scholarly journals Transcription amplification by nuclear speckle association

2019 ◽  
Author(s):  
Jiah Kim ◽  
Nimish Khanna ◽  
Andrew S. Belmont
Keyword(s):  
Heat Shock ◽  
Liquid Droplet ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Nuclear Compartment ◽  
Endogenous Genes ◽  
Amplification Mechanism ◽  
Transcriptional Bursting ◽  
Transcriptional Amplification ◽  
Chromosome Regions

AbstractA significant fraction of active chromosome regions and genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, we observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 °C. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through a “transcriptional amplification” mechanism distinct from transcriptional bursting.

scholarly journals Gene expression amplification by nuclear speckle association

2019 ◽  
pp. jcb.201904046 ◽  
Author(s):  
Jiah Kim ◽  
Neha Chivukula Venkata ◽  
Gabriela Andrea Hernandez Gonzalez ◽  
Nimish Khanna ◽  
Andrew S. Belmont
Keyword(s):  
Gene Expression ◽  
Rna Polymerase Ii ◽  
Liquid Droplet ◽  
Transcript Level ◽  
Rna Degradation ◽  
Nuclear Speckles ◽  
Nascent Transcript ◽  
Nuclear Compartment ◽  
Transcript Levels ◽  
Endogenous Genes

Many active genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that HSPA1B BAC transgenes and endogenous Hsp70 genes turn on 2–4 min after heat shock (HS), irrespective of their distance to speckles. However, both total HSPA1B mRNA counts and nascent transcript levels measured adjacent to the transgene are approximately twofold higher for speckle-associated alleles 15 min after HS. Nascent transcript level fold-increases for speckle-associated alleles are 12–56-fold and 3–7-fold higher 1–2 h after HS for HSPA1B transgenes and endogenous genes, respectively. Severalfold higher nascent transcript levels for several Hsp70 flanking genes also correlate with speckle association at 37°C. Live-cell imaging reveals that HSPA1B nascent transcript levels increase/decrease with speckle association/disassociation. Initial investigation reveals that increased nascent transcript levels accompanying speckle association correlate with reduced exosome RNA degradation and larger Ser2p CTD-modified RNA polymerase II foci. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through “gene expression amplification.”

scholarly journals Hsp70 gene association with nuclear speckles is Hsp70 promoter specific

2010 ◽  
Vol 191 (4) ◽  
pp. 711-719 ◽  
Author(s):  
Yan Hu ◽  
Matt Plutz ◽  
Andrew S. Belmont
Keyword(s):  
Heat Shock ◽  
Integration Site ◽  
Promoter Sequence ◽  
Artificial Chromosome ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Transcript Accumulation ◽  
Nuclear Speckle ◽  
Hsp70 Promoter ◽  
Acid Processing

Many mammalian genes localize near nuclear speckles, nuclear bodies enriched in ribonucleic acid–processing factors. In this paper, we dissect cis-elements required for nuclear speckle association of the heat shock protein 70 (Hsp70) locus. We show that speckle association is a general property of Hsp70 bacterial artificial chromosome transgenes, independent of the chromosome integration site, and can be recapitulated using a 2.8-kilobase HSPA1A gene fragment. Association of Hsp70 transgenes and their transcripts with nuclear speckles is transcription dependent, independent of the transcribed sequence identity, but dependent on the Hsp70 promoter sequence. Transgene speckle association does not correlate with the amount of transcript accumulation, with large transgene arrays driven by different promoters showing no speckle association, but smaller Hsp70 transgene arrays with lower transcript accumulation showing high speckle association. Moreover, despite similar levels of transcript accumulation, Hsp70 transgene speckle association is observed after heat shock but not cadmium treatment. We suggest that certain promoters may direct specific chromatin and/or transcript ribonucleoprotein modifications, leading to nuclear speckle association.

scholarly journals RNAs as proximity labeling media for identifying nuclear speckle positions relative to the genome

2018 ◽  
Author(s):  
Weizhong Chen ◽  
Zhangming Yan ◽  
Simin Li ◽  
Norman Huang ◽  
Xuerui Huang ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Genomic Sequence ◽  
Single Cells ◽  
Regulation Of Gene Expression ◽  
Nuclear Genome ◽  
Fold Increase ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Interaction Sites ◽  
Genomic Regions

AbstractNuclear speckles are interchromatin structures enriched in RNA splicing factors. Determining their relative positions with respect to the folded nuclear genome could provide critical information on co-and post-transcriptional regulation of gene expression. However, it remains challenging to identify which parts of the nuclear genome are in proximity to nuclear speckles, due to physical separation between nuclear speckle cores and chromatin. We hypothesized that noncoding RNAs including small nuclear RNAs, 7SK and Malat1, which accumulate at the periphery of nuclear speckles (nsaRNA,nuclearspeckleassociated RNA), may extend to sufficient proximity to the nuclear genome. Leveraging a transcriptome-genome interaction assay (MARGI), we identified nsaRNA-interacting genomic sequences, which exhibited clustering patterns (nsaPeaks) in the genome, suggesting existence of relatively stable interaction sites for nsaRNAs in nuclear genome. Posttranscriptional pre-mRNAs, which are known to be clustered to nuclear speckles, exhibited proximity to nsaPeaks but rarely to other genomic regions. Furthermore, CDK9 proteins that localize to the vicinity of nuclear speckles produced ChIP-seq peaks that overlapped with nsaPeaks. Our combined DNA FISH and immunofluorescence analysis in 182 single cells revealed a 3-fold increase in odds for nuclear speckles to localize near an nsaPeak than its neighboring genomic sequence. These data suggest a model that nsaRNAs locate in sufficient proximity to nuclear genome and leave identifiable genomic footprints, thus revealing the parts of genome proximal to nuclear speckles.

scholarly journals Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel Interaction with Nuclear Speckle Domains

2008 ◽  
Vol 83 (2) ◽  
pp. 701-711 ◽  
Author(s):  
Jana Schneider ◽  
Bianca Dauber ◽  
Krister Melén ◽  
Ilkka Julkunen ◽  
Thorsten Wolff
Keyword(s):  
Gene Expression ◽  
Protein Domain ◽  
Replication Capacity ◽  
Influenza B Virus ◽  
Viral Gene ◽  
Influenza B ◽  
Ns1 Protein ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
B Virus

ABSTRACT Many proteins that function in the transcription, maturation, and export of metazoan mRNAs are concentrated in nuclear speckle domains, indicating that the compartment is important for gene expression. Here, we show that the NS1 protein of influenza B virus (B/NS1) accumulates in nuclear speckles and causes rounding and morphological changes of the domains, indicating a disturbance in their normal functions. This property was located within the N-terminal 90 amino acids of the B/NS1 protein and was shown to be independent of any other viral gene product. Within this protein domain, we identified a monopartite importin α binding nuclear localization signal. Reverse-genetic analysis of this motif indicated that nuclear import and speckle association of the B/NS1 protein are required for the full replication capacity of the virus. In the late phase of virus infection, the B/NS1 protein relocated to the cytoplasm, which occurred in a CRM1-independent manner. The interaction of the B/NS1 protein with nuclear speckles may reflect a recruitment function to promote viral-gene expression. To our knowledge, this is the first functional description of a speckle-associated protein that is encoded by a negative-strand RNA virus.

scholarly journals Stress inhibits nucleocytoplasmic shuttling of heat shock protein hsc70

2005 ◽  
Vol 289 (4) ◽  
pp. C1034-C1041 ◽  
Author(s):  
Mohamed Kodiha ◽  
Angel Chu ◽  
Omar Lazrak ◽  
Ursula Stochaj
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Level ◽  
Nuclear Accumulation ◽  
Stress Exposure ◽  
Nucleocytoplasmic Shuttling ◽  
Nuclear Compartment ◽  
Intracellular Targeting ◽  
Shock Proteins

Heat shock proteins of the hsp/hsc70 family are essential chaperones, implicated in the stress response, aging, and a growing number of human diseases. At the molecular level, hsc70s are required for the proper folding and intracellular targeting of polypeptides as well as the regulation of apoptosis. Cytoplasmic members of the hsp/hsc70 family are believed to shuttle between nuclei and cytoplasm; they are found in both compartments of unstressed cells. Our experiments demonstrate that actin filament-destabilizing drugs trigger the nuclear accumulation of hsc70s in unstressed and heat-shocked cells recovering from stress. Using human-mouse heterokaryons, we show that stress inhibits shuttling and sequesters the chaperone in nuclei. The inhibition of hsc70 shuttling upon heat shock is only transient, and transport is reestablished when cells recover from stress. Hsc70 shuttling is controlled by hsc70 retention in the nucleus, a process that is mediated by two distinct mechanisms, ATP-sensitive binding of hsc70s to chaperone substrates and, furthermore, the association with nucleoli. The nucleolar protein fibrillarin and ribosomal protein rpS6 were identified as components that show an increased association with hsc70s in the nucleus upon stress exposure. Together, our data suggest that stress abolishes the exit of hsc70s from the nucleus to the cytoplasm, thereby limiting their function to the nuclear compartment. We propose that during recovery from stress hsc70s are released from nuclear and nucleolar anchors, which is a prerequisite to restore shuttling.

scholarly journals Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

2021 ◽  
Author(s):  
Evan Lester ◽  
Felicia K. Ooi ◽  
Nadine Bakkar ◽  
Jacob Ayers ◽  
Amanda L. Woerman ◽  
...  
Keyword(s):  
Cell Culture ◽  
Frontotemporal Dementia ◽  
Corticobasal Degeneration ◽  
Mrna Splicing ◽  
Protein Assemblies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Tau Aggregation ◽  
In Cells

AbstractTau 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 both cytosolic and nuclear tau aggregates contain RNA, with enrichment for snRNAs and snoRNAs. Nuclear tau aggregates colocalize with and alter the composition, dynamics, and organization of nuclear speckles, which are membraneless organelles involved in pre-mRNA splicing. Moreover, several nuclear speckle components, including SRRM2, mislocalize to cytosolic tau aggregates in cells, mouse brains, and patient brains with AD, frontotemporal dementia (FTD), and corticobasal degeneration (CBD). Consistent with these alterations we observe 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.

scholarly journals SON and SRRM2 are essential for nuclear speckle formation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
İbrahim Avşar Ilik ◽  
Michal Malszycki ◽  
Anna Katharina Lübke ◽  
Claudia Schade ◽  
David Meierhofer ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Complete Dissolution ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
The Core ◽  
Main Target ◽  
A Cell

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.

scholarly journals Revealing RCOR2 as a regulatory component of nuclear speckles

2021 ◽  
Author(s):  
Carlos Rivera ◽  
Daniel Verbel ◽  
Duxan Arancibia ◽  
Anna Lappala ◽  
Marcela González ◽  
...  
Keyword(s):  
System Development ◽  
Cell Types ◽  
Nervous System Development ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Multiple Cell ◽  
Rna Maturation ◽  
Nuclear Processes ◽  
The Brain

Abstract 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.

scholarly journals Revealing RCOR2 as a regulatory component of nuclear speckles

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Carlos Rivera ◽  
Daniel Verbel-Vergara ◽  
Duxan Arancibia ◽  
Anna Lappala ◽  
Marcela González ◽  
...  
Keyword(s):  
System Development ◽  
Cell Types ◽  
Nervous System Development ◽  
Nuclear Bodies ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Multiple Cell ◽  
Rna Maturation ◽  
Nuclear Processes ◽  
The Brain

Abstract 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.

scholarly journals Tyramide signal amplification mass spectrometry (TSA-MS) ratio identifies nuclear speckle proteins

2020 ◽  
Vol 219 (9) ◽  
Author(s):  
Joseph Dopie ◽  
Michael J. Sweredoski ◽  
Annie Moradian ◽  
Andrew S. Belmont
Keyword(s):  
Mass Spectrometry ◽  
Signal Amplification ◽  
Protein Composition ◽  
Nuclear Bodies ◽  
Ratio Method ◽  
Tyramide Signal Amplification ◽  
Label Free ◽  
Nuclear Speckles ◽  
Nuclear Speckle ◽  
Speckle Structure

We present a simple ratio method to infer protein composition within cellular structures using proximity labeling approaches but compensating for the diffusion of free radicals. We used tyramide signal amplification (TSA) and label-free mass spectrometry (MS) to compare proteins in nuclear speckles versus centromeres. Our “TSA-MS ratio” approach successfully identified known nuclear speckle proteins. For example, 96% and 67% of proteins in the top 30 and 100 sorted proteins, respectively, are known nuclear speckle proteins, including proteins that we validated here as enriched in nuclear speckles. We show that MFAP1, among the top 20 in our list, forms droplets under certain circumstances and that MFAP1 expression levels modulate the size, stability, and dynamics of nuclear speckles. Localization of MFAP1 and its binding partner, PRPF38A, in droplet-like nuclear bodies precedes formation of nuclear speckles during telophase. Our results update older proteomic studies of nuclear speckles and should provide a useful reference dataset to guide future experimental dissection of nuclear speckle structure and function.

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