Nuclear Compartments: An Incomplete Primer to Nuclear Compartments, Bodies, and Genome Organization Relative to Nuclear Architecture

Cold Spring Harbor Perspectives in Biology, Год журнала: 2021, Номер 14(7), С. a041268 - a041268

Опубликована: Авг. 16, 2021

This work reviews nuclear compartments, defined broadly to include distinct structures, bodies, and chromosome domains. It first summarizes original cytological observations before comparing concepts of compartments emerging from microscopy versus genomic approaches then introducing new multiplexed imaging that promise in the future meld both approaches. I discuss how previous models radial distribution chromosomes or binary division genome into A B are now being refined by recognition more complex compartmentalization. The poorly understood question these established maintained is discussed, including through modern perspective phase separation, moving on address possible functions using role speckles modulating gene expression as an example. Finally, review concludes with a discussion questions for this field.

Язык: Английский

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Nuclear speckles: dynamic hubs of gene expression regulation

FEBS Journal, Год журнала: 2021, Номер 289(22), С. 7234 - 7245

Опубликована: Июль 10, 2021

Complex, multistep biochemical reactions that routinely take place in our cells require high concentrations of enzymes, substrates, and other structural components to proceed efficiently typically chemical environments can inhibit their immediate vicinity. Eukaryotic solve these problems by restricting such into diffusion‐restricted compartments within the cell called organelles be separated from environment a lipid membrane, or membrane‐less form through liquid–liquid phase separation (LLPS). One most easily noticeable earliest discovered organelle is nucleus, which harbors genetic material where transcription RNA polymerases produces messenger RNAs plethora noncoding RNAs, turn are required for translation mRNAs cytoplasm. The interior nucleus not uniform soup biomolecules rather consists variety bodies, as nucleolus, nuclear speckles (NS), paraspeckles, Cajal histone locus more. In this review, we will focus on NS with an emphasis recent developments including own findings about formation two large IDR‐rich proteins SON SRRM2.

Язык: Английский

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Merging Established Mechanisms with New Insights: Condensates, Hubs, and the Regulation of RNA Polymerase II Transcription

Journal of Molecular Biology, Год журнала: 2021, Номер 434(1), С. 167216 - 167216

Опубликована: Авг. 30, 2021

The regulation of RNA polymerase II (pol II) transcription requires a complex and context-specific array proteins protein complexes, as well nucleic acids metabolites. Every major physiological process coordinated specific sets genes at the appropriate time, breakdown in this is hallmark human disease. A proliferation recent studies has revealed that many general components, including sequence-specific, DNA-binding factors, Mediator, pol itself, are capable liquid–liquid phase separation, to form condensates partition these factors away from bulk aqueous phase. These findings hold great promise for next-level understanding transcription; however, mechanistic aspects align with more conventional models, whether separation per se regulates activity cells remains controversial. In review, we describe condensate-dependent why their similarities differences important. We also compare contrast models context genome organization (initiation, elongation, termination), highlight central role processes. Finally, discuss mutations disrupt normal partitioning how may contribute

Язык: Английский

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SRRM2 organizes splicing condensates to regulate alternative splicing

Nucleic Acids Research, Год журнала: 2022, Номер 50(15), С. 8599 - 8614

Опубликована: Авг. 5, 2022

Abstract SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that forms biomolecular condensates satisfying most hallmarks of liquid-liquid phase separation, including spherical shape, dynamic rearrangement, coalescence and concentration dependence supported by in vitro experiments. Live-cell imaging shows organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present spliceosome structures, deficiency induces skipping cassette exons short introns weak splice sites, tending to change large protein domains. In THP-1 myeloid-like depletion compromises viability, upregulates differentiation markers, sensitizes cells anti-leukemia drugs. FES isoform attenuates innate inflammatory responses, MUC1 isoforms undergo shedding oncogenic properties. We conclude acts as scaffold organize speckles, regulating alternative immunity homeostasis.

Язык: Английский

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Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns

Molecular Cell, Год журнала: 2022, Номер 82(5), С. 1035 - 1052.e9

Опубликована: Фев. 18, 2022

Язык: Английский

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Nuclear speckles – a driving force in gene expression

Journal of Cell Science, Год журнала: 2022, Номер 135(13)

Опубликована: Июль 1, 2022

Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which splicing factors, that together display complex biophysical properties dictating nuclear speckle formation maintenance. Although these were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions modulation gene activity. Major advancements mapping techniques combined with microscopy approaches have enabled insights into roles may play enhancing expression, how positioning specific landmarks can regulate expression RNA processing. Some studies drawn a link between disease. Certain maladies either involve directly or dictate localization reorganization factors. This is most striking during viral infection, as viruses alter entire architecture highjack host machinery. As discussed this Review, represent fascinating target study not reveal links positioning, genome subcompartments activity, but also potential for therapeutics.

Язык: Английский

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Polyphasic linkage and the impact of ligand binding on the regulation of biomolecular condensates

Biophysics Reviews, Год журнала: 2021, Номер 2(2)

Опубликована: Июнь 1, 2021

Cellular matter can be spatially and temporally organized into membraneless biomolecular condensates. The current thinking is that these condensates form dissolve via phase transitions driven by one or more condensate-specific multivalent macromolecules known as scaffolds. Cells likely regulate condensate formation dissolution exerting control over the concentrations of regulatory molecules, which we refer to ligands. Wyman Gill introduced framework polyphasic linkage explain how ligands exert thermodynamic transitions. This review focuses on describing concepts relevance such a mechanism for controlling dissolution. We describe ligand-mediated scaffold behavior quantified experimentally. Further, build recent studies highlight features make them suppressors vs drivers separation. Finally, areas where advances are needed further understand in complex cellular environments. These include understanding effects networks modulate controlled different combinations homotypic heterotypic interactions among macromolecules. Insights gained from application should useful designing novel pharmaceutical

Язык: Английский

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RNA is required for the integrity of multiple nuclear and cytoplasmic membrane‐less RNP granules

The EMBO Journal, Год журнала: 2022, Номер 41(9)

Опубликована: Март 31, 2022

Article31 March 2022Open Access Source DataTransparent process RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules Carolyn J Decker orcid.org/0000-0002-2672-8919 Department Biochemistry, University Colorado Boulder, CO, USA Howard Hughes Medical Institute, Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, review & editing Search more papers by this author James M Burke orcid.org/0000-0002-5525-3641 Funding acquisition, Patrick K Mulvaney orcid.org/0000-0002-4641-0385 Roy Parker Corresponding Author [email protected] orcid.org/0000-0002-8412-4152 BioFrontiers Supervision, Information Decker1,2, Burke1, Mulvaney1 *,1,2,3 1Department 2Howard 3BioFrontiers *Corresponding author. Tel: +1 303 735 7780; E-mail: The EMBO Journal (2022)41:e110137https://doi.org/10.15252/embj.2021110137 PDFDownload PDF article text main figures. Peer ReviewDownload a summary editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures Info Abstract Numerous organelles, composed combination proteins, are observed in nucleus cytoplasm eukaryotic cells. These include stress (SGs), processing bodies (PBs), Cajal bodies, speckles. An unresolved question how frequently molecules either or cytosol. To address issue, we degraded intracellular cytosol activation RNase L examined impact loss on several granules. We find majority granules, SGs, speckles, nucleolus, altered degradation their components. In contrast, PBs super-enhancer complexes were largely not affected respective compartments. overall led apparent dissolution some whereas others reorganized into structures with morphology. findings highlight critical widespread role organization Synopsis It unclear whether all diverse types found Here, alter protein indicating organization. Loss has differential effect where P-bodies altered. Targeting provides an inducible means degrade RNA. alters assembly bodies. does size number transcriptional condensates. Introduction Eukaryotic cells contain many different compartments that bound membranes (Spector, 2006; Gomes Shorter, 2019). Many these organelles large assemblies protein, referred as ribonucleoprotein (RNP) (PBs) (Jain Parker, 2013), (SGs) (Anderson Kedersha, 2006), U-bodies (Liu Gall, 2007), IMP (Jønson et al, FMRP (Mazroui 2002), neuronal (Kiebler Bassell, germinal (Voronina 2011). Examples paraspeckles, SGs (Biamonti, 2004; Mao 2011a). Understanding non-membrane-bound assemble maintain will provide fundamental insight organizing cell important question. There evidence formation organelles. Several assembled specific nascent transcripts which act scaffolds nucleate assembly. For example, long non-coding (lncRNA), NEAT1_2, plays core architectural paraspeckles (Clemson 2009; Sasaki 2009). transcription precursor rRNA nucleoli. At end mitosis, nucleoli form around sites pre-rRNA nucleolar also requires 45S produced prior mitosis (Hernandez-Verdun, RNAs condensates cytoplasm. Formation non-translating mRNAs (Kedersha 1999; Teixeira 2005; Buchan 2008; Whether essential other less clear. involved biogenesis recycling classes small RNPs (snRNPs), at genomic individual body proteins concentrated due local snRNAs (Machyna 2013; Sawyer 2016), being artificially tethered (Kaiser 2008). therefore able speckles unclear. A called MALAT1 enriched but it (Hutchinson 2007). However, poly(A)+ present under conditions localization defective (Miyagawa 2012), so species remains be determined. stable maintenance once they formed issue. One way could promote scaffold RNA-binding then bind additional through homotypic heterotypic protein–protein interactions and/or higher order (Fox 2018). addition, intermolecular RNA–RNA can directly (Van Treek Thus, granule thought summation RNA–RNA, RNA–protein, contribution three may differ between same type conditions. recently reported differentially sensitive (Burke 2020) suggesting RNA–protein structural differs. antiviral endoribonuclease, ribonuclease (RNase L), activated response dsRNA leads 2019; Rath activity limits disassemble preformed 2020). dramatically affect suggests strongly dependent interactions, maybe interactions. Alternatively, within stably associated protected from comparison SGs. better understand cellular compartments, have variety presence integrity. quantified volume PBs. targeted induce determine if maintained structure disassembled. susceptible disassemble, morphology, resident RNAs. Taken together, our reveal Results P-body unaffected been inhibit 2019, Similarly, knockout (RL-KO) A549 poly(I:C), viral mimic activates pathway, was prevented wild-type active L, judged depletion (GAPDH RNA) (Fig 1A). WT cells, only G3BP foci 1A B), L-dependent (RLBs) RLBs distinct having composition forming independently kinase R Figure 1. GAPDH smFISH IF analysis using anti-G3BP antibody (G3BP) anti-Dcp1b (DCP1) (RL-WT) RL-KO mock transfected (mock) poly(I:C) (PIC) 5 h. Scale bar 10 microns. Graph fraction volumes PIC-treated RL-WT Number DCP1 per relative mock-treated Wilcoxon Signed Rank test, ns, non-significant. Average Dcp1 mock- One-way ANOVA Sidak's comparisons information: (C, D) Bar graphs show mean + SD N = 4 independent experiments. data available online figure. 1 [embj2021110137-sup-0002-SDataFig1.zip] Download figure PowerPoint did observe any poly(I:C)-treated average change significantly 1C possibility compared ineffective accessing test targeting would integrity, fused PB (Ingelfinger 2002) EV1A B). Activation Dcp1-RNase fusion PIC treatment caused EV1C) significant EV1C–E). results argue lack activating unable access Although cannot rule out residual responsible maintaining when degraded, persistence sufficient cell. Click here expand EV1. increase Full-length (DRL) expressed. Western anti-RNase whole lysates knock (RL-KO), transduced lentiviral vectors containing DCP1a (RL-KO DRL-WT) catalytic mutant L-R667A DRL-CM). Arrows left indicate migration endogenous (RL) (DRL). DRL co-localize P-bodies. anti-DCP1b detect anti-Flag Flag-tagged DCP1a-RNase proteins. DRL-WT active. DRL-CM expressing non-significant test. (D, E) lead changes microtubule intermediate filament networks EV2A B) consistent specifically affecting RNA-dependent assemblies. EV2. Degradation lamin PML A. microtubules (anti-alpha tubulin antibody) filaments (anti-pan keratin treated B. reduced C. D, E. nuclear-localized (D) Oligo(dT) FISH (anti-lamin antibody). (E) (anti-PML F. oligo(dT) signal. G. All images scale micron. (B, C) Mean SEM four experiments least 8 condition experiment analyzed. (F, G) unpaired two-tailed t-test, Method manner us require maintained. reasoned its innate immune response. cmyc signal (NLS) introduced modified (NLS-RL-WT) (NLS-RL-CM) via transduction, determined NLS target nucleus. By biochemical fractionation, approximately 30% NLS-RL-WT NLS-RL-CM detected fraction, while 90% 2A This demonstrates portion exogenous overexpressed > 40 fold should allow robustly both 2. Nuclear-localized degrades (N) (C) fractions (W) (RL WT), KO), sequence. Short exposure anti-RL antibody. used marker. Histone H3 depicting two values plotted. probes mRNA without median value intensity fluorescence nuclei divided indicated 43 analyzed condition. Kruskal–Wallis Dunn's ****P-value ≤ 0.0001, ***P-value ns significant. 2 [embj2021110137-sup-0003-SDataFig2.zip] examining FISH, resulted Specifically, poly(A) depleted 2C treatment. since D). increased observation inhibition export 2021). Consistent tagged cytosol, cytosolic assessed occurred following NLS-RL 2C). system allows living fashion. approach Nucleolar nucleolus ribosomal subunits occurs. organized subdomains, fibrillar center (FC) rDNA, dense component (DFC) rRNA, snoRNAs initial steps granular (GC) further processing, modification, occur (Lam 2005). monitor nucleolar-associated accessible RNA, snoRD3A, C/D box snoRNA localizes DFC GC (Gerbi Brovjagin, 1997) 5' 47S (ETS1) (Yao mock-transfected NLS-RL-WT, snoRD3A ETS1 (Figs 3A D EV3A B and, E), identified 3A). B, E); however, perhaps structure, protection continual production transcription. 3. nucleoli, Analysis nucleolar-localized nucleophosmin (NPM1). nuclei. 17 31 Fraction NPM1 ring classified dispersed nucleoplasm. ribosome factor (RPF1). 69 RPF1 speckle-localized anti-sc35 speckle SRRM2. 51 56 SRRM2 Mock 1526 136 20 foci. coilin. decreased distributions coilin protein. 90 192 significance testing. Mann–Whitney 0.0001. 3 [embj2021110137-sup-0004-SDataFig3.zip] EV3. Effect 0.0009. 21 28 24 lower than ave vol FBL 33 29 **P-value 0.0025. fibrillarin (FBL) 23 0.0008. 0.0001 total SON. ratio 14 SON *P-value 0.034 19 Examination (nucleophosmin 1), (Spector 1984), demonstrated lost degraded. nucleoplasm surrounding EV3A). based signal, C), had disrupted. Similar disruption although seen rings C). analyzing (ribosome 1) (fibrillarin). binds pre-60S (Wehner Baserga, 2002; Kater 2017). snoRNPs first sorting colocalized 3D EV3D). levels, F) requiring structure. depleted, one few well arising robust. No nuclear-targeted EV4A) EV4. Changes morphology SMN1 disperses RPF1. sc35 Ol

Язык: Английский

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Poison cassette exon splicing ofSRSF6regulates nuclear speckle dispersal and the response to hypoxia

Nucleic Acids Research, Год журнала: 2023, Номер 51(2), С. 870 - 890

Опубликована: Янв. 9, 2023

Abstract Hypoxia induces massive changes in alternative splicing (AS) to adapt cells the lack of oxygen. Here, we identify factor SRSF6 as a key AS response hypoxia. The level is strongly reduced acute hypoxia, which serves dual purpose: it allows for exon skipping and triggers dispersal nuclear speckles. Our data suggest that use speckles reprogram their gene expression during hypoxic adaptation plays an important role cohesion Down-regulation achieved through inclusion poison cassette (PCE) promoted by SRSF4. Removing PCE 3′ splice site using CRISPR/Cas9 abolishes reduction Aberrantly high levels hypoxia attenuate hypoxia-mediated impair As consequence, proliferation genomic instability are increased, while stress suppressed. SRSF4–PCE–SRSF6 axis active different cancer types, tumors correlates with poor prognosis. We propose ultra-conserved acts tumor suppressor its crucial reduce levels. This may prevent from entering metastatic route adaptation.

Язык: Английский

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Shell protein composition specified by the lncRNA NEAT1 domains dictates the formation of paraspeckles as distinct membraneless organelles

Nature Cell Biology, Год журнала: 2023, Номер 25(11), С. 1664 - 1675

Опубликована: Ноя. 1, 2023

Язык: Английский

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