Sequential Entry of Components of Gene Expression Machinery into Daughter Nuclei

In eukaryotic cells, RNA polymerase II (RNA pol II) transcription and pre-mRNA processing are coordinated events. We have addressed how individual components of the transcription and pre-mRNA processing machinery are organized during mitosis and subsequently recruited into the newly formed daughter nuclei. Interestingly, localization studies of numerous RNA pol II transcription and pre-mRNA processing factors revealed a nonrandom and sequential entry of these factors into daughter nuclei after nuclear envelope/lamina formation. The initiation competent form of RNA pol II and general transcription factors appeared in the daughter nuclei simultaneously, but prior to pre-mRNA processing factors, whereas the elongation competent form of RNA pol II was detected even later. The differential entry of these factors rules out the possibility that they are transported as a unitary complex. Telophase nuclei were competent for transcription and pre-mRNA splicing concomitant with the initial entry of the respective factors. In addition, our results revealed a low turnover rate of transcription and pre-mRNA splicing factors during mitosis. We provide evidence to support a model in which the entry of the RNA pol II gene expression machinery into newly forming daughter nuclei is a staged and ordered process.

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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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Cohesin removal reprograms gene expression upon mitotic entry

10.1101/678003 ◽

2019 ◽

Cited By ~ 1

Author(s):

Carlos Perea-Resa ◽

Leah Bury ◽

Iain Cheeseman ◽

Michael D. Blower

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Rna Polymerase Ii ◽

Chromosome Segregation ◽

List Type ◽

Mitotic Chromosomes ◽

Rna Pol Ii ◽

Pol Ii ◽

Mitotic Entry ◽

Nascent Transcripts

SummaryEntering mitosis, the genome is restructured to facilitate chromosome segregation, accompanied by dramatic changes in gene expression. However, the mechanisms that underlie mitotic transcriptional regulation are unclear. In contrast to transcribed genes, centromere regions retain transcriptionally active RNA Polymerase II (RNAPII) in mitosis. Here, we demonstrate that chromatin-bound cohesin is sufficient to retain RNAPII at centromeres while WAPL-mediated removal of cohesin during prophase is required for RNAPII dissociation from chromosome arms. Failure to remove cohesin from chromosome arms results in a failure to release elongating RNAPII and nascent transcripts from mitotic chromosomes and dramatically alters gene expression. We propose that prophase cohesin removal is the key step in reprogramming gene expression as cells transition from G2 to mitosis, and is temporally coupled with chromosome condensation to coordinate chromosome segregation with changes in gene expression.HighlightsMitotic centromere transcription requires cohesinCohesin removal releases elongating RNA Pol II and nascent RNA from chromatinThe prophase pathway reprograms gene expression during mitosis

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Maize RAMOSA3 accumulates in nuclear condensates enriched in RNA POLYMERASE II isoforms during the establishment of axillary meristem determinacy.

10.1101/2021.04.06.438639 ◽

2021 ◽

Author(s):

Edgar Demesa-Arevalo ◽

Maria Jazmin Abraham-Juarez ◽

Xiaosa Xu ◽

Madelaine Bartlett ◽

David Jackson

Keyword(s):

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Transcriptional Response ◽

Mrna Processing ◽

Nuclear Speckles ◽

Rna Pol Ii ◽

Pol Ii ◽

Imaging Processing ◽

Whole Mount ◽

Double Immunolabeling

Maize meristem determinacy is regulated by Trehalose-6-Phosphate Phosphatase (TPP) metabolic enzymes RAMOSA3 and TPP4. However, this function is independent of their enzymatic activity, suggesting they have an unpredicted, or moonlighting function. Using whole-mount double immunolabeling and imaging processing, we investigated the co-localization of RA3 nuclear speckles with markers for transcription, chromatin state and splicing. We find evidence for RA3 co-localization with RNA POL II, a transcription marker, and not with markers for promoter chromatin remodeling or mRNA processing, suggesting a function of nuclear RA3 in mediating a transcriptional response during meristem determinacy.

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c-Myc Is Required for the ChREBP-Dependent Activation of Glucose-Responsive Genes

Molecular Endocrinology ◽

10.1210/me.2009-0437 ◽

2010 ◽

Vol 24 (6) ◽

pp. 1274-1286 ◽

Cited By ~ 27

Author(s):

Pili Zhang ◽

Mallikarjurna R. Metukuri ◽

Sharell M. Bindom ◽

Edward V. Prochownik ◽

Robert M. O'Doherty ◽

...

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Time Course ◽

Target Genes ◽

Process Time ◽

Glucose Response ◽

Rna Pol Ii ◽

Pol Ii ◽

Molecular Events ◽

Regulated Gene Expression

Abstract Glucose regulates programs of gene expression that orchestrate changes in cellular phenotype in several metabolically active tissues. Carbohydrate response element-binding protein (ChREBP) and its binding partner, Mlx, mediate glucose-regulated gene expression by binding to carbohydrate response elements on target genes, such as the prototypical glucose-responsive gene, liver-type pyruvate kinase (Pklr). c-Myc is also required for the glucose response of the Pklr gene, although the relationship between c-Myc and ChREBP has not been defined. Here we describe the molecular events of the glucose-mediated activation of Pklr and determine the effects of decreasing the activity or abundance of c-Myc on this process. Time-course chromatin immunoprecipitation revealed a set of transcription factors [hepatocyte nuclear factor (HNF)1α, HNF4α, and RNA polymerase II (Pol II)] constitutively resident on the Pklr promoter, with a relative enrichment of acetylated histones 3 and 4 in the same region of the gene. Glucose did not affect HNF1α binding or the acetylation of histones H3 or H4. By contrast, glucose promoted the recruitment of ChREBP and c-Myc and increased the occupancy of HNF4α and RNA Pol II, which were coincident with the glucose-mediated increase in transcription as determined by a nuclear run-on assay. Depletion of c-Myc activity using a small molecule inhibitor (10058-F4/1RH) abolished the glucose-mediated recruitment of HNF4α, ChREBP, and RNA Pol II, without affecting basal gene expression, histone acetylation, and HNF1α or basal HNF4α occupancy. The activation and recruitment of ChREBP to several glucose-responsive genes were blocked by 1RH, indicating a general necessity for c-Myc in this process.

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Selective Requirement for SAGA in Hog1-Mediated Gene Expression Depending on the Severity of the External Osmostress Conditions

Molecular and Cellular Biology ◽

10.1128/mcb.00089-07 ◽

2007 ◽

Vol 27 (11) ◽

pp. 3900-3910 ◽

Cited By ~ 68

Author(s):

Meritxell Zapater ◽

Marc Sohrmann ◽

Matthias Peter ◽

Francesc Posas ◽

Eulàlia de Nadal

Keyword(s):

Gene Expression ◽

Rna Polymerase Ii ◽

Genetic Screening ◽

Essential Role ◽

Regulation Of Gene Expression ◽

Transcriptional Program ◽

Rna Pol Ii ◽

Pol Ii ◽

Genome Wide ◽

Transcriptional Complex

ABSTRACT Regulation of gene expression by the Hog1 stress-activated protein kinase is essential for proper cell adaptation to osmostress. Hog1 coordinates an extensive transcriptional program through the modulation of transcription. To identify systematically novel components of the transcriptional machinery required for osmostress-mediated gene expression, we performed an exhaustive genome-wide genetic screening, searching for mutations that render cells osmosensitive at high osmolarity and that are associated with reduced expression of osmoresponsive genes. The SAGA and Mediator complexes were identified as putative novel regulators of osmostress-mediated transcription. Interestingly, whereas Mediator is essential for osmostress gene expression, the requirement for SAGA is different depending on the strength of the extracellular osmotic conditions. At mild osmolarity, SAGA mutants show only very slight defects on RNA polymerase II (Pol II) recruitment and gene expression, whereas at severe osmotic conditions, SAGA mutants show completely impaired RNA Pol II recruitment and transcription of osmoresponsive genes. Thus, our results define an essential role for Mediator in osmostress gene expression and a selective role for SAGA under severe osmostress. Our results indicate that the requirement for a transcriptional complex to regulate a promoter might be determined by the strength of the stimuli perceived by the cell through the regulation of interactions between transcriptional complexes.

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Son Is Essential for Nuclear Speckle Organization and Cell Cycle Progression

Molecular Biology of the Cell ◽

10.1091/mbc.e09-02-0126 ◽

2010 ◽

Vol 21 (4) ◽

pp. 650-663 ◽

Cited By ~ 53

Author(s):

Alok Sharma ◽

Hideaki Takata ◽

Kei-ichi Shibahara ◽

Athanasios Bubulya ◽

Paula A. Bubulya

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Mrna Processing ◽

Mrna Splicing ◽

Scaffolding Protein ◽

Splicing Factors ◽

Nuclear Speckles ◽

Intact Cells ◽

Cycle Progression ◽

Processing Factors

Subnuclear organization and spatiotemporal regulation of pre-mRNA processing factors is essential for the production of mature protein-coding mRNAs. We have discovered that a large protein called Son has a novel role in maintaining proper nuclear organization of pre-mRNA processing factors in nuclear speckles. The primary sequence of Son contains a concentrated region of multiple unique tandem repeat motifs that may support a role for Son as a scaffolding protein for RNA processing factors in nuclear speckles. We used RNA interference (RNAi) approaches and high-resolution microscopy techniques to study the functions of Son in the context of intact cells. Although Son precisely colocalizes with pre-mRNA splicing factors in nuclear speckles, its depletion by RNAi leads to cell cycle arrest in metaphase and causes dramatic disorganization of small nuclear ribonuclear protein and serine-arginine rich protein splicing factors during interphase. Here, we propose that Son is essential for appropriate subnuclear organization of pre-mRNA splicing factors and for promoting normal cell cycle progression.

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TFIIF Facilitates Dissociation of RNA Polymerase II from Noncoding RNAs That Lack a Repression Domain

Molecular and Cellular Biology ◽

10.1128/mcb.01115-09 ◽

2009 ◽

Vol 30 (1) ◽

pp. 91-97 ◽

Cited By ~ 13

Author(s):

Stacey D. Wagner ◽

Jennifer F. Kugel ◽

James A. Goodrich

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Repression ◽

Noncoding Rnas ◽

Mrna Transcription ◽

Rna Pol Ii ◽

Pol Ii ◽

General Transcription Factors ◽

B2 Rna ◽

Repression Domain

ABSTRACT Noncoding RNAs (ncRNAs) have recently been found to regulate multiple steps in mammalian mRNA transcription. Mouse B2 RNA and human Alu RNA bind RNA polymerase II (Pol II) and repress mRNA transcription, using regions of the ncRNAs referred to as repression domains. Two other ncRNAs, mouse B1 RNA and human small cytoplasmic Alu (scAlu) RNA, bind Pol II with high affinity but lack repression domains and hence do not inhibit transcription. To better understand the interplay between ncRNAs that bind Pol II and their functions in transcription, we studied how Pol II binding and transcriptional repression are controlled by general transcription factors. We found that TFIIF associates with B1 RNA/Pol II and scAlu RNA/Pol II complexes and decreases their kinetic stability. Both subunits of TFIIF are required for this activity. Importantly, fusing a repression domain to B1 RNA stabilizes its interaction with Pol II in the presence of TFIIF. These results suggest a new role for TFIIF in regulating the interaction of ncRNAs with Pol II; specifically, it destabilizes interactions with ncRNAs that are not transcriptional repressors. These studies also identify a new function for ncRNA repression domains: they stabilize interactions of ncRNAs with Pol II in the presence of TFIIF.

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Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.62.2.465-503.1998 ◽

1998 ◽

Vol 62 (2) ◽

pp. 465-503 ◽

Cited By ~ 356

Author(s):

Michael Hampsey

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Regulatory Elements ◽

Transcriptional Repressors ◽

Rna Pol Ii ◽

Pol Ii ◽

The Core ◽

General Transcription Factors ◽

Transcriptional Machinery

SUMMARY Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

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Organization of transcription and pre-mRNA splicing within the mammalian cell nucleus

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014021x ◽

1995 ◽

Vol 53 ◽

pp. 768-769

Author(s):

D.L. Spector ◽

S. Huang ◽

S. Kaurin

Keyword(s):

Rna Polymerase Ii ◽

Cell Nucleus ◽

Functional Organization ◽

Mrna Splicing ◽

Splicing Factors ◽

Interchromatin Granule Clusters ◽

Interchromatin Granule ◽

Nuclear Regions ◽

Relationship Of ◽

Perichromatin Fibrils

We have been interested in the organization of RNA polymerase II transcription and pre-mRNA splicing within the cell nucleus. Several models have been proposed for the functional organization of RNA within the eukaryotic nucleus and for the relationship of this organization to the distribution of pre-mRNA splicing factors. One model suggests that RNAs which must be spliced are capable of recruiting splicing factors to the sites of transcription from storage and/or reassembly sites. When one examines the organization of splicing factors in the nucleus in comparison to the sites of chromatin it is clear that splicing factors are not localized in coincidence with heterochromatin (Fig. 1). Instead, they are distributed in a speckled pattern which is composed of both perichromatin fibrils and interchromatin granule clusters. The perichromatin fibrils are distributed on the periphery of heterochromatin and on the periphery of interchromatin granule clusters as well as being diffusely distributed throughout the nucleoplasm. These nuclear regions have been previously shown to represent initial sites of incorporation of 3H-uridine.

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The methyltransferase SETD2 couples transcription and splicing by engaging mRNA processing factors through its SHI domain

Nature Communications ◽

10.1038/s41467-021-21663-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Saikat Bhattacharya ◽

Michaella J. Levy ◽

Ning Zhang ◽

Hua Li ◽

Laurence Florens ◽

...

Keyword(s):

Rna Splicing ◽

Rna Binding ◽

Mrna Processing ◽

Key Factors ◽

Pol Ii ◽

Mrna Transcripts ◽

Hnrnp Protein ◽

Processing Factors

AbstractHeterogeneous ribonucleoproteins (hnRNPs) are RNA binding molecules that are involved in key processes such as RNA splicing and transcription. One such hnRNP protein, hnRNP L, regulates alternative splicing (AS) by binding to pre-mRNA transcripts. However, it is unclear what factors contribute to hnRNP L-regulated AS events. Using proteomic approaches, we identified several key factors that co-purify with hnRNP L. We demonstrate that one such factor, the histone methyltransferase SETD2, specifically interacts with hnRNP L in vitro and in vivo. This interaction occurs through a previously uncharacterized domain in SETD2, the SETD2-hnRNP Interaction (SHI) domain, the deletion of which, leads to a reduced H3K36me3 deposition. Functionally, SETD2 regulates a subset of hnRNP L-targeted AS events. Our findings demonstrate that SETD2, by interacting with Pol II as well as hnRNP L, can mediate the crosstalk between the transcription and the splicing machinery.

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