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Nucleic Acids Research Oct 2017The nucleoplasm is not homogenous; it consists of many types of nuclear bodies, also known as nuclear domains or nuclear subcompartments. These self-organizing... (Review)
Review
The nucleoplasm is not homogenous; it consists of many types of nuclear bodies, also known as nuclear domains or nuclear subcompartments. These self-organizing structures gather machinery involved in various nuclear activities. Nuclear speckles (NSs) or splicing speckles, also called interchromatin granule clusters, were discovered as sites for splicing factor storage and modification. Further studies on transcription and mRNA maturation and export revealed a more general role for splicing speckles in RNA metabolism. Here, we discuss the functional implications of the localization of numerous proteins crucial for epigenetic regulation, chromatin organization, DNA repair and RNA modification to nuclear speckles. We highlight recent advances suggesting that NSs facilitate integrated regulation of gene expression. In addition, we consider the influence of abundant regulatory and signaling proteins, i.e. protein kinases and proteins involved in protein ubiquitination, phosphoinositide signaling and nucleoskeletal organization, on pre-mRNA synthesis and maturation. While many of these regulatory proteins act within NSs, direct evidence for mRNA metabolism events occurring in NSs is still lacking. NSs contribute to numerous human diseases, including cancers and viral infections. In addition, recent data have demonstrated close relationships between these structures and the development of neurological disorders.
Topics: Cell Nucleus; Chromatin; Disease; Epigenesis, Genetic; Humans; Interphase; Macromolecular Substances; Microscopy, Fluorescence; Nuclear Proteins; RNA Precursors; RNA Splicing; RNA-Binding Proteins
PubMed: 28977640
DOI: 10.1093/nar/gkx759 -
Nature Chemical Biology Oct 2011We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A)...
We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m(6)A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m(6)A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m(6)A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m(6)A in nuclear RNA is a major physiological substrate of FTO.
Topics: Adenosine; Alpha-Ketoglutarate-Dependent Dioxygenase FTO; Cell Nucleus; HeLa Cells; Humans; Methylation; Obesity; Oxidation-Reduction; Proteins; RNA, Messenger; Substrate Specificity
PubMed: 22002720
DOI: 10.1038/nchembio.687 -
Nucleic Acids Research Aug 2022SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in...
SRRM2 is a nuclear-speckle marker containing multiple disordered domains, whose dysfunction is associated with several human diseases. Using mainly EGFP-SRRM2 knock-in HEK293T cells, we show that SRRM2 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 that SRRM2 organizes nuclear speckles along the cell cycle. As bona-fide splicing factor present in spliceosome structures, SRRM2 deficiency induces skipping of cassette exons with short introns and weak splice sites, tending to change large protein domains. In THP-1 myeloid-like cells, SRRM2 depletion compromises cell viability, upregulates differentiation markers, and sensitizes cells to anti-leukemia drugs. SRRM2 induces a FES splice isoform that attenuates innate inflammatory responses, and MUC1 isoforms that undergo shedding with oncogenic properties. We conclude that SRRM2 acts as a scaffold to organize nuclear speckles, regulating alternative splicing in innate immunity and cell homeostasis.
Topics: Alternative Splicing; Exons; HEK293 Cells; Humans; Introns; Protein Isoforms; RNA Splicing; RNA-Binding Proteins
PubMed: 35929045
DOI: 10.1093/nar/gkac669 -
Cell Sep 2020The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation...
The 3D organization of chromatin regulates many genome functions. Our understanding of 3D genome organization requires tools to directly visualize chromatin conformation in its native context. Here we report an imaging technology for visualizing chromatin organization across multiple scales in single cells with high genomic throughput. First we demonstrate multiplexed imaging of hundreds of genomic loci by sequential hybridization, which allows high-resolution conformation tracing of whole chromosomes. Next we report a multiplexed error-robust fluorescence in situ hybridization (MERFISH)-based method for genome-scale chromatin tracing and demonstrate simultaneous imaging of more than 1,000 genomic loci and nascent transcripts of more than 1,000 genes together with landmark nuclear structures. Using this technology, we characterize chromatin domains, compartments, and trans-chromosomal interactions and their relationship to transcription in single cells. We envision broad application of this high-throughput, multi-scale, and multi-modal imaging technology, which provides an integrated view of chromatin organization in its native structural and functional context.
Topics: Algorithms; Cell Line; Cell Nucleus; Chromatin; Chromosomes, Human; DNA; Genomics; High-Throughput Screening Assays; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Molecular Conformation; Multimodal Imaging; Nucleolus Organizer Region; RNA; Single-Cell Analysis; Software
PubMed: 32822575
DOI: 10.1016/j.cell.2020.07.032 -
Molecular Cell Mar 2022The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these...
The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
Topics: Cell Nucleus; Gene Expression Regulation; Humans; Introns; RNA Splicing; RNA-Binding Proteins
PubMed: 35182477
DOI: 10.1016/j.molcel.2021.12.010 -
ELife Oct 2020Nuclear speckles (NS) are among the most prominent biomolecular condensates. Despite their prevalence, research on the function of NS is virtually restricted to...
Nuclear speckles (NS) are among the most prominent biomolecular condensates. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified. The monoclonal antibody SC35, raised against a spliceosomal extract, is frequently used to mark NS. Unexpectedly, we found that this antibody was mischaracterized and the main target of SC35 mAb is SRRM2, a spliceosome-associated protein that sharply localizes to NS. Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while co-depletion of SON and SRRM2 or depletion of SON in a cell-line where intrinsically disordered regions (IDRs) of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.
Topics: Antibodies; Antibodies, Monoclonal; Blotting, Western; Cell Line; Cell Nucleus; DNA-Binding Proteins; Gene Expression Regulation; Gene Knock-In Techniques; Humans; Minor Histocompatibility Antigens; Phylogeny; RNA Interference; RNA-Binding Proteins; Serine-Arginine Splicing Factors
PubMed: 33095160
DOI: 10.7554/eLife.60579 -
Neuron May 2021Tau aggregates contribute to neurodegenerative diseases, including frontotemporal dementia and Alzheimer's disease (AD). Although RNA promotes tau aggregation in vitro,...
Tau aggregates contribute to neurodegenerative diseases, including frontotemporal dementia and Alzheimer's disease (AD). Although RNA promotes tau aggregation in vitro, whether tau aggregates in cells contain RNA is unknown. We demonstrate, in cell culture and mouse brains, that cytosolic and nuclear tau aggregates contain RNA with enrichment for small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs). Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and brains of individuals with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations, we observe that the presence of tau aggregates is sufficient to alter pre-mRNA splicing. This work identifies tau alteration of nuclear speckles as a feature of tau aggregation that may contribute to the pathology of tau aggregates.
Topics: Alzheimer Disease; Animals; Cell Nucleus; Cytosol; Female; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Protein Binding; Protein Transport; RNA Splicing; RNA, Small Nucleolar; RNA-Binding Proteins; tau Proteins
PubMed: 33848474
DOI: 10.1016/j.neuron.2021.03.026 -
Cell Jul 2018Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation....
Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.
Topics: Cell Nucleolus; Cell Nucleus; Chromosome Mapping; Chromosomes; DNA; Eukaryota; Genome; Humans; Structure-Activity Relationship
PubMed: 29887377
DOI: 10.1016/j.cell.2018.05.024 -
Cold Spring Harbor Perspectives in... Feb 2011Nuclear speckles, also known as interchromatin granule clusters, are nuclear domains enriched in pre-mRNA splicing factors, located in the interchromatin regions of the... (Review)
Review
Nuclear speckles, also known as interchromatin granule clusters, are nuclear domains enriched in pre-mRNA splicing factors, located in the interchromatin regions of the nucleoplasm of mammalian cells. When observed by immunofluorescence microscopy, they usually appear as 20-50 irregularly shaped structures that vary in size. Speckles are dynamic structures, and their constituents can exchange continuously with the nucleoplasm and other nuclear locations, including active transcription sites. Studies on the composition, structure, and dynamics of speckles have provided an important paradigm for understanding the functional organization of the nucleus and the dynamics of the gene expression machinery.
Topics: Cell Cycle; Cell Nucleus; Cell Nucleus Structures; Microscopy, Fluorescence; Nuclear Proteins; RNA Splicing; Transcription, Genetic
PubMed: 20926517
DOI: 10.1101/cshperspect.a000646 -
Current Opinion in Genetics &... Apr 2019Higher eukaryotic cell nuclei are highly compartmentalized into bodies and structural assemblies of specialized functions. Nuclear speckles/IGCs are one of the most... (Review)
Review
Higher eukaryotic cell nuclei are highly compartmentalized into bodies and structural assemblies of specialized functions. Nuclear speckles/IGCs are one of the most prominent nuclear bodies, yet their functional significance remains largely unknown. Recent advances in sequence-based mapping of nuclear genome organization now provide genome-wide analysis of chromosome organization relative to nuclear speckles. Here we review older microscopy-based studies on a small number of genes with the new genomic mapping data suggesting a significant fraction of the genome is almost deterministically positioned near nuclear speckles. Both microscopy and genomic-based approaches support the concept of the nuclear speckle periphery as a major active chromosomal compartment which may play an important role in fine-tuning gene regulation.
Topics: Animals; Cell Nucleus; Chromosomes; Gene Expression Regulation; Genome; Humans; Nuclear Proteins; Transcription, Genetic
PubMed: 31394307
DOI: 10.1016/j.gde.2019.06.008