Rate Of rRNA Synthesis Research Articles

KMT2D Deficiency Promotes Myeloid Leukemias which Is Vulnerable to Ribosome Biogenesis Inhibition.

KMT2C and KMT2D are the most frequently mutated epigenetic genes in human cancers. While KMT2C is identified as a tumor suppressor in acute myeloid leukemia (AML), the role of KMT2D remains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported that KMT2D is downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells with Kmt2d loss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found that KMT2D deficiency leads to the activation of the mTOR pathway in both mouse and human AML cells. Kmt2d directly regulates the expression of Ddit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX-5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML with Kmt2d loss in vivo and extends the survival of leukemic mice. These studies validate KMT2D as a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

Glucose triggers strong taxon-specific responses in microbial growth and activity: insights from DNA and RNA qSIP.

Growth of soil microorganisms is often described as carbon limited, and adding labile carbon to soil often results in a transient and large increase in respiration. In contrast, soil microbial biomass changes little, suggesting that growth and respiration are decoupled in response to a carbon pulse. Alternatively, measuring bulk responses of the entire community (total respiration and biomass) could mask ecologically important variation among taxa in response to the added carbon. Here, we assessed taxon-specific variation in cellular growth (measured as DNA synthesis) and metabolic activity (measured as rRNA synthesis) following glucose addition to soil using quantitative stable isotope probing with H218 O. We found that glucose addition altered rates of DNA and rRNA synthesis, but the effects were strongly taxon specific: glucose stimulated growth and rRNA transcription for some taxa, and suppressed these for others. These contrasting taxon-specific responses could explain the small and transient changes in total soil microbial biomass. Responses to glucose were not well predicted by a priori assignments of taxa into copiotrophic or oligotrophic categories. Across all taxa, rates of DNA and rRNA synthesis changed in parallel, indicating that growth and activity were coupled, and the degree of coupling was unaffected by glucose addition. This pattern argues against the idea that labile carbon addition causes a large reduction in metabolic growth efficiency; rather, the large pulse of respiration observed with labile substrate addition is more likely to be the result of rapid turnover of microbial biomass, possibly due to trophic interactions. Our results support a strong connection between rRNA synthesis and bacterial growth, and indicate that taxon-specific responses among soil bacteria can buffer responses at the scale of the whole community.

Discovery of novel inhibitors of ribosome biogenesis by innovative high throughput screening strategies.

Over the past two decades, ribosome biogenesis has emerged as an attractive target for cancer treatment. In this study, two high-throughput screens were used to identify ribosome biogenesis inhibitors. Our primary screen made use of the HaloTag selective labeling strategy to identify compounds that decreased the abundance of newly synthesized ribosomes in A375 malignant melanoma cells. This screen identified 5786 hit compounds. A subset of those initial hit compounds were tested using a secondary screen that directly measured pre-ribosomal RNA (pre-rRNA) abundance as a reporter of rRNA synthesis rate, using quantitative RT-PCR. From the secondary screen, we identified two structurally related compounds that are potent inhibitors of rRNA synthesis. These two compounds, Ribosome Biogenesis Inhibitors 1 and 2 (RBI1 and RBI2), induce a substantial decrease in the viability of A375 cells, comparable to the previously published ribosome biogenesis inhibitor CX-5461. Anchorage-independent cell growth assays further confirmed that RBI2 inhibits cell growth and proliferation. Thus, the RBI compounds have promising properties for further development as potential cancer chemotherapeutics.

Nop-7-associated 2 (NSA2) is required for ribosome biogenesis and protein synthesis

Ribosome biogenesis is a fundamental cellular process and occurs mainly in the nucleolus in eukaryotes. The process is exceptionally complex and highly regulated by numerous ribosomal and non-ribosomal factors. A recent discovery strengthened the link between ribosome biogenesis and malignant transformation. Here, we determined that Nop-7-associated 2 (NSA2) is a nucleolar protein required for ribosome biogenesis. NSA2 knockdown reduced the rate of rRNA synthesis, diminishing the 60S ribosomal subunit. Moreover, we demonstrated that depletion of NSA2 suppressed protein synthesis. To investigate the signaling pathway affected by NSA2, NSA2 was depleted, which triggered the inactivation of the mTOR signaling pathway. Taken together, our findings reveal a novel function of NSA2 and provide insight into the regulation of ribosome biogenesis by NSA2.

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.

The Regulation of rRNA Gene Transcription during Directed Differentiation of Human Embryonic Stem Cells.

It has become increasingly clear that proper cellular control of pluripotency and differentiation is related to the regulation of rRNA synthesis. To further our understanding of the role that the regulation of rRNA synthesis has in pluripotency we monitored rRNA synthesis during the directed differentiation of human embryonic stem cells (hESCs). We discovered that the rRNA synthesis rate is reduced ~50% within 6 hours of ACTIVIN A treatment. This precedes reductions in expression of specific stem cell markers and increases in expression of specific germ layer markers. The reduction in rRNA synthesis is concomitant with dissociation of the Pol I transcription factor, UBTF, from the rRNA gene promoter and precedes any increase to heterochromatin throughout the rRNA gene. To directly investigate the role of rRNA synthesis in pluripotency, hESCs were treated with the Pol I inhibitor, CX-5461. The direct reduction of rRNA synthesis by CX-5461 induces the expression of markers for all three germ layers, reduces the expression of pluripotency markers, and is overall similar to the ACTIVIN A induced changes. This work indicates that the dissociation of UBTF from the rRNA gene, and corresponding reduction in transcription, represent early regulatory events during the directed differentiation of pluripotent stem cells.

ComGA-RelA interaction and persistence in the Bacillus subtilis K-state.

The bistably expressed K-state of Bacillus subtilis is characterized by two distinct features; transformability and arrested growth when K-state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA(+) protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA-dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)ppGpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)ppGpp in K-state cells, which are thus trapped in a non-growing state until ComGA is degraded. We show that some K-state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet-hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B. subtilis in the soil.

Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs).

CRM1 and its ribosome export adaptor NMD3 localize to the nucleolus and affect rRNA synthesis

CRM1 is an export factor that together with its adaptor NMD3 transports numerous cargo molecules from the nucleus to cytoplasm through the nuclear pore. Previous studies have suggested that CRM1 and NMD3 are detected in the nucleolus. However, their localization with subnucleolar domains or participation in the activities of the nucleolus are unclear. We demonstrate here biochemically and using imaging analyses that CRM1 and NMD3 co-localize with nucleolar marker proteins in the nucleolus. In particular, their nucleolar localization is markedly increased by inhibition of RNA polymerase I (Pol I) transcription by actinomycin D or by silencing Pol I catalytic subunit, RPA194. We show that CRM1 nucleolar localization is dependent on its activity and the expression of NMD3, whereas NMD3 nucleolar localization is independent of CRM1. This suggests that NMD3 provides nucleolar tethering of CRM1. While inhibition of CRM1 by leptomycin B inhibited processing of 28S ribosomal (r) RNA, depletion of NMD3 did not, suggesting that their effects on 28S rRNA processing are distinct. Markedly, depletion of NMD3 and inhibition of CRM1 reduced the rate of pre-47S rRNA synthesis. However, their inactivation did not lead to nucleolar disintegration, a hallmark of Pol I transcription stress, suggesting that they do not directly regulate transcription. These results indicate that CRM1 and NMD3 have complex functions in pathways that couple rRNA synthetic and processing engines and that the rRNA synthesis rate may be adjusted according to proficiency in rRNA processing and export.

P19ARF and RasV12 Offer Opposing Regulation of DHX33 Translation To Dictate Tumor Cell Fate

DHX33 is a pivotal DEAH-box RNA helicase in the multistep process of RNA polymerase I-directed transcription of the ribosomal DNA locus. We explored the regulation of DHX33 expression by Ras(V12) and ARF to determine DHX33's role in sensing these opposing signals to regulate ribosome biogenesis. In wild-type primary fibroblasts, Ras(V12) infection induced a transient increase in DHX33 protein level, as well as an rRNA transcriptional rate that was eventually suppressed by a delayed activation of the ARF/p53 pathway. DHX33 expression was exclusively controlled at the level of translation. ARF caused a dramatic reduction in polysome-associated DHX33 mRNAs, while Ras(V12) led to a complete shift of existing DHX33 mRNAs to actively translating polysomes. The translation of DHX33 by Ras(V12) was sensitive to inhibitors of phosphatidylinositol 3-kinase, mTOR, and mitogen-activated protein and was pivotal for enhanced rRNA transcription and enhanced overall cellular protein translation. In addition, DHX33 knockdown abolished Ras(V12)-induced rRNA transcription and protein translation and prevented both the in vitro and in vivo transforming properties of oncogenic Ras(V12). Our results directly implicate DHX33 as a crucial player in establishing rRNA synthesis rates in the face of Ras(V12) or ARF signals, adjusting ribosome biogenesis to match the appropriate growth or antigrowth signals.

The SWI/SNF Chromatin Remodeling Complex Influences Transcription by RNA Polymerase I in Saccharomyces cerevisiae

SWI/SNF is a chromatin remodeling complex that affects transcription initiation and elongation by RNA polymerase II. Here we report that SWI/SNF also plays a role in transcription by RNA polymerase I (Pol I) in Saccharomyces cerevisiae. Deletion of the genes encoding the Snf6p or Snf5p subunits of SWI/SNF was lethal in combination with mutations that impair Pol I transcription initiation and elongation. SWI/SNF physically associated with ribosomal DNA (rDNA) within the coding region, with an apparent peak near the 5′ end of the gene. In snf6Δ cells there was a ∼2.5-fold reduction in rRNA synthesis rate compared to WT, but there was no change in average polymerase occupancy per gene, the number of rDNA gene repeats, or the percentage of transcriptionally active rDNA genes. However, both ChIP and EM analyses showed a small but reproducible increase in Pol I density in a region near the 5′ end of the gene. Based on these data, we conclude that SWI/SNF plays a positive role in Pol I transcription, potentially by modifying chromatin structure in the rDNA repeats. Our findings demonstrate that SWI/SNF influences the most robust transcription machinery in proliferating cells.

The Paf1 complex is required for efficient transcription elongation by RNA polymerase I

Regulation of RNA polymerase I (Pol I) transcription is critical for controlling ribosome synthesis. Most previous investigations into Pol I transcription regulation have focused on transcription initiation. To date, the factors involved in the control of Pol I transcription elongation are poorly understood. The Paf1 complex (Paf1C) is a well-defined factor that influences polymerase II (Pol II) transcription elongation. We found that Paf1C associates with rDNA. Deletion of genes for Paf1C subunits (CDC73, CTR9, or PAF1) reduces the rRNA synthesis rate; however, there is no significant alteration of rDNA copy number or Pol I occupancy of the rDNA. Furthermore, EM analysis revealed a substantial increase in the frequency of large gaps between transcribing polymerases in ctr9Delta mutant cells compared with WT. Together, these data indicate that Paf1C promotes Pol I transcription through the rDNA by increasing the net rate of elongation. Thus, the multifunctional, conserved transcription factor Paf1C plays an important role in transcription elongation by Pol I in vivo.

Transcription of Multiple Yeast Ribosomal DNA Genes Requires Targeting of UAF to the Promoter by Uaf30

Upstream activating factor (UAF) is a multisubunit complex that functions in the activation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I). Cells lacking the Uaf30 subunit of UAF reduce the rRNA synthesis rate by approximately 70% compared to wild-type cells and produce rRNA using both Pol I and Pol II. Miller chromatin spreads demonstrated that even though there is an overall reduction in rRNA synthesis in uaf30 mutants, the active rDNA genes in such strains are overloaded with polymerases. This phenotype was specific to defects in Uaf30, as mutations in other UAF subunits resulted in a complete absence of rDNA genes with high or even modest Pol densities. The lack of Uaf30 prevented UAF from efficiently binding to the rDNA promoter in vivo, leading to an inability to activate a large number of rDNA genes. The relatively few genes that did become activated were highly transcribed, apparently to compensate for the reduced rRNA synthesis capacity. The results show that Uaf30p is a key targeting factor for the UAF complex that facilitates activation of a large proportion of rDNA genes in the tandem array.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Chromatin structure of ribosomal genes in Chironomus thummi (Diptera: Chironomidae): tissue specificity and behaviour under drug treatment

In eukaryotes the ribosomal gene population shows two different states in terms of chromatin structure. One subset is organized as nucleosomes (silent copies) while the other has a non-nucleosomal configuration (active copies). Insect cells are not the exception and this bimodal distribution of ribosomal chromatin also occurs in salivary gland cells, and cells of other larval tissues, of the midge Chironomus thummi. In run-on experiments on salivary glands cells we confirmed that transcribed rRNA genes show a non-nucleosomal configuration. The proportion of rRNA genes adopting an open, non-nucleosomal configuration was found to be tissue-dependent, suggesting that the population of unfolded ribosomal chromatin in C. thummi was established during cell differentiation. We propose that cell differentiation determines the fraction of non-nucleosomal rRNA gene copies and thus defines the range of possible rRNA synthesis rates in a particular cell type. In the salivary gland the fraction of unfolded chromatin was not significantly affected when transcription was repressed. However, transcription activation by pilocarpine led to a moderate increase in this fraction. These findings indicate that, in addition to a possible increase in the number of RNA-polymerases per transcribing rDNA unit, the proportion of transcribed ribosomal genes in differentiated cells can be modulated in response to an exceptional rRNA synthesis requirement.

Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30 degrees C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.