domains.

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