Regulation Of Ribosome Biogenesis Research Articles

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

SUMMARYA central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena—from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.

Ribosome biogenesis and the cellular energy economy

Cell growth relies upon the ability to produce new proteins, which requires energy and chemical precursors, and an adequate supply of the molecular machines for protein synthesis - ribosomes. Although not widely appreciated, ribosomes are remarkably abundant in all cells. For example, in a rapidly growing yeast cell there are ∼2-4 x 105 ribosomes, produced and exported to the cytoplasm at a rate of ∼2,000-4,000 per minute, with ribosomal proteins making up ∼50% of total cellular protein number and ∼30% of cellular protein mass. Even in a typical human cell ribosomal proteins constitute ∼4-6% of total protein mass, and ribosomes are present at ∼107 per cell. We begin this primer by exploring the tight relationship between ribosome production and cell growth, which has important implications not just for the cell's global protein expression profile and maximum growth rate, but also for the molecular composition of the ribosome itself. We then discuss how and to what extent the expression of the RNA and protein components of ribosomes is fine-tuned to match the cell's needs and minimise waste. Finally, we highlight the importance of coordinated ribosomal RNA (rRNA) and ribosomal protein expression in eukaryotes and explore how defects in this process are associated with proteotoxicity and disease. A central underlying question addressed throughout is whether regulation of ribosome biogenesis has evolved to optimise energy efficiency or is instead (or in addition) driven by other goals, such as maximising cell growth rate, promoting adaptation to changing environmental conditions, or maintaining the stability of the cellular proteome.

TRAP1 regulates the response of colorectal cancer cells to hypoxia and inhibits ribosome biogenesis under conditions of oxygen deprivation.

Metabolic rewiring fuels rapid cancer cell proliferation by promoting adjustments in energetic resources, and increasing glucose uptake and its conversion into lactate, even in the presence of oxygen. Furthermore, solid tumors often contain hypoxic areas and can rapidly adapt to low oxygen conditions by activating hypoxia inducible factor (HIF)-1α and several downstream pathways, thus sustaining cell survival and metabolic reprogramming. Since TNF receptor-associated protein 1 (TRAP1) is a HSP90 molecular chaperone upregulated in several human malignancies and is involved in cancer cell adaptation to unfavorable environments and metabolic reprogramming, in the present study, its role was investigated in the adaptive response to hypoxia in human colorectal cancer (CRC) cells and organoids. In the present study, glucose uptake, lactate production and the expression of key metabolic genes were evaluated in TRAP1-silenced CRC cell models under conditions of hypoxia/normoxia. Whole genome gene expression profiling was performed in TRAP1-silenced HCT116 cells exposed to hypoxia to establish the role of TRAP1 in adaptive responses to oxygen deprivation. The results revealed that TRAP1 was involved in regulating hypoxia-induced HIF-1α stabilization and glycolytic metabolism and that glucose transporter 1 expression, glucose uptake and lactate production were partially impaired in TRAP1-silenced CRC cells under hypoxic conditions. At the transcriptional level, the gene expression reprogramming of cancer cells driven by HIF-1α was partially inhibited in TRAP1-silenced CRC cells and organoids exposed to hypoxia. Moreover, Gene Set Enrichment Analysis of TRAP1-silenced HCT116 cells exposed to hypoxia demonstrated that TRAP1 was involved in the regulation of ribosome biogenesis and this occurred with the inhibition of the mTOR pathway. Therefore, as demonstrated herein, TRAP1 is a key factor in maintaining HIF-1α-induced genetic/metabolic program under hypoxic conditions and may represent a promising target for novel metabolic therapies.

RRS1 knockdown inhibits the proliferation of neuroblastoma cell via PI3K/Akt/NF-κB pathway.

RRS1 plays an important role in regulating ribosome biogenesis. Recently, RRS1 has emerged as an oncoprotein involved in tumorigenicity of some cancers. However its role in neuroblastoma remains unknown. RRS1 expression was detected in pediatric neuroblastoma patients' tissues and cell lines. The effects of RRS1 knockdown on proliferation, apoptosis, and cell cycle were evaluated in neuroblastoma cell lines. RRS1-related survival pathway was analyzed by co-immunoprecipitation (Co-IP), mass spectrometry, reverse transcription-quantitative real-time PCR (RT-qPCR), and western blot. Protein-protein interaction (PPI) network was constructed using Cytoscape software and the STRING databases. Increased RRS1 level was found in neuroblastoma cases (35.6%) and cell lines. High RRS1 expression levels were associated with poor prognosis. RRS1 knockdown inhibited cell proliferation, induced apoptosis, and caused cell cycle arrest in SK-N-AS and SH-SY5Y cells. Co-IP and mass spectrometry analysis showed that RRS1 affects PI3K/Akt and nuclear factor κB (NF-κB) pathways. RT-qPCR and western blot results revealed that RRS1 knockdown inhibited the PI3K/Akt/NF-κB pathway through dephosphorylation of key proteins. In PPI network, AKT, PI3K, and P65 connected RRS1 with differentially expressed proteins more closely. This study suggests RRS1 knockdown may inhibit neuroblastoma cell proliferation by the PI3K/Akt/NF-κB pathway. Therefore, RRS1 may be a potential target for neuroblastoma treatment. RRS1 is involved in the progression of neuroblastoma. Knockdown of RRS1 contributes to inhibit the survival of neuroblastoma cells. RRS1 is associated with the PI3K/Akt/NF-κB signaling pathway in neuroblastoma cells. RRS1 may be a promising target for neuroblastoma therapy.

The pathophysiology of cobalamin (vitamin B12) deficiency syndromes impacting development

CblC is the most common inborn error of intracellular cobalamin metabolism and due to mutations in MMACHC. Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC‐like disease. However, additional de‐regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated the first mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

DNA Methylation Analysis of Ribosomal DNA in Adults With Down Syndrome.

Control of ribosome biogenesis is a critical aspect of the regulation of cell metabolism. As ribosomal genes (rDNA) are organized in repeated clusters on chromosomes 13, 14, 15, 21, and 22, trisomy of chromosome 21 confers an excess of rDNA copies to persons with Down syndrome (DS). Previous studies showed an alteration of ribosome biogenesis in children with DS, but the epigenetic regulation of rDNA genes has not been investigated in adults with DS so far. In this study, we used a targeted deep-sequencing approach to measure DNA methylation (DNAm) of rDNA units in whole blood from 69 adults with DS and 95 euploid controls. We further evaluated the expression of the precursor of ribosomal RNAs (RNA45S) in peripheral blood mononuclear cells (PBMCs) from the same subjects. We found that the rDNA promoter tends to be hypermethylated in DS concerning the control group. The analysis of epihaplotypes (the combination of methylated and unmethylated CpG sites along the same DNA molecule) showed a significantly lower intra-individual diversity in the DS group, which at the same time was characterized by a higher interindividual variability. Finally, we showed that RNA45S expression is lower in adults with DS. Collectively, our results suggest a rearrangement of the epigenetic profile of rDNA in DS, possibly to compensate for the extranumerary rDNA copies. Future studies should assess whether the regulation of ribosome biogenesis can contribute to the pathogenesis of DS and explain the clinical heterogeneity characteristic of the syndrome.

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions.

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

Ribosomal Proteins and Cancer

The translation process consists of translation factors and ribosomes. Ribosomal components include ribosomal proteins (RP) and ribosomal RNA. Many RPs are involved in assembling ribosomal particles and or stabilizing important regions of rRNA. Besides their conventional roles, RPs have been reported to exhibit secondary functions that have not yet been fully characterized in other cellular processes such as DNA repair, apoptosis, drug resistance, proliferation, and growth inhibition. Since cancer cells require a large amount of protein, they need ribosomes that work much more efficiently than normal cells. Several tumor suppressors and oncogenic proteins control the progression of cancer cells by regulating ribosome biogenesis and protein synthesis. Interestingly, free RPs also have diverse roles in tumorigenesis or tumor suppression. The physiological link between RPs and cancers has been extensively reviewed and elucidated on several pathways, including their interaction with the p53-MDM2 complex. The first evidence of an association between RPs and cancer came from observing the haploinsufficiency of eS4 in Turner Syndrome and eS19 mutation in Diamond-Blackfan Anemia. In the following years, the roles of different RPs in various cancer types such as colorectal cancer, breast cancer, lung adenocarcinoma, T-cell acute lymphoblastic leukemia, prostate cancer, breast cancer, gastric carcinomas, ovarian cancer, and liver cancer have been the subject of research. Apart from their effects on carcinogenesis, it was also emphasized that RPs could be evaluated as predictive biomarkers for diagnosis, prognosis, and treatment for some cancer types. In addition, some studies have been conducted on the use of these proteins in cancer treatment. Identifying novel extra-ribosomal functions of some RPs has identified these proteins as a new class of oncogenic or tumor suppressor factors. Suppression and stimulation of the expression of these novel oncogenic and tumor suppressor proteins, respectively, are considered could open up new therapeutic strategies in cancer therapy.

Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

Krüppel-like factor 7 influences translation and pathways involved in ribosomal biogenesis in breast cancer

BackgroundRibosomal biogenesis and ribosomal proteins have attracted attention in the context of tumor biology in recent years. Instead of being mere translational machineries, ribosomes might play an active role in tumor initiation and progression. Despite its importance, regulation of ribosomal biogenesis is still not completely understood.MethodsUsing Gene Set Enrichment Analysis of RNA sequencing and proteomical mass spectrometry data in breast cancer cells expressing Krüppel-like factor 7 (KLF7), we identified processes altered by this transcription factor. In silico analyses of a cohort of breast cancer patients in The Cancer Genome Atlas confirmed our finding. We further verified the role of KLF7 the identified ribosomal processes in in vitro assays of mammary carcinoma cell lines and analyses of breast cancer patients’ tissue slices.ResultsWe identified the transcription factor Krüppel-like factor 7 (KLF7) as a regulator of ribosomal biogenesis and translation in breast cancer cells and tissue. Highly significant overlapping processes related to ribosomal biogenesis were identified in proteomics and transcriptomics data and confirmed in patients’ breast cancer RNA Seq data. Further, nucleoli, the sites of ribosomal biogenesis, were morphologically altered and quantitatively increased in KLF7-expressing cells. Pre-rRNA processing was identified as one potential process affected by KLF7. In addition, an increase in global translation independent from proliferation and transcription was observed upon exogenous KLF7 expression in vitro. Importantly, in a cohort of breast cancer patients, KLF7-expression levels correlated with aggressiveness of the intrinsic breast cancer subtype and tumor grading. Moreover, KLF7 correlated with nucleolar characteristics in human breast tumor tissue, indicating a role for KLF7 in ribosomal biogenesis.ConclusionIn mammary carcinoma, KLF7 is involved in ribosomal biogenesis. Alterations of ribosomal biogenesis has far reaching quantitative and qualitative implications for the proteome of the cancer cells. This might influence the aggressiveness of cancer cells.

ВКЛАД mTORC1 И GSK-3 В РЕГУЛЯЦИЮ БИОГЕНЕЗА РИБОСОМ В ПОСТУРАЛЬНОЙ МЫШЦЕ КРЫСЫ В УСЛОВИЯХ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ

The investigation was aimed to reveal a potential role of mechanistic target of rapamycin (mTORC1) and glycogen synthase kinase (GSK-3) in the regulation of ribosome biogenesis and protein synthesis in rat soleus muscle during simulated gravitational unloading. Wistar rats were subject to 7-day tail-suspension (hindlimb unloading) and treated with rapamycin (mTORC1 inhibitor) or AR-A014418 (GSK-3 inhibitor). Protein synthesis was measured with SUnSET. Phosphorylation of glycogen synthase-1 (GS-1) and ribosomal protein S6 (rpS6) was measured with the use of Western-blotting. Levels of 28S and 18S rRNA were determined using agarose gel electrophoresis. Expression of 47S pre-rRNA and c-Myc was assessed with RT-PCR. Gravitational unloading led to an increase in phosphorylation of GS-1 (a marker of GSK-3 activity) and inhibited phosphorylation of rpS6 (a marker of mTORC1 activity), expression of the ribosome biogenesis markers (total RNA РНК, 28S and 18S rRNA, 47S pre-rRNA, с-Myc) and protein synthesis in soleus muscle. GSK-3 inhibition prevented an increase in GS-1 phosphorylation and, partly, a reduction in the markers of both ribosomal biogenesis and protein synthesis. The mTORC1 inhibitor contributed to the phospho-rpS6 reduction but did not substantially influence the parameters of translational capacity and protein synthesis. To sum up, unlike mTORC1, an elevated GSK-3 activity can contribute to the inhibition of ribosome biogenesis and protein synthesis in rat soleus muscle under 7-day gravitational unloading.

Meioc-Piwil1 Complexes Regulate Ribosome Biogenesis for the Differentiation of Germline Stem Cells

Meioc-Piwil1 Complexes Regulate Ribosome Biogenesis for the Differentiation of Germline Stem Cells

Airway Basal Cells Mediate Hypoxia-Induced EMT by Increasing Ribosome Biogenesis.

Excessive secretion of airway mucus and fluid accumulation are the common features of many respiratory diseases, which, in turn, induce cell hypoxia in the airway epithelium, resulting in epithelial–mesenchymal transition (EMT) and ultimately fibrosis. However, the mechanisms of EMT induced by hypoxia in the airway are currently unclear. To mimic the status of edematous fluid retention in the airway, we cultured primary mouse tracheal epithelial cells (MTECs) in a liquid–liquid interface (LLI) mode after full differentiation in a classic air–liquid interface (ALI) culture system. The cell hypoxia was verified by the physical characteristics and lactate production in cultured medium as well as HIF expression in MTECs cultured by LLI mode. EMT was evidenced and mainly mediated by basal cells, supported by flow cytometry and immunofluorescence assay. The differently expressed genes of basal and other airway epithelial cells were found to be enriched in the ribosome by our analysis of an MTEC single-cell RNA sequencing data set and Myc, the global regulator of ribosome biogenesis was identified to be highly expressed in basal cells. We next separated basal cells from bulk MTECs by flow cytometry, and the real-time PCR results showed that ribosome biogenesis was significantly upregulated in basal cells, whereas the inhibition of ribosome biogenesis alleviated the phosphorylation of the mammalian target of rapamycin/AKT and abrogated hypoxia-induced EMT in MTECs. Collectively, these observations strongly suggest that basal cells in the airway epithelium may mediate the process of hypoxia-induced EMT, partly through enhancing ribosome biogenesis.

Oxidative damage-induced hyperactive ribosome biogenesis participates in tumorigenesis of offspring by cross-interacting with the Wnt and TGF-\u03b21 pathways in IVF embryos

In vitro fertilization (IVF) increases the risk of tumorigenesis in offspring. The increased oxidative damage during IVF may be involved in tumor formation. However, the molecular mechanisms underlying this phenomenon remain largely unclear. Using a well-established model of oxidatively damaged IVF mouse embryos, we applied the iTRAQ method to identify proteins differentially expressed between control and oxidatively damaged zygotes and explored the possible tumorigenic mechanisms, especially with regard to the effects of oxidative damage on ribosome biogenesis closely related to tumorigenesis. The iTRAQ results revealed that ribosomal proteins were upregulated by oxidative stress through the Nucleolin/β-Catenin/n-Myc pathway, which stimulated ribosomes to synthesize an abundance of repair proteins to correct the damaged DNA/chromosomes in IVF-derived embryos. However, the increased percentages of γH2AX-positive cells and apoptotic cells in the blastocyst suggested that DNA repair was insufficient, resulting in aberrant ribosome biogenesis. Overexpression of ribosomal proteins, particularly Rpl15, which gradually increased from the 1-cell to 8-cell stages, indicated persistent hyperactivation of ribosome biogenesis, which promoted tumorigenesis in offspring derived from oxidatively damaged IVF embryos by selectively enhancing the translation of β-Catenin and TGF-β1. The antioxidant epigallocatechin-3-gallate (EGCG) was added to the in vitro culture medium to protect embryos from oxidative damage, and the expression of ribosome-/tumor-related proteins returned to normal after EGCG treatment. This study suggests that regulation of ribosome biogenesis by EGCG may be a means of preventing tumor formation in human IVF-derived offspring, providing a scientific basis for optimizing in vitro culture conditions and improving human-assisted reproductive technology.

Dyskerin: an essential pseudouridine synthase with multifaceted roles in ribosome biogenesis, splicing, and telomere maintenance.

Dyskerin and its homologs are ancient and conserved enzymes that catalyze the most common post-transcriptional modification found in cells, pseudouridylation. The resulting pseudouridines provide stability to RNA molecules and regulate ribosome biogenesis and splicing events. Dyskerin does not act independently—it is the core component of a protein heterotetramer, which associates with RNAs that contain the H/ACA motif. The variety of H/ACA RNAs that guide the function of this ribonucleoprotein (RNP) complex highlights the diversity of cellular processes in which dyskerin participates. When associated with small nucleolar (sno) RNAs, it regulates ribosomal (r) RNAs and ribosome biogenesis. By interacting with small Cajal body (sca) RNAs, it targets small nuclear (sn) RNAs to regulate pre-mRNA splicing. As a component of the telomerase holoenzyme, dyskerin binds to the telomerase RNA to modulate telomere maintenance. In a disease context, dyskerin malfunction can result in multiple detrimental phenotypes. Mutations in DKC1, the gene that encodes dyskerin, cause the premature aging syndrome X-linked dyskeratosis congenita (X-DC), a still incurable disorder that typically leads to bone marrow failure. In this review, we present the classical and most recent findings on this essential protein, discussing the evolutionary, structural, and functional aspects of dyskerin and the H/ACA RNP. The latest research underscores the role that dyskerin plays in the regulation of gene expression, translation efficiency, and telomere maintenance, along with the impacts that defective dyskerin has on aging, cell proliferation, haematopoietic potential, and cancer.

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes

Simple SummaryHMGB1 over-expression is associated to prostate and ovary cancers: in this work, using a proteomic approach, we aimed to discover new protein interactions that might contribute to understand the oncogenic function of HMGB1 in cancers models. Our findings show that HMGB1 interacts with components of the NuRD, THOC and septin complexes, revealing new connections of HMGB1 functions to RNA processing and ribosome biogenesis. Results might contribute to consider the components of these interactomes as targets for diagnosis and therapy in future studies.This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.

The exon-junction complex helicase eIF4A3 controls cell fate via coordinated regulation of ribosome biogenesis and translational output.

Eukaryotic initiation factor 4A-III (eIF4A3), a core helicase component of the exon junction complex, is essential for splicing, mRNA trafficking, and nonsense-mediated decay processes emerging as targets in cancer therapy. Here, we unravel eIF4A3's tumor-promoting function by demonstrating its role in ribosome biogenesis (RiBi) and p53 (de)regulation. Mechanistically, eIF4A3 resides in nucleoli within the small subunit processome and regulates rRNA processing via R-loop clearance. EIF4A3 depletion induces cell cycle arrest through impaired RiBi checkpoint-mediated p53 induction and reprogrammed translation of cell cycle regulators. Multilevel omics analysis following eIF4A3 depletion pinpoints pathways of cell death regulation and translation of alternative mouse double minute homolog 2 (MDM2) transcript isoforms that control p53. EIF4A3 expression and subnuclear localization among clinical cancer specimens correlate with the RiBi status rendering eIF4A3 an exploitable vulnerability in high-RiBi tumors. We propose a concept of eIF4A3's unexpected role in RiBi, with implications for cancer pathogenesis and treatment.

Genetic and epigenetic regulation of skeletal muscle ribosome biogenesis with exercise.

Ribosome biogenesis and MYC transcription are associated with acute resistance exercise (RE) and are distinct from endurance exercise in human skeletal muscle throughout a 24h time course of recovery. A PCR-based method for relative ribosomal DNA (rDNA) copy number estimation was validated by whole genome sequencing and revealed that rDNA dosage is positively correlated with ribosome biogenesis in response to RE. Acute RE modifies rDNA methylation patterns in enhancer, intergenic spacer and non-canonical MYC-associated regions, but not the promoter. Myonuclear-specific rDNA methylation patterns with acute mechanical overload in mice corroborate and expand on rDNA findings with RE in humans. A genetic predisposition for hypertrophic responsiveness may exist based on rDNA gene dosage. Ribosomes are the macromolecular engines of protein synthesis. Skeletal muscle ribosome biogenesis is stimulated by exercise, although the contribution of ribosomal DNA (rDNA) copy number and methylation to exercise-induced rDNA transcription is unclear. To investigate the genetic and epigenetic regulation of ribosome biogenesis with exercise, a time course of skeletal muscle biopsies was obtained from 30 participants (18men and 12 women; 31±8 years, 25±4kgm-2 ) at rest and 30min, 3h, 8h and 24h after acute endurance (n=10, 45min cycling, 70% ) or resistance exercise (n=10, 4 × 7 × 2 exercises); 10 control participants underwent biopsies without exercise. rDNA transcription and dosage were assessed using quantitative PCR and whole genome sequencing. rDNA promoter methylation was investigated using massARRAY EpiTYPER and global rDNA CpG methylation was assessed using reduced-representation bisulphite sequencing. Ribosome biogenesis and MYC transcription were associated primarily with resistance but not endurance exercise, indicating preferential up-regulation during hypertrophic processes. With resistance exercise, ribosome biogenesis was associated with rDNA gene dosage, as well as epigenetic changes in enhancer and non-canonical MYC-associated areas in rDNA, but not the promoter. A mouse model of in vivo metabolic RNA labelling and genetic myonuclear fluorescence labelling validated the effects of an acute hypertrophic stimulus on ribosome biogenesis and Myc transcription, and also corroborated rDNA enhancer and Myc-associated methylation alterations specifically in myonuclei. The present study provides the first information on skeletal muscle genetic and rDNA gene-wide epigenetic regulation of ribosome biogenesis in response to exercise, revealing novel roles for rDNA dosage and CpG methylation.

Sp1-Mediated circRNA circHipk2 Regulates Myogenesis by Targeting Ribosomal Protein Rpl7.

Circular RNAs (circRNAs) represent a class of covalently closed single-stranded RNA molecules that are emerging as essential regulators of various biological processes. The circRNA circHipk2 originates from exon 2 of the Hipk2 gene in mice and was reported to be involved in acute promyelocytic leukemia and myocardial injury. However, the functions and mechanisms of circHipk2 in myogenesis are largely unknown. Here, to deepen our knowledge about the role of circHipk2, we studied the expression and function of circHipk2 during skeletal myogenesis. We found that circHipk2 was mostly distributed in the cytoplasm, and dynamically and differentially expressed in various myogenesis systems in vitro and in vivo. Functionally, overexpression of circHipk2 inhibited myoblast proliferation and promoted myotube formation in C2C12 cells, whereas the opposite effects were observed after circHipk2 knockdown. Mechanistically, circHipk2 could directly bind to ribosomal protein Rpl7, an essential 60S preribosomal assembly factor, to inhibit ribosome translation. In addition, we verified that transcription factor Sp1 directly bound to the promoter of circHipk2 and affected the expression of Hipk2 and circHipk2 in C2C12 myoblasts. Collectively, these findings identify circHipk2 as a candidate circRNA regulating ribosome biogenesis and myogenesis proliferation and differentiation.

The Role of tRNA Derived Small RNAs in Gene Regulation in Normal Tissues and Cancer

The function of tRNAs is not merely to provide the specific amino acids for a growing peptide chain during protein synthesis. Precursor and mature tRNAs can be enzymatically cleaved into various tRNA derived small RNAs. While there are thousands of different RNA species that can be generated, the function of most are not known. I will provide a brief introduction into this field and describe the mechanism of how at least one specific tsRNA derived from the 3′end of a mature tRNA is involved in regulating ribosome biogenesis (Kim et al. Nature 2017). This 3′tsRNA is upregulated in many tumors. Reducing the tsRNA in patient derived hepatocellular carcinomas using antisense oligonucleotides induced apoptosis and tumor shrinkage in mice. More recently, we have discovered the 3′tsRNAs are aminoacylated suggesting they are generated in the cytoplasm and perhaps on the ribosome. I will provide an update on current approaches we are developing to help us identify, quantify, and define the targets of the myriad of various tsRNAs present in various cell types. This will further allow investigation as to their role in maintaining health and disease states.