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1.
Nature ; 629(8014): 1165-1173, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38720076

RESUMO

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.


Assuntos
Genoma , Salpicos Nucleares , Precursores de RNA , Splicing de RNA , RNA Mensageiro , Spliceossomos , Animais , Humanos , Masculino , Camundongos , Genes , Genoma/genética , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Salpicos Nucleares/genética , Salpicos Nucleares/metabolismo , Precursores de RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/metabolismo , Transcrição Gênica
2.
Cell Rep ; 43(3): 113827, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38381607

RESUMO

Nuclear speckles (NSs) are nuclear biomolecular condensates that are postulated to form by macromolecular phase separation, although the detailed underlying forces driving NS formation remain elusive. SRRM2 and SON are 2 non-redundant scaffold proteins for NSs. How each individual protein governs assembly of the NS protein network and the functional relationship between SRRM2 and SON are largely unknown. Here, we uncover immiscible multiphases of SRRM2 and SON within NSs. SRRM2 and SON are functionally independent, specifically regulating alternative splicing of subsets of mRNA targets, respectively. We further show that SRRM2 forms multicomponent liquid phases in cells to drive NS subcompartmentalization, which is reliant on homotypic interaction and heterotypic non-selective protein-RNA complex coacervation-driven phase separation. SRRM2 serine/arginine-rich (RS) domains form higher-order oligomers and can be replaced by oligomerizable synthetic modules. The serine residues within the RS domains, however, play an irreplaceable role in fine-tuning the liquidity of NSs.


Assuntos
Salpicos Nucleares , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Separação de Fases , Processamento Alternativo/genética , Serina/metabolismo
3.
J Cell Biol ; 222(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-37956386

RESUMO

Current models posit that nuclear speckles (NSs) serve as reservoirs of splicing factors and facilitate posttranscriptional mRNA processing. Here, we discovered that ribotoxic stress induces a profound reorganization of NSs with enhanced recruitment of factors required for splice-site recognition, including the RNA-binding protein TIAR, U1 snRNP proteins and U2-associated factor 65, as well as serine 2 phosphorylated RNA polymerase II. NS reorganization relies on the stress-activated p38 mitogen-activated protein kinase (MAPK) pathway and coincides with splicing activation of both pre-existing and newly synthesized pre-mRNAs. In particular, ribotoxic stress causes targeted excision of retained introns from pre-mRNAs of immediate early genes (IEGs), whose transcription is induced during the stress response. Importantly, enhanced splicing of the IEGs ZFP36 and FOS is accompanied by relocalization of the corresponding nuclear mRNA foci to NSs. Our study reveals NSs as a dynamic compartment that is remodeled under stress conditions, whereby NSs appear to become sites of IEG transcription and efficient cotranscriptional splicing.


Assuntos
Genes Precoces , Salpicos Nucleares , Splicing de RNA , Íntrons , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Humanos
4.
J Cell Biol ; 222(12)2023 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-37988026

RESUMO

Excision of introns during splicing regulates gene expression. In this issue, work by Sung et al. (https://doi.org/10.1083/jcb.202111151) demonstrates that the timing of intron removal in response to stress is coordinated in nuclear speckles, adding a component of spatial regulation to co-/post-transcriptional splicing.


Assuntos
Processamento Alternativo , Splicing de RNA , Íntrons/genética , Splicing de RNA/genética , Salpicos Nucleares
5.
Nat Commun ; 14(1): 4504, 2023 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-37587144

RESUMO

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein's Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity.


Assuntos
Aptâmeros de Nucleotídeos , Proteínas do Complexo SMN , Condensados Biomoleculares , Carbocianinas , Salpicos Nucleares , Domínio Tudor
6.
Exp Cell Res ; 427(1): 113571, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37003559

RESUMO

Nuclear speckles are subcellular structures originally characterized by punctate immunofluorescence staining of the monoclonal antibody SC35, which recognizes an epitope on SRRM2 (serine/arginine repetitive matrix protein 2) and Sfrs2, a member of the SR (serine/arginine-rich) family of splicing factors. Galectin-3 co-localizes with SC35 in nuclear speckles, which represent one group of nuclear bodies that include the nucleolus, Cajal bodies and gems, paraspeckles, etc. Although they appear to have well-delineated physical boundaries, these nuclear bodies are not membrane-bound structures but represent macromolecular assemblies arising from a phenomenon called liquid-liquid phase separation. There has been much recent interest in liquid phase condensation as a newly recognized mechanism by which a cell can organize and compartmentalize subcellular structures with distinct composition. The punctate/speckled staining of galectin-3 with SC3 demonstrates their co-localization in a phase-separated body in vivo, under conditions endogenous to the cell. The purpose of the present review is to summarize the studies that document three key features of galectin-3 for its localization in liquid phase condensates: (a) an intrinsically disordered domain; (b) oligomer formation for multivalent binding; and (c) association with RNA and ribonucleoprotein complexes.


Assuntos
Galectina 3 , Salpicos Nucleares , Galectina 3/metabolismo , Nucléolo Celular , Ribonucleoproteínas/metabolismo , Arginina/metabolismo
7.
J Am Chem Soc ; 145(17): 9571-9583, 2023 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-37062072

RESUMO

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.


Assuntos
Trifosfato de Adenosina , Doença de Huntington , Humanos , Salpicos Nucleares , Proteína Huntingtina/metabolismo , Adenina , RNA/metabolismo , RNA Mensageiro , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos
8.
Plant Physiol ; 192(2): 1449-1465, 2023 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-36869668

RESUMO

Plants can sense the shade from neighboring plants by detecting a reduction of the red:far-red light (R:FR) ratio. Phytochrome B (phyB) is the primary photoreceptor that perceives shade light and regulates jasmonic acid (JA) signaling. However, the molecular mechanisms underlying phyB and JA signaling integration in shade responses remain largely unknown. Here, we show the interaction of phyB and FAR-RED INSENSITIVE 219 (FIN219)/JASMONATE RESISTANT1 (JAR1) in a functional demand manner in Arabidopsis (Arabidopsis thaliana) seedling development. Genetic evidence and interaction studies indicated that phyB and FIN219 synergistically and negatively regulate shade-induced hypocotyl elongation. Moreover, phyB interacted with various isoforms of FIN219 under high and low R:FR light. Methyl jasmonate (MeJA) treatment, FIN219 mutation, and PHYBOE digalactosyldiacylglycerol synthase1-1 (dgd1-1) plants, which show increased levels of JA, altered the patterns of phyB-associated nuclear speckles under the same conditions. Surprisingly, PHYBOE dgd1-1 showed a shorter hypocotyl phenotype than its parental mutants under shade conditions. Microarray assays using PHYBOE and PHYBOE fin219-2 indicated that PHYB overexpression substantially affects defense response-related genes under shade light and coregulates expression of auxin-responsive genes with FIN219. Thus, our findings reveal that phyB substantially crosstalks with JA signaling through FIN219 to modulate seedling development under shade light.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fitocromo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Hipocótilo , Luz , Mutação/genética , Salpicos Nucleares , Fitocromo/metabolismo , Fitocromo A/genética , Fitocromo A/metabolismo , Fitocromo B/genética , Fitocromo B/metabolismo
9.
Nucleic Acids Res ; 51(2): 870-890, 2023 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-36620874

RESUMO

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


Assuntos
Hipóxia Celular , Salpicos Nucleares , Splicing de RNA , Fatores de Processamento de Serina-Arginina , Humanos , Processamento Alternativo , Éxons/genética , Fosfoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Células HeLa
10.
RNA Biol ; 19(1): 1244-1255, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-36412986

RESUMO

Intracellular and intercellular signalling networks play an essential role in optimizing cellular homoeostasis and are thought to be partly reflected in nuclear mRNA dynamics. However, the regulation of nuclear mRNA dynamics by intracellular and intercellular signals remains largely unexplored, and research tools are lacking. Through an original screening based on the mRNA metabolic mechanism, we discovered that eight well-known inhibitors cause significant nuclear poly(A)+ RNA accumulation. Among these inhibitors, we discovered a new mRNA metabolic response in which the addition of antimycin A, an inhibitor of mitochondrial respiratory-chain complex III (complex III), resulted in a marked accumulation of poly(A)+ RNA near the nuclear speckles. Furthermore, dihydroorotate dehydrogenase (DHODH) inhibitors, a rate-limiting enzyme in the intracellular de novo pyrimidine synthesis reaction that specifically exchanges electrons with complex III, also caused a remarkable accumulation of nuclear poly(A)+ RNA adjacent to the nuclear speckles, which was abolished by extracellular uridine supply, indicating that the depletion of intracellular pyrimidine affects poly(A)+ RNA metabolism. Further analysis revealed that ataxia telangiectasia mutated (ATM), a serine and threonine kinase and a master regulator of DNA double-strand break (DSB) and nucleolar stress, is required for this poly(A)+ RNA nuclear accumulation phenomenon. This study reports new insights into novel aspects of nuclear poly(A)+ RNA metabolism, especially the relationship between mitochondrial respiratory-chain functions, pyrimidine metabolism, and nuclear RNA metabolism.


Assuntos
Ataxia Telangiectasia , Humanos , Di-Hidro-Orotato Desidrogenase , Poli A , RNA , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Salpicos Nucleares , RNA Mensageiro/genética , Pirimidinas , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(32): e2206216119, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914133

RESUMO

The eukaryotic genome is partitioned into distinct topological domains separated by boundary elements. Emerging data support the concept that several well-established nuclear compartments are ribonucleoprotein condensates assembled through the physical process of phase separation. Here, based on our demonstration that chemical disruption of nuclear condensate assembly weakens the insulation properties of a specific subset (∼20%) of topologically associated domain (TAD) boundaries, we report that the disrupted boundaries are characterized by a high level of transcription and striking spatial clustering. These topological boundary regions tend to be spatially associated, even interchromosomally, segregate with nuclear speckles, and harbor a specific subset of "housekeeping" genes widely expressed in diverse cell types. These observations reveal a previously unappreciated mode of genome organization mediated by conserved boundary elements harboring highly and widely expressed transcription units and associated transcriptional condensates.


Assuntos
Compartimento Celular , Núcleo Celular , Eucariotos , Ribonucleoproteínas , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromossomos/genética , Eucariotos/citologia , Eucariotos/genética , Genes Essenciais , Genoma/genética , Salpicos Nucleares/genética , Ribonucleoproteínas/metabolismo , Transcrição Gênica
12.
J Cell Sci ; 135(13)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35788677

RESUMO

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


Assuntos
Corpos Nucleares , Salpicos Nucleares , Biofísica , Núcleo Celular/genética , Expressão Gênica
13.
Cells ; 11(12)2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35741072

RESUMO

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). Maternally expressed gene 3 (MEG3) is a nuclear retained lncRNA found to associate with nuclear speckles. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells, we generated a fluorescently tagged MEG3 transcript that could be detected in real time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Transcription or splicing inactivation conditions, known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with metastasis-associated lung adenocarcinoma (MALAT1) lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions, MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches, we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.


Assuntos
RNA Longo não Codificante , Núcleo Celular/metabolismo , Salpicos Nucleares , Splicing de RNA/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
14.
Proc Natl Acad Sci U S A ; 119(25): e2206046119, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35704758

RESUMO

Nuclear speckles are non-membrane-bound organelles known as storage sites for messenger RNA (mRNA) processing and splicing factors. More recently, nuclear speckles have also been implicated in splicing and export of a subset of mRNAs, including the influenza virus M mRNA that encodes proteins required for viral entry, trafficking, and budding. However, little is known about how nuclear speckles are assembled or regulated. Here, we uncovered a role for the cellular protein kinase TAO2 as a constituent of nuclear speckles and as a factor required for the integrity of these nuclear bodies and for their functions in pre-mRNA splicing and trafficking. We found that a nuclear pool of TAO2 is localized at nuclear speckles and interacts with nuclear speckle factors involved in RNA splicing and nuclear export, including SRSF1 and Aly/Ref. Depletion of TAO2 or inhibition of its kinase activity disrupts nuclear speckle structure, decreasing the levels of several proteins involved in nuclear speckle assembly and splicing, including SC35 and SON. Consequently, splicing and nuclear export of influenza virus M mRNA were severely compromised and caused a disruption in the virus life cycle. In fact, low levels of TAO2 led to a decrease in viral protein levels and inhibited viral replication. Additionally, depletion or inhibition of TAO2 resulted in abnormal expression of a subset of mRNAs with key roles in viral replication and immunity. Together, these findings uncovered a function of TAO2 in nuclear speckle formation and function and revealed host requirements and vulnerabilities for influenza infection.


Assuntos
Núcleo Celular , Salpicos Nucleares , Proteínas Quinases , Splicing de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/enzimologia , Células HeLa , Humanos , Proteínas Quinases/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Processamento de Serina-Arginina/genética
15.
RNA ; 28(6): 878-894, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35351812

RESUMO

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Using high-throughput sequencing of cellular fractions, we now demonstrate that IPA transcripts require the zinc finger protein ZFC3H1 for their nuclear retention and degradation. Using reporter mRNAs, we demonstrate that ZFC3H1 promotes the nuclear retention of mRNAs with intact 5'SS motifs by sequestering them into nuclear speckles. Furthermore, we find that U1-70K, a component of the spliceosomal U1 snRNP, is also required for the nuclear retention of these reporter mRNAs and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of reporter mRNAs with 5'SS motifs. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs by sequestering them into nuclear speckles.


Assuntos
Sítios de Splice de RNA , Ribonucleoproteína Nuclear Pequena U1 , Salpicos Nucleares , Sítios de Splice de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismo
16.
Nucleus ; 13(1): 58-73, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35220893

RESUMO

Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a "putative stable association" whose coupling might be subject to the combined regulation from both chromatin and NS changes.Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.


Assuntos
Núcleo Celular , Cromatina , Animais , Núcleo Celular/metabolismo , Cromatina/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Salpicos Nucleares , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo
17.
FEBS J ; 289(22): 7234-7245, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-34245118

RESUMO

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


Assuntos
Núcleo Celular , Salpicos Nucleares , Núcleo Celular/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Citoplasma , Regulação da Expressão Gênica
18.
Front Immunol ; 12: 752482, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745125

RESUMO

Although considered the ternary inflammasome structure, whether the singular, perinuclear NLRP3:ASC speck is synonymous with the NLRP3 inflammasome is unclear. Herein, we report that the NLRP3:ASC speck is not required for nigericin-induced inflammasome activation but facilitates and maximizes IL-1ß processing. Furthermore, the NLRP3 agonists H2O2 and MSU elicited IL-1ß maturation without inducing specks. Notably, caspase-1 activity is spatially distinct from the speck, occurring at multiple cytoplasmic sites. Additionally, caspase-1 activity negatively regulates speck frequency and speck size, while speck numbers and IL-1ß processing are negatively correlated, cyclical and can be uncoupled by NLRP3 mutations or inhibiting microtubule polymerization. Finally, when specks are present, caspase-1 is likely activated after leaving the speck structure. Thus, the speck is not the NLRP3 inflammasome itself, but is instead a dynamic structure which may amplify the NLRP3 response to weak stimuli by facilitating the formation and release of small NLRP3:ASC complexes which in turn activate caspase-1.


Assuntos
Proteínas Adaptadoras de Sinalização CARD , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Células Cultivadas , Humanos , Salpicos Nucleares
19.
Nat Commun ; 12(1): 6648, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34789764

RESUMO

The U6 snRNA, the core catalytic component of the spliceosome, is extensively modified post-transcriptionally, with 2'-O-methylation being most common. However, how U6 2'-O-methylation is regulated remains largely unknown. Here we report that TFIP11, the human homolog of the yeast spliceosome disassembly factor Ntr1, localizes to nucleoli and Cajal Bodies and is essential for the 2'-O-methylation of U6. Mechanistically, we demonstrate that TFIP11 knockdown reduces the association of U6 snRNA with fibrillarin and associated snoRNAs, therefore altering U6 2'-O-methylation. We show U6 snRNA hypomethylation is associated with changes in assembly of the U4/U6.U5 tri-snRNP leading to defects in spliceosome assembly and alterations in splicing fidelity. Strikingly, this function of TFIP11 is independent of the RNA helicase DHX15, its known partner in yeast. In sum, our study demonstrates an unrecognized function for TFIP11 in U6 snRNP modification and U4/U6.U5 tri-snRNP assembly, identifying TFIP11 as a critical spliceosome assembly regulator.


Assuntos
Fatores de Processamento de RNA/metabolismo , Splicing de RNA/fisiologia , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Nucléolo Celular/metabolismo , Sobrevivência Celular , Corpos Enovelados/metabolismo , Células HeLa , Humanos , Metilação , Mitose , Proteínas Nucleares/metabolismo , Salpicos Nucleares/metabolismo , Ligação Proteica , Estabilidade Proteica , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/genética , RNA Nucleolar Pequeno/metabolismo , Spliceossomos/metabolismo
20.
Epigenetics Chromatin ; 14(1): 51, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34819154

RESUMO

BACKGROUND: Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here, we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. RESULTS: Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. CONCLUSION: Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.


Assuntos
Proteínas Correpressoras/genética , Corpos Nucleares , Salpicos Nucleares , Núcleo Celular , Humanos , Proteínas Nucleares/genética , RNA
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