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

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.

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

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.

Unbiased K-mer Analysis Reveals Changes in Copy Number of Highly Repetitive Sequences During Maize Domestication and Improvement

The major component of complex genomes is repetitive elements, which remain recalcitrant to characterization. Using maize as a model system, we analyzed whole genome shotgun (WGS) sequences for the two maize inbred lines B73 and Mo17 using k-mer analysis to quantify the differences between the two genomes. Significant differences were identified in highly repetitive sequences, including centromere repeats, 45S ribosomal DNA (rDNA), knob, and telomere repeats. Previously unknown genotype specific 45S rDNA sequences were discovered. The B73-specific 45S rDNA is not only located on the nucleolus organizer region (NOR) on chromosome 6 but also dispersed on all the chromosomes in B73, indicating the relatively recent spread of 45S rDNA from the NOR. The B73 and Mo17 polymorphic k-mers were used to examine allele-specific expression of 45S rDNA. Although Mo17 contains higher copy number than B73, equivalent levels of overall 45S rDNA expression indicates that dosage compensation operates for the 45S rDNA in the hybrids. Using WGS sequences of B73xMo17 double haploids (DHs), genomic locations showing differential repetitive contents were genetically mapped. Analysis of WGS sequences of HapMap2 lines, including maize wild progenitor teosintes, landraces, and improved lines, decreases and increases in abundance of additional sets of k-mers associated with centromere repeats, 45S rDNA, knob, and retrotransposon sequences were found between teosinte and maize lines, revealing global evolutionary trends of genomic repeats during maize domestication and improvement.

Nucleolar dominance and maternal control of 45S rDNA expression

Using a system of interspecies hybrids, trihybrids, and recombinants with varying proportions of genomes from three distinct Xenopus species, we provide evidence for de novo epigenetic silencing of paternal 45S ribosomal ribonucleic acid (rRNA) genes and their species-dependent expression dominance that escapes transcriptional inactivation after homologous recombination. The same pattern of imprinting is maintained in the offspring from mothers being genetic males (ZZ) sex-reversed to females, indicating that maternal control of ribosomal deoxyribonucleic acid (rDNA) expression is not sex-chromosome linked. Nucleolar dominance (nucleolus underdevelopment) in Xenopus hybrids appears to be associated with a major non-Mendelian reduction in the number of 45S rDNA gene copies rather than a specific pattern of their expression. The loss of rRNA gene copies in F 1 hybrids was non-random with respect to the parental species, with the transcriptionally dominant variant preferentially removed from hybrid zygotes. This dramatic disruption in the structure and function of 45S rDNA impacts transcriptome patterns of small nucleolar RNAs and messenger RNAs, with genes from the ribosome and oxidative stress pathways being among the most affected. Unorthodoxies of rDNA inheritance and expression may be interpreted as hallmarks of genetic conflicts between parental genomes, as well as defensive epigenetic mechanisms employed to restore genome integrity.

Mutational Analysis of the Structure and Localization of the Nucleolus in the Yeast Saccharomyces cerevisiae

The nucleolus in Saccharomyces cerevisiae is a crescent-shaped structure that makes extensive contact with the nuclear envelope. In different chromosomal rDNA deletion mutants that we have analyzed, the nucleolus is not organized into a crescent structure, as determined by immunofluorescence microscopy, fluorescence in situ hybridization, and electron microscopy. A strain carrying a plasmid with a single rDNA repeat transcribed by RNA polymerase I (Pol I) contained a fragmented nucleolus distributed throughout the nucleus, primarily localized at the nuclear periphery. A strain carrying a plasmid with the 35S rRNA coding region fused to the GAL7 promoter and transcribed by Pol II contained a rounded nucleolus that often lacked extensive contact with the nuclear envelope. Ultrastructurally distinct domains were observed within the round nucleolus. A similar rounded nucleolar morphology was also observed in strains carrying the Pol I plasmid in combination with mutations that affect Pol I function. In a Pol I–defective mutant strain that carried copies of the GAL7-35S rDNA fusion gene integrated into the chromosomal rDNA locus, the nucleolus exhibited a round morphology, but was more closely associated with the nuclear envelope in the form of a bulge. Thus, both the organization of the rDNA genes and the type of polymerase involved in rDNA expression strongly influence the organization and localization of the nucleolus.

Meiotic imbalance in laboratory-produced hybrid males of Chorthippus parallelus parallelus and Chorthippus parallelus erythropus

SummaryThe grasshopper Chorthippus parallelus has two quite distinct subspecies, which meet along the Pyrenees forming a hybrid zone. Using silver staining we show that on the French side Cp. parallelus has three nucleolar organizer regions, on the L2, L3 and X chromosomes, while on the Spanish side Cp. erythropus has only two NORs, on the L2 and L3. Laboratory F1 hybrid males show reciprocal differences in the expression of NORs. When a Cp. erythropus is female parent the male progeny show four active NORs in mitotic cells and two silver precipitates in meiotic cells, as expected. But when a Cp. parallelus female donates the X with a NOR, her male offspring have a variable disrupted nucleolar expression. Some NORs are not expressed and extra sites of cryptic rDNA are revealed. Meiosis is more disturbed in this latter F1 cross with higher levels of polyploidy, but both Fls show around 90% spermatid abnormality. Such variation in rDNA expression is also found in individuals collected from the hybrid zone, and the role of this disturbance in affecting fitness is discussed.