Gene expression amplification by nuclear speckle association

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.”

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Transcription amplification by nuclear speckle association

10.1101/604298 ◽

2019 ◽

Cited By ~ 3

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.

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ZFC3H1 prevents RNA trafficking into nuclear speckles through condensation

Nucleic Acids Research ◽

10.1093/nar/gkab774 ◽

2021 ◽

Vol 49 (18) ◽

pp. 10630-10643

Author(s):

Yimin Wang ◽

Jing Fan ◽

Jianshu Wang ◽

Yi Zhu ◽

Lin Xu ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Rna Degradation ◽

Nuclear Speckles ◽

Systematic Mapping ◽

Normal Cells ◽

Rna Trafficking ◽

Nuclear Rna ◽

Condensation Activity ◽

Polyadenylated Rnas

Abstract Controlling proper RNA pool for nuclear export is important for accurate gene expression. ZFC3H1 is a key controller that not only facilitates nuclear exosomal degradation, but also retains its bound polyadenylated RNAs in the nucleus upon exosome inactivation. However, how ZFC3H1 retains RNAs and how its roles in RNA retention and degradation are related remain largely unclear. Here, we found that upon degradation inhibition, ZFC3H1 forms nuclear condensates to prevent RNA trafficking to nuclear speckles (NSs) where many RNAs gain export competence. Systematic mapping of ZFC3H1 revealed that it utilizes distinct domains for condensation and RNA degradation. Interestingly, ZFC3H1 condensation activity is required for preventing RNA trafficking to NSs, but not for RNA degradation. Considering that no apparent ZFC3H1 condensates are formed in normal cells, our study suggests that nuclear RNA degradation and retention are two independent mechanisms with different preference for controlling proper export RNA pool—degradation is preferred in normal cells, and condensation retention is activated upon degradation inhibition.

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TASOR epigenetic repressor cooperates with a CNOT1 RNA degradation pathway to repress HIV

10.1101/2020.11.22.386656 ◽

2020 ◽

Author(s):

Roy Matkovic ◽

Marina Morel ◽

Pauline Larrous ◽

Benjamin Martin ◽

Fabienne Bejjani ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Degradation ◽

Degradation Pathway ◽

Transcriptional Level ◽

Heterochromatin Formation ◽

Nascent Transcripts ◽

Ribosomal Dnas

AbstractThe Human Silencing Hub (HUSH) complex constituted of TASOR, MPP8 and Periphilin is involved in the spreading of H3K9me3 repressive marks across genes and transgenes such as ZNF encoding genes, ribosomal DNAs, LINE-1, Retrotransposons and Retroelements or the integrated HIV provirus1–5. The deposit of these repressive marks leads to heterochromatin formation and inhibits gene expression. The precise mechanisms of silencing mediated by HUSH is still poorly understood. Here, we show that TASOR depletion increases the accumulation of transcripts derived from the HIV-1 LTR promoter at a post-transcriptional level. By counteracting HUSH, Vpx from HIV-2 mimics TASOR depletion. With the use of a Yeast-Two-Hybrid screen, we identified new TASOR partners involved in RNA metabolism including the RNA deadenylase CCR4-NOT complex scaffold CNOT1. TASOR and CNOT1 interact in vivo and synergistically repress HIV expression from its LTR. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex presents structural homology with HUSH. During transcription elongation by RNA polymerase II, RITS recruits a TRAMP-like RNA degradation complex composed of CNOT1 partners, MTR4 and the exosome, to ultimately repress gene expression via H3K9me3 deposit. Similarly, we show that TASOR interacts and cooperates with MTR4 and the exosome, in addition to CNOT1. We also highlight an interaction between TASOR and RNA Polymerase II, predominantly under its elongating state, and between TASOR and some HUSH-targeted nascent transcripts. Furthermore, we show that TASOR overexpression facilitates the association of the aforementioned RNA degradation proteins with RNA polymerase II. Altogether, we propose that HUSH operates at the transcriptional and post-transcriptional levels to repress HIV proviral gene expression.

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CDK13 Mutations Drive Melanoma via Accumulation of Prematurely Terminated Transcripts

10.1101/824193 ◽

2019 ◽

Author(s):

Megan L. Insco ◽

Brian J. Abraham ◽

Sara J. Dubbury ◽

Sofia Dust ◽

Constance Wu ◽

...

Keyword(s):

Gene Expression ◽

Overall Survival ◽

Rna Polymerase Ii ◽

Kinase Activity ◽

Prognostic Significance ◽

Rna Degradation ◽

Poor Overall Survival ◽

Cyclin Dependent Kinases ◽

Nuclear Rna ◽

Melanoma Patients

AbstractTranscriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

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Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

eLife ◽

10.7554/elife.37663 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 21

Author(s):

Sarah Gilbertson ◽

Joel D Federspiel ◽

Ella Hartenian ◽

Ileana M Cristea ◽

Britt Glaunsinger

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Mrna Decay ◽

Binding Proteins ◽

Rna Binding ◽

Nuclear Translocation ◽

Rna Binding Proteins ◽

Binding Protein ◽

Protein Localization ◽

Rna Degradation

Alterations in global mRNA decay broadly impact multiple stages of gene expression, although signals that connect these processes are incompletely defined. Here, we used tandem mass tag labeling coupled with mass spectrometry to reveal that changing the mRNA decay landscape, as frequently occurs during viral infection, results in subcellular redistribution of RNA binding proteins (RBPs) in human cells. Accelerating Xrn1-dependent mRNA decay through expression of a gammaherpesviral endonuclease drove nuclear translocation of many RBPs, including poly(A) tail-associated proteins. Conversely, cells lacking Xrn1 exhibited changes in the localization or abundance of numerous factors linked to mRNA turnover. Using these data, we uncovered a new role for relocalized cytoplasmic poly(A) binding protein in repressing recruitment of TATA binding protein and RNA polymerase II to promoters. Collectively, our results show that changes in cytoplasmic mRNA decay can directly impact protein localization, providing a mechanism to connect seemingly distal stages of gene expression.

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Expression of Ethylene Biosynthetic Genes in the Gynoecium and Receptacle Associated With Sepal Abscission During Senescence in Delphinium Grandiflorum

10.21203/rs.3.rs-975326/v1 ◽

2021 ◽

Author(s):

Mitsutoshi Okamoto ◽

Tomoko Niki ◽

Mirai Azuma ◽

Kenichi Shibuya ◽

Kazuo Ichimura

Keyword(s):

Gene Expression ◽

Carboxylic Acid ◽

Ethylene Production ◽

Acc Oxidase ◽

Acc Synthase ◽

Transcript Level ◽

Flower Senescence ◽

Biosynthetic Genes ◽

Transcript Levels ◽

Highly Sensitive

Abstract Delphinium flowers are highly sensitive to ethylene and its sepals abscise during senescence, which is associated with increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO) activities and ethylene production in gynoecium and receptacle. Three ACS genes (DgACS1, DgACS2, and DgACS3) and three ACO genes (DgACO1, DgACO2, and DgACO3) were cloned from Delphinium grandiflorum cv. Super Grand Blue. To investigate the contribution of these genes to ethylene production, their expression was analyzed in these genes in the gynoecium and receptacle during natural senescence and following ethylene exposure and pollination. Ethylene production in the gynoecium and receptacle increased during natural flower senescence. The transcript levels of the ACS and ACO genes in these organs, excluding DgACS2 in the receptacle, increased during senescence. Exposure to ethylene accelerated sepal abscission and more strongly increased ethylene production in the receptacle than in the gynoecium. DgACS1 transcript levels in the gynoecium and DgACS2 and DgACO3 transcript levels in the receptacle were increased by ethylene exposure. Pollination accelerated sepal abscission and increased ethylene production in the gynoecium and receptacle. Pollination slightly affected ACS and ACO transcript levels in the gynoecium, whereas DgACO3 transcript level in the receptacle were markedly increased. These results reveal that ACS and ACO gene expression is differently regulated in the gynoecium and receptacle, and some of these genes are more strongly upregulated by ethylene exposure and pollination in the receptacle than in the gynoecium, suggesting the significance of the receptacle to sepal abscission.

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eQTL regulating Transcript Levels Associated with Diverse Biological Processes in Tomato

10.1101/040592 ◽

2016 ◽

Cited By ~ 2

Author(s):

Aashish Ranjan ◽

Jessica M. Budke ◽

Steven D. Rowland ◽

Daniel H. Chitwood ◽

Ravi Kumar ◽

...

Keyword(s):

Gene Expression ◽

Leaf Development ◽

Expression Patterns ◽

Transcript Level ◽

Phenotypic Traits ◽

Biological Processes ◽

Hypocotyl Length ◽

Solanum Pennellii ◽

Abundance Pattern ◽

Transcript Levels

AbstractVariation in gene expression, in addition to sequence polymorphisms, is known to influence developmental, physiological and metabolic traits in plants. Genetic mapping populations have facilitated identification of expression Quantitative Trait Loci (eQTL), the genetic determinants of variation in gene expression patterns. We used an introgression population developed from the wild desert-adapted Solanum pennellii and domesticated tomato Solanum lycopersicum to identify the genetic basis of transcript level variation. We established the effect of each introgression on the transcriptome, and identified ~7,200 eQTL regulating the steady state transcript levels of 5,300 genes. Barnes-Hut t-distributed stochastic neighbor embedding clustering identified 42 modules revealing novel associations between transcript level patterns and biological processes. The results showed a complex genetic architecture of global transcript abundance pattern in tomato. Several genetic hotspots regulating a large number of transcript level patterns relating to diverse biological processes such as plant defense and photosynthesis were identified. Important eQTL regulating transcript level patterns were related to leaf number and complexity, and hypocotyl length. Genes associated with leaf development showed an inverse correlation with photosynthetic gene expression but eQTL regulating genes associated with leaf development and photosynthesis were dispersed across the genome. This comprehensive expression QTL analysis details the influence of these loci on plant phenotypes, and will be a valuable community resource for investigations on the genetic effects of eQTL on phenotypic traits in tomato.

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Evolved Differences in cis and trans Regulation Between the Maternal and Zygotic mRNA Complements in the Drosophila Embryo

Genetics ◽

10.1534/genetics.120.303626 ◽

2020 ◽

Vol 216 (3) ◽

pp. 805-821

Author(s):

Emily L. Cartwright ◽

Susan E. Lott

Keyword(s):

Gene Expression ◽

Developmental Stages ◽

Transcript Level ◽

Drosophila Embryo ◽

Gene Products ◽

Regulatory Evolution ◽

Transcript Levels ◽

Maternal Transcript ◽

Zygotic Transcription ◽

Trans Regulation

How gene expression can evolve depends on the mechanisms driving gene expression. Gene expression is controlled in different ways in different developmental stages; here we ask whether different developmental stages show different patterns of regulatory evolution. To explore the mode of regulatory evolution, we used the early stages of embryonic development controlled by two different genomes, that of the mother and that of the zygote. During embryogenesis in all animals, initial developmental processes are driven entirely by maternally provided gene products deposited into the oocyte. The zygotic genome is activated later, when developmental control is handed off from maternal gene products to the zygote during the maternal-to-zygotic transition. Using hybrid crosses between sister species of Drosophila (D. simulans, D. sechellia, and D. mauritiana) and transcriptomics, we find that the regulation of maternal transcript deposition and zygotic transcription evolve through different mechanisms. We find that patterns of transcript level inheritance in hybrids, relative to parental species, differ between maternal and zygotic transcripts, and maternal transcript levels are more likely to be conserved. Changes in transcript levels occur predominantly through differences in trans regulation for maternal genes, while changes in zygotic transcription occur through a combination of both cis and trans regulatory changes. Differences in the underlying regulatory landscape in the mother and the zygote are likely the primary determinants for how maternal and zygotic transcripts evolve.

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SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles

Molecular Biology of the Cell ◽

10.1091/mbc.e12-03-0206 ◽

2012 ◽

Vol 23 (18) ◽

pp. 3694-3706 ◽

Cited By ~ 58

Author(s):

Vidisha Tripathi ◽

David Y. Song ◽

Xinying Zong ◽

Sergey P. Shevtsov ◽

Stephen Hearn ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Cell Nucleus ◽

Interphase Nucleus ◽

Sr Proteins ◽

Mrna Processing ◽

Splicing Factors ◽

Nuclear Speckles ◽

Nuclear Speckle ◽

Processing Factors

The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.

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POINT Technology Illuminates the Processing of Polymerase-Associated Intact Nascent Transcripts

10.1101/2020.11.09.374108 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rui Sousa-Luis ◽

Gwendal Dujardin ◽

Inna Zukher ◽

Hiroshi Kimura ◽

Maria Carmo-Fonseca ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Rna Processing ◽

Rna Degradation ◽

List Type ◽

Nascent Transcript ◽

Pol Ii ◽

Human Genes ◽

Nascent Rna ◽

Full Length Transcript ◽

Nascent Transcripts

SUMMARYMammalian chromatin is the site of both RNA polymerase II (Pol II) transcription and coupled RNA processing. However, molecular details of such co-transcriptional mechanisms remain obscure, partly due to technical limitations in purifying authentic nascent transcripts. We present a new approach to purify and profile nascent RNA, called Polymerase Intact Nascent Transcript (POINT) technology. This three-pronged methodology maps nascent RNA 5’ends (POINT-5), establishes the kinetics of co-transcriptional splicing patterns (POINT-nano) and profiles whole transcription units (POINT-seq). In particular we show by depletion of the nuclear exonuclease Xrn2 that this activity acts selectively on cleaved 5’P-RNA at polyadenylation sites. Furthermore POINT-nano reveals that splicing occurs either immediately after splice site transcription or is delayed until Pol II transcribes downstream sequences. Finally, we connect RNA cleavage and splicing with either premature or full-length transcript termination. We anticipate that POINT technology will afford full dissection of the complexity of co-transcriptional RNA processing.HIGHLIGHTSPOINT methodology dissects intact nascent RNA processingSpecificity of Xrn2 exonuclease in co-transcriptional RNA degradationSplicing suppresses Xrn2-dependent premature terminationDifferent kinetic classes of co-transcriptional splicing in human genes

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