Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster

AbstractRibosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

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Repression of interrupted and intact rDNA by the SUMO pathway in Drosophila melanogaster

eLife ◽

10.7554/elife.52416 ◽

2020 ◽

Vol 9 ◽

Author(s):

Yicheng Luo ◽

Elena Fefelova ◽

Maria Ninova ◽

Yung-Chia Ariel Chen ◽

Alexei A Aravin

Keyword(s):

Drosophila Melanogaster ◽

Single Cells ◽

Rrna Genes ◽

Cell Heterogeneity ◽

Rdna Genes ◽

Variable Expression ◽

Essential Components ◽

Rdna Expression ◽

Sumo Pathway ◽

High Level

Ribosomal RNAs (rRNAs) are essential components of the ribosome and are among the most abundant macromolecules in the cell. To ensure high rRNA level, eukaryotic genomes contain dozens to hundreds of rDNA genes, however, only a fraction of the rRNA genes seems to be active, while others are transcriptionally silent. We found that individual rDNA genes have high level of cell-to-cell heterogeneity in their expression in Drosophila melanogaster. Insertion of heterologous sequences into rDNA leads to repression associated with reduced expression in individual cells and decreased number of cells expressing rDNA with insertions. We found that SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 are required for efficient repression of interrupted rDNA units and variable expression of intact rDNA. Disruption of the SUMO pathway abolishes discrimination of interrupted and intact rDNAs and removes cell-to-cell heterogeneity leading to uniformly high expression of individual rDNA in single cells. Our results suggest that the SUMO pathway is responsible for both repression of interrupted units and control of intact rDNA expression.

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Expression and amplification of the genes for ribosomal RNA in bobbed mutants of Drosophila melanogaster

Genetics Research ◽

10.1017/s0016672300022084 ◽

1985 ◽

Vol 45 (2) ◽

pp. 155-165 ◽

Cited By ~ 3

Author(s):

F. Lee Dutton ◽

Hallie M. Krider

Keyword(s):

Drosophila Melanogaster ◽

Sexual Dimorphism ◽

Ribosomal Rna ◽

Dosage Compensation ◽

Rrna Genes ◽

Rdna Genes ◽

Diploid Genome ◽

Minimum Dose ◽

Phenotypic Character ◽

Male Genotype

SummaryWe have employed stocks bearing clonally derived X chromosomes to investigate several features of the bobbed mutant syndrome, and the amplification of rDNA genes in D. melanogaster. We report that posterior macroscutellar bristle length correlates well with the rDNA content (i.e. dose of ivs–, or uninterrupted genes) in cloned X derivative strains. X/O males and X/X females with statistically indistinguishable rDNA contents have virtually identical bristle lengths. This indicates that (with respect to this phenotypic character) the rDNAs in these two genotypes are expressed equally, without apparent sexual dimorphism or dosage compensation. However, the severity of bobbed phenotype in terms of bristle morphology, turgite etching, and delayed eclosion is greater in the Xbb/XNO− female than in the Xbb/O male genotype for the alleles examined. We estimate the minimum dose of functioning rRNA genes required for viability at 26 δC to be 70 genes per diploid genome. We have examined the capacity of several X chromosomes which bear bobbed mutant alleles to compensate in X/O males, and find that disproportionate replication of these rDNAs does not take place. In contrast, at least one of the non-compensating bobbed alleles does appear to undergo rDNA magnification.

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Heterochromatic Stellate Gene Cluster in Drosophila melanogaster: Structure and Molecular Evolution

Genetics ◽

10.1093/genetics/146.1.253 ◽

1997 ◽

Vol 146 (1) ◽

pp. 253-262 ◽

Cited By ~ 2

Author(s):

Alexei V Tulin ◽

Galina L Kogan ◽

Dominik Filipp ◽

Maria D Balakireva ◽

Vladimir A Gvozdev

Keyword(s):

Drosophila Melanogaster ◽

Molecular Evolution ◽

Protein Kinase Ck2 ◽

Unknown Origin ◽

Open Reading Frames ◽

Β Subunit ◽

Retrotransposon Insertion ◽

High Level ◽

Kinase Ck2 ◽

Reading Frames

The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 β-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (∼14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.

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Non-Coding, RNAPII-Dependent Transcription at the Promoters of rRNA Genes Regulates Their Chromatin State in S. cerevisiae

Non-Coding RNA ◽

10.3390/ncrna7030041 ◽

2021 ◽

Vol 7 (3) ◽

pp. 41

Author(s):

Emma Lesage ◽

Jorge Perez-Fernandez ◽

Sophie Queille ◽

Christophe Dez ◽

Olivier Gadal ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Tandem Repeats ◽

Chromatin State ◽

Rrna Genes ◽

Rdna Genes ◽

Regulatory Pathways ◽

Chromatin States ◽

Non Coding Rnas ◽

Pervasive Transcription

Pervasive transcription is widespread in eukaryotes, generating large families of non-coding RNAs. Such pervasive transcription is a key player in the regulatory pathways controlling chromatin state and gene expression. Here, we describe long non-coding RNAs generated from the ribosomal RNA gene promoter called UPStream-initiating transcripts (UPS). In yeast, rDNA genes are organized in tandem repeats in at least two different chromatin states, either transcribed and largely depleted of nucleosomes (open) or assembled in regular arrays of nucleosomes (closed). The production of UPS transcripts by RNA Polymerase II from endogenous rDNA genes was initially documented in mutants defective for rRNA production by RNA polymerase I. We show here that UPS are produced in wild-type cells from closed rDNA genes but are hidden within the enormous production of rRNA. UPS levels are increased when rDNA chromatin states are modified at high temperatures or entering/leaving quiescence. We discuss their role in the regulation of rDNA chromatin states and rRNA production.

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Multiplexed profiling of single-cell extracellular vesicles secretion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814348116 ◽

2019 ◽

Vol 116 (13) ◽

pp. 5979-5984 ◽

Cited By ~ 22

Author(s):

Yahui Ji ◽

Dongyuan Qi ◽

Linmei Li ◽

Haoran Su ◽

Xiaojie Li ◽

...

Keyword(s):

Single Cell ◽

Cell Lines ◽

Extracellular Vesicles ◽

Single Cell Analysis ◽

Comprehensive Evaluation ◽

Single Cells ◽

Deep Understanding ◽

Principal Component ◽

Cell Heterogeneity ◽

Indispensable Tool

Extracellular vesicles (EVs) are important intercellular mediators regulating health and diseases. Conventional methods for EV surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EV secretion. Herein, by using spatially patterned antibody barcodes, we realized multiplexed profiling of single-cell EV secretion from more than 1,000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to a deep understanding of previously undifferentiated single-cell heterogeneity underlying EV secretion. Notably, we observed that the decrement of certain EV phenotypes (e.g.,CD63+EV) was associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EV secretion and cytokines secretion simultaneously from the same single cells to investigate the multidimensional spectrum of cellular communications, from which we resolved tiered functional subgroups with distinct secretion profiles by visualized clustering and principal component analysis. In particular, we found that different cell subgroups dominated EV secretion and cytokine secretion. The technology introduced here enables a comprehensive evaluation of EV secretion heterogeneity at single-cell level, which may become an indispensable tool to complement current single-cell analysis and EV research.

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Multiplexed Profiling of Single-cell Extracellular Vesicles Secretion

10.1101/459438 ◽

2018 ◽

Author(s):

Yahui Ji ◽

Dongyuan Qi ◽

Linmei Li ◽

Haoran Su ◽

Xiaojie Li ◽

...

Keyword(s):

Single Cell ◽

Cell Lines ◽

Extracellular Vesicles ◽

Single Cells ◽

Cell Communication ◽

Deep Understanding ◽

Surface Markers ◽

Cell Heterogeneity ◽

Health And Disease ◽

Cytokines Secretion

AbstractExtracellular vesicles (EVs) are important intercellular mediators regulating health and disease. Conventional EVs surface marker profiling, which was based on population measurements, masked the cell-to-cell heterogeneity in the quantity and phenotypes of EVs secretion. Herein, by using spatially patterned antibodies barcode, we realized multiplexed profiling of single-cell EVs secretion from more than 1000 single cells simultaneously. Applying this platform to profile human oral squamous cell carcinoma (OSCC) cell lines led to deep understanding of previously undifferentiated single cell heterogeneity underlying EVs secretion. Notably, we observed the decrement of certain EV phenotypes (e.g. CD63+EVs) were associated with the invasive feature of both OSCC cell lines and primary OSCC cells. We also realized multiplexed detection of EVs secretion and cytokines secretion simultaneously from the same single cells to investigate multidimensional spectrum of intercellular communications, from which we resolved three functional subgroups with distinct secretion profiles by visualized clustering. In particular, we found EVs secretion and cytokines secretion were generally dominated by different cell subgroups. The technology introduced here enables comprehensive evaluation of EVs secretion heterogeneity at single cell level, which may become an indispensable tool to complement current single cell analysis and EV research.SignificanceExtracellular vesicles (EVs) are cell derived nano-sized particles medicating cell-cell communication and transferring biology information molecules like nucleic acids to regulate human health and disease. Conventional methods for EV surface markers profiling can’t tell the differences in the quantity and phenotypes of EVs secretion between cells. To address this need, we developed a platform for profiling an array of surface markers on EVs from large numbers of single cells, enabling more comprehensive monitoring of cellular communications. Single cell EVs secretion assay led to previously unobserved cell heterogeneity underlying EVs secretion, which might open up new avenues for studying cell communication and cell microenvironment in both basic and clinical research.

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A new approach reveals syncytia within the visceral musculature ofDrosophila melanogaster

Development ◽

10.1242/dev.128.13.2517 ◽

2001 ◽

Vol 128 (13) ◽

pp. 2517-2524 ◽

Cited By ~ 1

Author(s):

Robert Klapper ◽

Sandra Heuser ◽

Thomas Strasser ◽

Wilfried Janning

Keyword(s):

Drosophila Melanogaster ◽

Single Cell ◽

Cell Transplantation ◽

Reporter Gene ◽

Mononuclear Cells ◽

Single Cells ◽

New Approach ◽

Transplantation Technique ◽

Somatic Musculature ◽

Somatic Muscles

In order to reveal syncytia within the visceral musculature of Drosophila melanogaster, we have combined the GAL4/UAS system with the single-cell transplantation technique. After transplantation of single cells from UAS-GFP donor embryos into ubiquitously GAL4-expressing recipients, the expression of the reporter gene was exclusively activated in syncytia containing both donor- and recipient-derived nuclei. In the first trial, we tested the system in the larval somatic musculature, which is already known to consist of syncytia. By this means we could show that most of the larval somatic muscles are generated by clonally non-related cells. Moreover, using this approach we were able to detect syncytia within the visceral musculature – a tissue that has previously been described as consisting of mononuclear cells. Both the longitudinal visceral musculature of the midgut and the circular musculature of the hindgut consist of syncytia and persist through metamorphosis. This novel application of the transplantation technique might be a powerful tool to trace syncytia in any organism using the GAL4/UAS system.

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Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila

Development ◽

10.1242/dev.114.1.165 ◽

1992 ◽

Vol 114 (1) ◽

pp. 165-171 ◽

Cited By ~ 5

Author(s):

G. Hime ◽

R. Saint

Keyword(s):

Drosophila Melanogaster ◽

Developmental Regulation ◽

Single Cells ◽

Embryonic Cell ◽

Cell Cycles ◽

Drosophila Embryogenesis ◽

Zygotic Transcription ◽

Homozygous Mutant ◽

Mitotic Figures

Mutations at the pebble locus of Drosophila melanogaster result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis revealed the presence of fewer and larger cells which contained enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining showed that the first thirteen rapid syncytial nuclear divisions proceeded normally in pebble mutant embryos. Following cellularization, the postblastoderm nuclear divisions occurred (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures were seen within single cells at the time of the cycle 15 mitoses. We conclude that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. We postulate that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis.

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Evidence That Runt Acts as a Counter-Repressor of Groucho During Drosophila melanogaster Primary Sex Determination

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401384 ◽

2020 ◽

Vol 10 (7) ◽

pp. 2487-2496

Author(s):

Sharvani Mahadeveraju ◽

Young-Ho Jung ◽

James W. Erickson

Keyword(s):

Drosophila Melanogaster ◽

Sex Determination ◽

Transcriptional Repression ◽

Biological Effects ◽

Regulatory Gene ◽

Animal Cells ◽

Interaction Domain ◽

Link Type ◽

High Level ◽

Runx Proteins

Runx proteins are bifunctional transcription factors that both repress and activate transcription in animal cells. Typically, Runx proteins work in concert with other transcriptional regulators, including co-activators and co-repressors to mediate their biological effects. In Drosophila melanogaster the archetypal Runx protein, Runt, functions in numerous processes including segmentation, neurogenesis and sex determination. During primary sex determination Runt acts as one of four X-linked signal element (XSE) proteins that direct female-specific activation of the establishment promoter (Pe) of the master regulatory gene Sex-lethal (Sxl). Successful activation of SxlPe requires that the XSE proteins overcome the repressive effects of maternally deposited Groucho (Gro), a potent co-repressor of the Gro/TLE family. Runx proteins, including Runt, contain a C-terminal peptide, VWRPY, known to bind to Gro/TLE proteins to mediate transcriptional repression. We show that Runt’s VWRPY co-repressor-interaction domain is needed for Runt to activate SxlPe. Deletion of the Gro-interaction domain eliminates Runt-ability to activate SxlPe, whereas replacement with a higher affinity, VWRPW, sequence promotes Runt-mediated transcription. This suggests that Runt may activate SxlPe by antagonizing Gro function, a conclusion consistent with earlier findings that Runt is needed for Sxl expression only in embryonic regions with high Gro activity. Surprisingly we found that Runt is not required for the initial activation of SxlPe. Instead, Runt is needed to keep SxlPe active during the subsequent period of high-level Sxl transcription suggesting that Runt helps amplify the difference between female and male XSE signals by counter-repressing Gro in female, but not in male, embryos.

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