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23 pages, 9962 KiB

Open AccessArticle

The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells

by Ella Alkalay, Chen Gam Ze Letova Refael, Irit Shoval, Noa Kinor, Ronit Sarid and Yaron Shav-Tal

Cells 2020, 9(9), 1958; https://doi.org/10.3390/cells9091958 - 25 Aug 2020

Cited by 4 | Viewed by 4452

Abstract

RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to [...] Read more.

RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi’s sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles. Since splicing is required for the maturation of certain KSHV transcripts, we suggest that the infected cell does not dismantle nuclear speckles but rearranges their components at the nuclear periphery to possibly serve in splicing and transport of viral RNAs into the cytoplasm. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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21 pages, 3988 KiB

Open AccessArticle

Addressing the Molecular Mechanism of Longitudinal Lamin Assembly Using Chimeric Fusions

by Giel Stalmans, Anastasia V. Lilina, Pieter-Jan Vermeire, Jan Fiala, Petr Novák and Sergei V. Strelkov

Cells 2020, 9(7), 1633; https://doi.org/10.3390/cells9071633 - 7 Jul 2020

Cited by 11 | Viewed by 3526

Abstract

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the [...] Read more.

The molecular architecture and assembly mechanism of intermediate filaments have been enigmatic for decades. Among those, lamin filaments are of particular interest due to their universal role in cell nucleus and numerous disease-related mutations. Filament assembly is driven by specific interactions of the elementary dimers, which consist of the central coiled-coil rod domain flanked by non-helical head and tail domains. We aimed to investigate the longitudinal ‘head-to-tail’ interaction of lamin dimers (the so-called A_CN interaction), which is crucial for filament assembly. To this end, we prepared a series of recombinant fragments of human lamin A centred around the N- and C-termini of the rod. The fragments were stabilized by fusions to heterologous capping motifs which provide for a correct formation of parallel, in-register coiled-coil dimers. As a result, we established crystal structures of two N-terminal fragments one of which highlights the propensity of the coiled-coil to open up, and one C-terminal rod fragment. Additional studies highlighted the capacity of such N- and C-terminal fragments to form specific complexes in solution, which were further characterized using chemical cross-linking. These data yielded a molecular model of the A_CN complex which features a 6.5 nm overlap of the rod ends. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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17 pages, 7759 KiB

Open AccessArticle

Super-Resolution Localisation of Nuclear PI(4)P and Identification of Its Interacting Proteome

by Veronika Fáberová, Ilona Kalasová, Alžběta Krausová and Pavel Hozák

Cells 2020, 9(5), 1191; https://doi.org/10.3390/cells9051191 - 11 May 2020

Cited by 20 | Viewed by 4408

Abstract

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do [...] Read more.

Phosphoinositides are glycerol-based phospholipids, and they play essential roles in cellular signalling, membrane and cytoskeletal dynamics, cell movement, and the modulation of ion channels and transporters. Phosphoinositides are also associated with fundamental nuclear processes through their nuclear protein-binding partners, even though membranes do not exist inside of the nucleus. Phosphatidylinositol 4-phosphate (PI(4)P) is one of the most abundant cellular phosphoinositides; however, its functions in the nucleus are still poorly understood. In this study, we describe PI(4)P localisation in the cell nucleus by super-resolution light and electron microscopy, and employ immunoprecipitation with a specific anti-PI(4)P antibody and subsequent mass spectrometry analysis to determine PI(4)P’s interaction partners. We show that PI(4)P is present at the nuclear envelope, in nuclear lamina, in nuclear speckles and in nucleoli and also forms multiple small foci in the nucleoplasm. Nuclear PI(4)P undergoes re-localisation to the cytoplasm during cell division; it does not localise to chromosomes, nucleolar organising regions or mitotic interchromatin granules. When PI(4)P and PI(4,5)P2 are compared, they have different nuclear localisations during interphase and mitosis, pointing to their functional differences in the cell nucleus. Mass spectrometry identified hundreds of proteins, including 12 potentially novel PI(4)P interactors, most of them functioning in vital nuclear processes such as pre-mRNA splicing, transcription or nuclear transport, thus extending the current knowledge of PI(4)P’s interaction partners. Based on these data, we propose that PI(4)P also plays a role in essential nuclear processes as a part of protein–lipid complexes. Altogether, these observations provide a novel insight into the role of PI(4)P in nuclear functions and provide a direction for further investigation. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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22 pages, 10255 KiB

Open AccessArticle

Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids

by Francisco Guillen-Chable, Ulises Rodríguez Corona, Alejandro Pereira-Santana, Andrea Bayona, Luis Carlos Rodríguez-Zapata, Cecilia Aquino, Lenka Šebestová, Nicolas Vitale, Pavel Hozak and Enrique Castano

Cells 2020, 9(5), 1143; https://doi.org/10.3390/cells9051143 - 6 May 2020

Cited by 16 | Viewed by 5689

Abstract

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear [...] Read more.

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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14 pages, 2595 KiB

Open AccessArticle

The Actin-Family Protein Arp4 Is a Novel Suppressor for the Formation and Functions of Nuclear F-Actin

by Shota Yamazaki, Christian Gerhold, Koji Yamamoto, Yuya Ueno, Robert Grosse, Kei Miyamoto and Masahiko Harata

Cells 2020, 9(3), 758; https://doi.org/10.3390/cells9030758 - 19 Mar 2020

Cited by 13 | Viewed by 5225

Abstract

The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation [...] Read more.

The crosstalk between actin and actin-related proteins (Arps), namely Arp2 and Arp3, plays a central role in facilitating actin polymerization in the cytoplasm and also in the nucleus. Nuclear F-actin is required for transcriptional regulation, double-strand break repair, and nuclear organization. The formation of nuclear F-actin is highly dynamic, suggesting the involvement of positive and negative regulators for nuclear actin polymerization. While actin assembly factors for nuclear F-actin have been recently described, information about inhibitory factors is still limited. The actin-related protein Arp4 which is predominantly localized in the nucleus, has been previously identified as an integral subunit of multiple chromatin modulation complexes, where it forms a heterodimer with monomeric actin. Therefore, we tested whether Arp4 functions as a suppressor of nuclear F-actin formation. The knockdown of Arp4 (Arp4 KD) led to an increase in nuclear F-actin formation in NIH3T3 cells, and purified Arp4 potently inhibited F-actin formation in mouse nuclei transplanted into Xenopus laevis oocytes. Consistently, Arp4 KD facilitated F-actin-inducible gene expression (e.g., OCT4) and DNA damage repair. Our results suggest that Arp4 has a critical role in the formation and functions of nuclear F-actin. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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Review

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31 pages, 1918 KiB

Open AccessReview

MeCP2 and Chromatin Compartmentalization

by Annika Schmidt, Hui Zhang and M. Cristina Cardoso

Cells 2020, 9(4), 878; https://doi.org/10.3390/cells9040878 - 3 Apr 2020

Cited by 32 | Viewed by 5908

Abstract

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional epigenetic reader playing a role in transcriptional regulation and chromatin structure, which was linked to Rett syndrome in humans. Here, we focus on its isoforms and functional domains, interactions, modifications and mutations found in Rett [...] Read more.

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional epigenetic reader playing a role in transcriptional regulation and chromatin structure, which was linked to Rett syndrome in humans. Here, we focus on its isoforms and functional domains, interactions, modifications and mutations found in Rett patients. Finally, we address how these properties regulate and mediate the ability of MeCP2 to orchestrate chromatin compartmentalization and higher order genome architecture. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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16 pages, 1037 KiB

Open AccessReview

Role of the Nuclear Lamina in Age-Associated Nuclear Reorganization and Inflammation

by Lidya Kristiani, Miri Kim and Youngjo Kim

Cells 2020, 9(3), 718; https://doi.org/10.3390/cells9030718 - 14 Mar 2020

Cited by 22 | Viewed by 4449

Abstract

Aging is characterized by the gradual loss of tissue function and integrity. Activation of inflammatory responses accelerates the deterioration of cells and tissues. Many studies have shown that alteration of the components of the nuclear lamina is associated with inflammation, both in vivo [...] Read more.

Aging is characterized by the gradual loss of tissue function and integrity. Activation of inflammatory responses accelerates the deterioration of cells and tissues. Many studies have shown that alteration of the components of the nuclear lamina is associated with inflammation, both in vivo and in vitro. However, the mechanism by which the nuclear lamina regulates inflammation is largely unknown. Recent studies have suggested that the nuclear lamina regulates both organization of the three-dimensional chromatin structure at the nuclear periphery and global gene expression, such as the expression of inflammatory response genes. Here, we discuss the current updates in the research on nuclear lamina alteration, activation of inflammation, and nuclear reorganization in models of cellular senescence and organismal aging. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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15 pages, 2382 KiB

Open AccessReview

Nuclear Inositides and Inositide-Dependent Signaling Pathways in Myelodysplastic Syndromes

by Jie Xian, Eric Owusu Obeng, Stefano Ratti, Isabella Rusciano, Maria Vittoria Marvi, Antonietta Fazio, Alessia De Stefano, Sara Mongiorgi, Alessandra Cappellini, Giulia Ramazzotti, Lucia Manzoli, Lucio Cocco and Matilde Yung Follo

Cells 2020, 9(3), 697; https://doi.org/10.3390/cells9030697 - 12 Mar 2020

Cited by 11 | Viewed by 4612

Abstract

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by peripheral blood cytopenia and abnormal myeloproliferation, as well as a variable risk of evolution into acute myeloid leukemia (AML). The nucleus is a highly organized organelle with several distinct domains where [...] Read more.

Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by peripheral blood cytopenia and abnormal myeloproliferation, as well as a variable risk of evolution into acute myeloid leukemia (AML). The nucleus is a highly organized organelle with several distinct domains where nuclear inositides localize to mediate essential cellular events. Nuclear inositides play a critical role in the modulation of erythropoiesis or myelopoiesis. Here, we briefly review the nuclear structure, the localization of inositides and their metabolic enzymes in subnuclear compartments, and the molecular aspects of nuclear inositides in MDS. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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19 pages, 2612 KiB

Open AccessFeature PaperReview

Nuclear Phosphoinositides—Versatile Regulators of Genome Functions

by Enrique Castano, Sukriye Yildirim, Veronika Fáberová, Alžběta Krausová, Lívia Uličná, Darina Paprčková, Martin Sztacho and Pavel Hozák

Cells 2019, 8(7), 649; https://doi.org/10.3390/cells8070649 - 28 Jun 2019

Cited by 26 | Viewed by 4964

Abstract

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in [...] Read more.

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), modulate nuclear processes. We focus on PI(4,5)P2’s role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets—recently described nucleoplasmic PI(4,5)P2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide–protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies. Full article

(This article belongs to the Special Issue Nuclear Architecture, Lipids, and Phase Separation)

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