UBF Is An Epigenetic Factor Required For Ribosomal RNA Gene Activity And Preinitiation Complex Assembly But Not For Nucleolar Body Formation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE55450 | GEO | 2014/08/19
SECONDARY ACCESSION(S): PRJNA239664
REPOSITORIES: GEO
Similar Datasets
Project description: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 conditional gene deletion in mouse, we now show that UBF is indeed essential for transcription of the rRNA genes. Unexpectedly, arrest of rRNA synthesis does not affect RPIII transcription of 5S or tRNA genes, nor RPII expression of the hundreds of mRNAs implicated in ribosome biogenesis, but does upreguglate snRNAs and snoRNAs. Thus, rRNA gene activity does not coordinate global gene expression required for ribosome biogenesis. Conditional UBF flox/flox MEFs and immortalized MEFs and isogenic controls all carrying homozygous ER-Cre alleles were treated with 4-HT and mRNA analyzedat 0h and at 24h intervals posttreatment over 4 days.
2014-08-19 | E-GEOD-55450 | biostudies-arrayexpress
Project description:In recent years, several small molecule cytotoxic drugs have been identified as potential inhibitors of ribosome biogenesis (Drygin et al., 2011; Peltonen et al., 2014a; Peltonen et al., 2014b). CX-5461 is one such drug that has also demonstrated anticancer potential for a wide range of malignancies (Bywater et al., 2012; Cornelison et al., 2017; Devlin et al., 2015; Drygin et al., 2011; Hald et al., 2019; Hein et al., 2017; Ismael et al., 2019; Lawrence et al., 2018; Lee et al., 2017; Negi and Brown, 2015; Taylor et al., 2019; Xu et al., 2017; Yan et al., 2017) (Haddach et al., 2012), and is presently under phase I trials for the treatment of both hematological cancers and solid tumours (Group, 2016; Khot et al., 2019). CX-5461 was initially characterized as an inhibitor of RNA Polymerase I (RPI/PolR1/PolI) that is responsible for the synthesis of the major ribosomal RNAs and the initial step in ribosome biogenesis (Drygin et al., 2011). Since RPI and its corresponding core transcription factors are dedicated to this task alone, they present ideal molecular targets by which to modulate ribosome biogenesis. However, the specificity of CX-5461 has been questioned and it has been suggested that this drug may also act by stabilizing DNA G-quadruplexes or by “poisoning” topoisomerase II (Topo II). Thus, the primary target of this drug and its mode of action are still in doubt. Here we used Deconvolution-ChIP-Seq in NIH3T3 and HEK293T cells treated for different times with CX-5461. The data show that the primary target of CX5461 is the initiation of ribosomal RNA gene (rDNA) transcription. CX-5461 blocks transcription initiation in vitro and in vivo by arresting RNA polymerase I (RPI/Pol1) within the preinitiation complex. In contrast to previous suggestions, CX-5461 does not effect recruitment of the TBP-TAF complex SL1 to the rDNA promoter, the recruitment of the initiation competent RPI-Rrn3 complex or ongoing transcription elongation, arguing against a role for G-quadruplex stabilization or topoisomerase II poisoning. Inhibition of transcription by CX-5461 is not reversible, the RPI-Rrn3 complex remains arrested in the preinitiation complex even after drug removal. This leads to nucleolar stress, extensive DNA damage and cell senescence. Our data show that the cytotoxicity of CX-5461 is the downstream result of the highly specific inhibition of rDNA transcription. The observation that this inhibition is irreversible will be important for the future design of chemotherapeutic strategies and the avoidance of drug resistance.
2021-04-07 | E-MTAB-9242 | biostudies-arrayexpress
Project description:The 5S RNP is assembled from its three components (5S rRNA, Rpl5/uL18, and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2–p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-EM structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1–uL18–uL5, and upon further recruitment of two nucleolar factors Rpf2–Rrs1 develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unraveled how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.
2023-06-13 | PXD040087 | Pride
Project description:The 120-nt long 5S rRNA, is an indispensable component of cytoplasmic ribosomes in all living organisms. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether maintaining 5S rRNA as an independent molecule is critical for ribosome function and cell survival. By fusing circularly permutated 5S rRNA (cp5S) with 23S rRNA and deleting all wild type 5S rRNA genes, we generated an Escherichia coli strain completely devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is not required for cell growth at 37°C and is unlikely to have essential functions outside the ribosome. The fully-assembled ribosomes carrying 23S-cp5S hybrid rRNA and lacking free 5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits that are unable to form stable 70S ribosomes. Cryo-EM analysis revealed a dramatically malformed peptidyl transferase center in the misassembled 50S subunits. The results of our experiments argue that the key evolutionary force preserving the autonomous nature of the smallest rRNA is its role in ribosome biogenesis.
2020-06-18 | PXD019058 | Pride
Project description:The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.
2020-09-14 | GSE157208 | GEO
Project description:The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.
2020-09-14 | GSE137951 | GEO
Project description:Anabolic activities such as ribosome biogenesis and protein synthesis are linked to aging. Ribosomal RNA (rRNA) synthesis is the limiting step of ribosome biogenesis, thus dictating the number of ribosomes in cells and, consequently, the capacity for mRNA translation. Knockdown of the rRNA synthesis repressor, NCL-1, accelerated aging, whereas knocking down the transcription initiation factor C36E8.1 promoted longevity. This suggested that rRNA synthesis has a negative correlation with lifespan. To investigate the metabolic changes upon manipulation of rRNA synthesis the proteome of NCL-1 KD and C36E8.1 KD were analyzed at young, middle, and old age (AD2, AD6, AD12).
2024-01-11 | PXD028600 | Pride
Project description:In all domains of life, the rate of protein synthesis is directly linked to the rates of cell growth and proliferation. Consequently, highly proliferative cancer cells are especially sensitive to perturbations in ribosome biogenesis. While ribosome synthesis and cancer have a well-established relationship, only recently has ribosome biogenesis drawn interest as a cancer therapeutic target. Here, we have exploited the relationship between ribosome biogenesis and cancer cell proliferation by using a potent ribosome biogenesis inhibitor: RBI2 (Ribosome Biogenesis Inhibitor 2) to perturb cancer cell growth and viability. We demonstrate that RBI2 significantly decreases cell viability in malignant melanoma cells and breast cancer cell lines. Treatment with RBI2 dramatically and rapidly decreases ribosomal RNA (rRNA) synthesis, without affecting the occupancy of RNA polymerase I (Pol I) on the ribosomal DNA template. Next-generation RNA sequencing (RNA-seq) reveals that RBI2 and previously described ribosome biogenesis inhibitor CX-5461 induce distinct changes in the transcriptome. Investigation of the content of the pre-rRNAs through RT-qPCR reveals an increase in polyadenylation of cellular rRNA after treatment with RBI2, a known pathway by which rRNA degradation occurs. Northern blotting revealed that RBI2 does not appear to impair or alter rRNA processing. Collectively, these data suggest that RBI2 inhibits rRNA synthesis distinctly from other previously described ribosome biogenesis inhibitors, potentially through a novel pathway that upregulates turnover of premature rRNAs.
2021-11-08 | GSE188262 | GEO
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA. CLIP-seq was performed in Flag-SIRT7-293T cells.
2016-01-07 | E-GEOD-76578 | biostudies-arrayexpress
Project description:SIRT7 is an NAD+-dependent protein deacetylase with important roles in ribosome biogenesis and cell proliferation. Previous studies have established that SIRT7 is associated with RNA polymerase I, interacts with pre-rRNA and promotes rRNA synthesis. Here we show that SIRT7 is also associated with snoRNAs that are involved in pre-rRNA processing and rRNA maturation. Knockdown of SIRT7 impairs U3 snoRNA-dependent early cleavage steps that are necessary for generation of 18S rRNA. Mechanistically, SIRT7 deacetylates U3-55k, a core component of the U3 snoRNP complex, and reversible acetylation of U3-55k modulates the association of U3-55k with U3 snoRNA. Deacetylation by SIRT7 enhances U3-55k binding to U3 snoRNA, which is a prerequisite for pre-rRNA processing. Under stress conditions, SIRT7 is released from nucleoli, leading to hyperacetylation of U3-55k and attenuation of prerRNA processing. The results reveal a multifaceted role of SIRT7 in ribosome biogenesis, regulating both transcription and processing of rRNA.
2016-01-07 | GSE76578 | GEO