Role of the RNA-binding protein ZC3H41 in the regulation of ribosomal protein messenger RNAs in trypanosomes.

BACKGROUND: Trypanosomes are single-celled eukaryotes that rely heavily on post-transcriptional mechanisms to regulate gene expression. RNA-binding proteins play essential roles in regulating the fate, abundance and translation of messenger RNAs (mRNAs). Among these, zinc finger proteins of the cysteine3histidine (CCCH) class have been shown to be key players in cellular processes as diverse as differentiation, regulation of the cell cycle and translation. ZC3H41 is an essential zinc finger protein that has been described as a component of spliced leader RNA granules and nutritional stress granules, but its role in RNA metabolism is unknown. METHODS: Cell cycle analysis in ZC3H41- and Z41AP-depleted cells was carried out using 4',6-diamidino-2-phenylindole staining, microscopic examination and flow cytometry. The identification of ZC3H41 protein partners was done using tandem affinity purification and mass spectrometry. Next-generation sequencing was used to evaluate the effect of ZC3H41 depletion on the transcriptome of procyclic Trypanosoma brucei cells, and also to identify the cohort of mRNAs associated with the ZC3H41/Z41AP complex. Levels of 5S ribosomal RNA (rRNA) species in ZC3H41- and Z41AP-depleted cells were assessed by quantitative reverse transcription-polymerase chain reaction. Surface sensing of translation assays were used to monitor global translation. RESULTS: We showed that depletion of the zinc finger protein ZC3H41 resulted in marked cell cycle defects and abnormal cell morphologies. ZC3H41 was found associated with an essential protein, which we named Z41AP, forming a stable heterodimer, and also with proteins of the poly(A)-binding protein 1 complex. The identification of mRNAs associated with the ZC3H41/Z41AP complex revealed that it is primarily composed of ribosomal protein mRNAs, and that binding to target transcripts is diminished upon nutritional stress. In addition, we observed that mRNAs encoding several proteins involved in the maturation of 5S rRNA are also associated with the ZC3H41/Z41AP complex. Finally, we showed that depletion of either ZC3H41 or Z41AP led to the accumulation of 5S rRNA precursors and a decrease of protein translation. CONCLUSIONS: We propose that ZC3H41 and Z41AP play important roles in controlling the fate of ribosomal components in response to environmental cues.

Features of smaller ribosomes in candidate phyla radiation (CPR) bacteria revealed with a molecular evolutionary analysis.

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100-150 amino acids, whereas the position of the peak varies in the range of 100-300 amino acids in free-living non-CPR bacteria, and at around 100-200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

Proteome-wide Capture of Co-translational Protein Dynamics in Bacillus subtilis Using TnDR, a Transposable Protein-Dynamics Reporter.

Dynamic protein maturation, such as localization, folding, and complex formation, can occur co-translationally. To what extent do nascent polypeptides engage in the co-translational dynamics to produce the functional proteome's complement? We address this question using a protein-dynamics reporter (DR) module comprising a force-sensitive arrest sequence (Bacillus subtilis MifM) followed in frame by LacZ. An engineered transposon, TnDR, carrying DR, is transposed into the B. subtilis chromosome to create translational fusions between N-terminal regions of proteins and the C-terminal DR module. By looking for LacZ+ colonies, we identify hundreds of proteins that cancel the elongation arrest, most probably reflecting their ability to initiate the maturation/localization process co-translationally. Case studies identify B. subtilis proteins that initiate assembly with a partner molecule before completion of translation. These results suggest that co-translational maturation is a frequently occurring event in protein biogenesis.

Nucleolar disruption, activation of P53 and premature senescence in POLR3A-mutated Wiedemann-Rautenstrauch syndrome fibroblasts.

Recently, mutations in the RNA polymerase III subunit A (POLR3A) have been described as the cause of the neonatal progeria or Wiedemann-Rautenstrauch syndrome (WRS). POLR3A has important roles in transcription regulation of small RNAs, including tRNA, 5S rRNA, and 7SK rRNA. We aim to describe the cellular and molecular features of WRS fibroblasts. Cultures of primary fibroblasts from one WRS patient [monoallelic POLR3A variant c.3772_3773delCT (p.Leu1258Glyfs*12)] and one control patient were cultured in vitro. The mutation caused a decrease in the expression of wildtype POLR3A mRNA and POLR3A protein and a sharp increase in mutant protein expression. In addition, there was an increase in the nuclear localization of the mutant protein. These changes were associated with an increase in the number and area of nucleoli and to a high increase in the expression of pP53 and pH2AX. All these changes were associated with premature senescence. The present observations add to our understanding of the differences between Hutchinson-Gilford progeria syndrome and WRS and opens new alternatives to study cell senesce and human aging.

MicroRNAs and long non-coding RNAs as novel regulators of ribosome biogenesis.

Ribosome biogenesis is the fine-tuned, essential process that generates mature ribosomal subunits and ultimately enables all protein synthesis within a cell. Novel regulators of ribosome biogenesis continue to be discovered in higher eukaryotes. While many known regulatory factors are proteins or small nucleolar ribonucleoproteins, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) are emerging as a novel modulatory layer controlling ribosome production. Here, we summarize work uncovering non-coding RNAs (ncRNAs) as novel regulators of ribosome biogenesis and highlight their links to diseases of defective ribosome biogenesis. It is still unclear how many miRNAs or lncRNAs are involved in phenotypic or pathological disease outcomes caused by impaired ribosome production, as in the ribosomopathies, or by increased ribosome production, as in cancer. In time, we hypothesize that many more ncRNA regulators of ribosome biogenesis will be discovered, which will be followed by an effort to establish connections between disease pathologies and the molecular mechanisms of this additional layer of ribosome biogenesis control.

Cancer-associated mutations in the ribosomal protein L5 gene dysregulate the HDM2/p53-mediated ribosome biogenesis checkpoint.

Perturbations in ribosome biogenesis have been associated with cancer. Such aberrations activate p53 through the RPL5/RPL11/5S rRNA complex-mediated inhibition of HDM2. Studies using animal models have suggested that this signaling pathway might constitute an important anticancer barrier. To gain a deeper insight into this issue in humans, here we analyze somatic mutations in RPL5 and RPL11 coding regions, reported in The Cancer Genome Atlas and International Cancer Genome Consortium databases. Using a combined computational and statistical approach, complemented by a range of biochemical and functional analyses in human cancer cell models, we demonstrate the existence of several mechanisms by which RPL5 mutations may impair wild-type p53 upregulation and ribosome biogenesis. Unexpectedly, the same approach provides only modest evidence for a similar role of RPL11, suggesting that RPL5 represents a preferred target during human tumorigenesis in cancers with wild-type p53. Furthermore, we find that several functional cancer-associated RPL5 somatic mutations occur as rare germline variants in general population. Our results shed light on the so-far enigmatic role of cancer-associated mutations in genes encoding ribosomal proteins, with implications for our understanding of the tumor suppressive role of the RPL5/RPL11/5S rRNA complex in human malignancies.

Oncogenic MYC Induces the Impaired Ribosome Biogenesis Checkpoint and Stabilizes p53 Independent of Increased Ribosome Content.

The role of MYC in regulating p53 stability as a function of increased ribosome biogenesis is controversial. On the one hand, it was suggested that MYC drives the overexpression of ribosomal proteins (RP)L5 and RPL11, which bind and inhibit HDM2, stabilizing p53. On the other, it has been proposed that increased ribosome biogenesis leads the consumption of RPL5/RPL11 into nascent ribosomes, reducing p53 levels and enhancing tumorigenesis. Here, we show that the components that make up the recently described impaired ribosome biogenesis checkpoint (IRBC) complex, RPL5, RPL11, and 5S rRNA, are reduced following MYC silencing. This leads to a rapid reduction in p53 protein half-life in an HDM2-dependent manner. In contrast, MYC induction leads to increased ribosome biogenesis and p53 protein stabilization. Unexpectedly, there is no change in free RPL5/RPL11 levels, but there is a striking increase in IRBC complex bound to HDM2. Our data support a cell-intrinsic tumor-suppressor response to MYC expression, which is presently being exploited to treat cancer. SIGNIFICANCE: Oncogenic MYC induces the impaired ribosome biogenesis checkpoint, which could be potentially targeted for cancer treatment.

Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-mediated immunity.

The sensor RIG-I detects double-stranded RNA derived from RNA viruses. Although RIG-I is also known to have a role in the antiviral response to DNA viruses, physiological RNA species recognized by RIG-I during infection with a DNA virus are largely unknown. Using next-generation RNA sequencing (RNAseq), we found that host-derived RNAs, most prominently 5S ribosomal RNA pseudogene 141 (RNA5SP141), bound to RIG-I during infection with herpes simplex virus 1 (HSV-1). Infection with HSV-1 induced relocalization of RNA5SP141 from the nucleus to the cytoplasm, and virus-induced shutoff of host protein synthesis downregulated the abundance of RNA5SP141-interacting proteins, which allowed RNA5SP141 to bind RIG-I and induce the expression of type I interferons. Silencing of RNA5SP141 strongly dampened the antiviral response to HSV-1 and the related virus Epstein-Barr virus (EBV), as well as influenza A virus (IAV). Our findings reveal that antiviral immunity can be triggered by host RNAs that are unshielded following depletion of their respective binding proteins by the virus.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna.

BACKGROUND: Polyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies. RESULTS: Molecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor. CONCLUSIONS: The obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Phylogeny of Helieae (Gentianaceae): Resolving taxonomic chaos in a Neotropical clade.

The monophyletic and Neotropical tribe Helieae of the worldwide family Gentianaceae (Gentianales, Asterids, Angiospermae) is well known for its problematic generic classifications. An initial phylogenetic analysis of Helieae shed light onto the relationships between genera, and indicated that traditional generic limits did not correspond to monophyletic groups. In order to obtain a more thorough understanding of generic relationships within the group, we enhanced sampling within the so-called Symbolanthus clade and performed phylogenetic analyses from DNA sequences from one plastid region (matK) and two nuclear regions (ITS and 5S-NTS), plus 112 morphological characters, which were analyzed separately and in combination, using parsimony and Bayesian approaches. A total of 83 individuals representing 20 genera and 51 species of Helieae were sampled; 13 species were included in this study solely based on their morphological characters. Ancestral character reconstructions were performed to identify potential synapomorphies of clades and patterns of homoplasy in the morphological dataset. Our results demonstrate that Prepusa is sister to the remainder of Helieae. Furthermore, the Macrocarpaea clade, the Irlbachia clade and the Symbolanthus clade were also recovered. Within the Symbolanthus clade, our results confirm that Calolisianthus and Chelonanthus are not monophyletic, and also contest the monophyly of Irlbachia as currently circumscribed. Specifically, two species of Calolisianthus group with the type species of Chelonanthus, while the other Calolisianthus species are more closely related to Tetrapollinia and Symbolanthus. Moreover, the green-white-flowered Chelonanthus species and Adenolisianthus are undoubtedly related to Helia and several analyses support Irlbachia pratensis as more closely related to the lineage including the type species of Chelonanthus described above The addition of new characters and taxa led to higher confidence in the relative position of some clades, as well as provided further support for a new generic circumscription of Calolisianthus, Chelonanthus, and Helia. Even though several morphological characters traditionally used in the taxonomy of the group were shown to be homoplasious, most clades can be diagnosed by a combination of morphological character states.

SIRT7 Is an RNA-Activated Protein Lysine Deacylase.

Mammalian SIRT7 is a member of the sirtuin family that regulates multiple biological processes including genome stability, metabolic pathways, stress responses, and tumorigenesis. SIRT7 has been shown to be important for ribosome biogenesis and transcriptional regulation. SIRT7 knockout mice exhibit complications associated with fatty liver and increased aging in hematopoietic stem cells. However, the molecular basis for its biological function remains unclear, in part due to the lack of efficient enzymatic activity in vitro. Previously, we have demonstrated that double-stranded DNA could activate SIRT7's deacetylase activity in vitro, allowing it to deacetylate H3K18 in the context of chromatin. Here, we show that RNA can increase the catalytic efficiency of SIRT7 even better and that SIRT7 can remove long chain fatty acyl groups more efficiently than removing acetyl groups. Truncation and mutagenesis studies revealed residues at both the amino and carboxyl termini of SIRT7 that are involved in RNA-binding and important for activity. RNA immunoprecipitation-sequencing (RIP-seq) identified ribosomal RNA (rRNA) as the predominant RNA binding partner of SIRT7. The associated RNA was able to effectively activate the deacetylase and defatty-acylase activities of SIRT7. Knockdown of SIRT7 increased the lysine fatty acylation of several nuclear proteins based on metabolic labeling with an alkyne-tagged fatty acid analog, supporting that the defatty-acylase activity of SIRT7 is physiologically relevant. These findings provide important insights into the biological functions of SIRT7, as well as an improved platform to develop SIRT7 modulators.

The pre-existing population of 5S rRNA effects p53 stabilization during ribosome biogenesis inhibition.

Pre-ribosomal complex RPL5/RPL11/5S rRNA (5S RNP) is considered the central MDM2 inhibitory complex that control p53 stabilization during ribosome biogenesis inhibition. Despite its role is well defined, the dynamic of 5S RNP assembly still requires further characterization. In the present work, we report that MDM2 inhibition is dependent by a pre-existing population of 5S rRNA.

Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress.

The nucleolus is a potent disease biomarker and a target in cancer therapy. Ribosome biogenesis is initiated in the nucleolus where most ribosomal (r-) proteins assemble onto precursor rRNAs. Here we systematically investigate how depletion of each of the 80 human r-proteins affects nucleolar structure, pre-rRNA processing, mature rRNA accumulation and p53 steady-state level. We developed an image-processing programme for qualitative and quantitative discrimination of normal from altered nucleolar morphology. Remarkably, we find that uL5 (formerly RPL11) and uL18 (RPL5) are the strongest contributors to nucleolar integrity. Together with the 5S rRNA, they form the late-assembling central protuberance on mature 60S subunits, and act as an Hdm2 trap and p53 stabilizer. Other major contributors to p53 homeostasis are also strictly late-assembling large subunit r-proteins essential to nucleolar structure. The identification of the r-proteins that specifically contribute to maintaining nucleolar structure and p53 steady-state level provides insights into fundamental aspects of cell and cancer biology.

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

Down-regulation of 5S rRNA by miR-150 and miR-383 enhances c-Myc-rpL11 interaction and inhibits proliferation of esophageal squamous carcinoma cells.

5S rRNA plays an important part in ribosome biology and is over-expression in multiple cancers. In this study, we found that 5S rRNA is a direct target of miR-150 and miR-383 in esophageal squamous cell carcinoma (ESCC). Overexpression of miR-150 and miR-383 inhibited ESCC cell proliferation in vitro and in vivo. Moreover, 5S rRNA silencing by miR-150 and miR-383 might intensify rpL11-c-Myc interaction, which attenuated role of c-Myc as an oncogenic transcriptional factor and dysregulation of multiple c-Myc target genes. Taken together, our results highlight the involvement of miRNAs in ribosomal regulation during tumorigenesis.

Chaperoning 5S RNA assembly.

In eukaryotes, three of the four ribosomal RNAs (rRNAs)­the 5.8S, 18S, and 25S/28S rRNAs­are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into preribosomes. In mammals, the 5S RNP is also a central regulator of the homeostasis of the tumor suppressor p53. The nucleolar localization of the 5S RNP and its assembly into preribosomes are performed by a specialized complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterization of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA, and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialized 5S RNA E-loop-binding module, contacts the Rpl5 protein, and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in preribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.

Perturbation of ribosome biogenesis drives cells into senescence through 5S RNP-mediated p53 activation.

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.

The 5S RNP couples p53 homeostasis to ribosome biogenesis and nucleolar stress.

Several proto-oncogenes and tumor suppressors regulate the production of ribosomes. Ribosome biogenesis is a major consumer of cellular energy, and defects result in p53 activation via repression of mouse double minute 2 (MDM2) homolog by the ribosomal proteins RPL5 and RPL11. Here, we report that RPL5 and RPL11 regulate p53 from the context of a ribosomal subcomplex, the 5S ribonucleoprotein particle (RNP). We provide evidence that the third component of this complex, the 5S rRNA, is critical for p53 regulation. In addition, we show that the 5S RNP is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene overexpression. Our data show that the abundance of the 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation and ribosome integration, including the tumor suppressor PICT1. The 5S RNP therefore emerges as the critical coordinator of signaling pathways that couple cell proliferation with ribosome production.

5S ribosomal RNA is an essential component of a nascent ribosomal precursor complex that regulates the Hdm2-p53 checkpoint.

Recently, we demonstrated that RPL5 and RPL11 act in a mutually dependent manner to inhibit Hdm2 and stabilize p53 following impaired ribosome biogenesis. Given that RPL5 and RPL11 form a preribosomal complex with noncoding 5S ribosomal RNA (rRNA) and the three have been implicated in the p53 response, we reasoned they may be part of an Hdm2-inhibitory complex. Here, we show that small interfering RNAs directed against 5S rRNA have no effect on total or nascent levels of the noncoding rRNA, though they prevent the reported Hdm4 inhibition of p53. To achieve efficient inhibition of 5S rRNA synthesis, we targeted TFIIIA, a specific RNA polymerase III cofactor, which, like depletion of either RPL5 or RPL11, did not induce p53. Instead, 5S rRNA acts in a dependent manner with RPL5 and RPL11 to inhibit Hdm2 and stabilize p53. Moreover, depletion of any one of the three components abolished the binding of the other two to Hdm2, explaining their common dependence. Finally, we demonstrate that the RPL5/RPL11/5S rRNA preribosomal complex is redirected from assembly into nascent 60S ribosomes to Hdm2 inhibition as a consequence of impaired ribosome biogenesis. Thus, the activation of the Hdm2-inhibitory complex is not a passive but a regulated event, whose potential role in tumor suppression has been recently noted.

Regulation of SirT1-nucleomethylin binding by rRNA coordinates ribosome biogenesis with nutrient availability.

Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of pre-rRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators.