Ribosome Production Research Articles

Intermedin (adrenomedullin 2) promotes breast cancer metastasis via Src/c-Myc-mediated ribosome production and protein translation.

Breast cancer is the most frequently diagnosed cancer and is the leading cause of cancer-associated mortality in women worldwide. Intermedin (IMD, also known as Adrenomedullin 2, ADM2) is an endogenous peptide that belongs to the calcitonin gene-related peptide family and has been reported to play important roles in several types of cancers, including breast cancer. In this study, we sought to investigate how IMD affects the behavior of breast cancer cells, the underlying mechanism of these effects, and whether blockade of IMD has a therapeutic effect against breast cancer. Transcriptome sequencing (RNA-Seq), cell biological experiments, Western blotting, immunoprecipitation, and animal tumor models were used. IMD expression was significantly increased in breast cancer samples, and the IMD level was positively correlated with lymph node metastasis and Ki67 expression. Cell biological experiments showed that IMD promoted the anchorage-independent growth, migration, and invasive ability of breast cancer cells. Inhibiting IMD activity with an anti-IMD monoclonal antibody blocked these tumor-promoting effects. In addition, blockade of IMD reduced in situ tumor growth and significantly decreased lung metastasis of 4T1 breast cancer in vivo. IMD induced Src kinase phosphorylation, which triggered the transcription of c-Myc, a major oncoprotein controlling the expression of genes that encode ribosomal components. Our data suggest that IMD is involved in breast cancer cell invasion and metastasis, potentially through increasing ribosome biogenesis and protein translation via the Src/c-Myc signaling pathway. These results suggest that IMD may be a novel target for the treatment of breast cancer.

Studies of mutations of assembly factor Hit1 in budding yeast suggest translation defects as the molecular basis for PEHO syndrome

Regulation of protein synthesis is critical for control of gene expression in all cells. Ribosomes are ribonucleoprotein machines responsible for translating cellular proteins. Defects in ribosome production, function, or regulation are detrimental to the cell and cause human diseases, such as progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO) syndrome. PEHO syndrome is a devastating neurodevelopmental disorder caused by mutations in the ZNHIT3 gene, which encodes an evolutionarily conserved nuclear protein. The precise mechanisms by which ZNHIT3 mutations lead to PEHO syndrome are currently unclear. Studies of the human zinc finger HIT-type containing protein 3 homolog in budding yeast (Hit1) revealed that this protein is critical for formation of small nucleolar ribonucleoprotein complexes that are required for rRNA processing and 2′-O-methylation. Here, we use budding yeast as a model system to reveal the basis for the molecular pathogenesis of PEHO syndrome. We show that missense mutations modeling those found in PEHO syndrome patients cause a decrease in steady-state Hit1 protein levels, a significant reduction of box C/D snoRNA levels, and subsequent defects in rRNA processing and altered cellular translation. Using RiboMethSeq analysis of rRNAs isolated from actively translating ribosomes, we reveal site-specific changes in the rRNA modification pattern of PEHO syndrome mutant yeast cells. Our data suggest that PEHO syndrome is a ribosomopathy and reveal potential new aspects of the molecular basis of this disease in translation dysregulation.

Transcriptomic Profiling of JEG-3 cells using human leiomyoma derived matrix

Oxygen tension varies during placental and fetal development. Although hypoxia drives early trophoblast invasion, low placental oxygen levels during pregnancy show association with pregnancy complications including fetal growth restriction and preeclampsia. JEG-3 cells are often used as a trophoblast model. We studied transcriptional changes of JEG-3 cells on a uterine leiomyoma derived matrix Myogel. This might be the closest condition to the real uterine environment that we can get for an in vitro model. We observed that culturing JEG-3 cells on the leiomyoma matrix leads to strong stimulation of ribosomal pathways, energy metabolism, and ATP production. Furthermore, Myogel improved JEG-3 cell adherence in comparison to tissue culture treated plastic. We also included PDMS microchip hypoxia creation, and observed changes in oxidative phosphorylation, oxygen related genes and several hypoxia genes. Our study highlights the effects of Myogel matrix on growing JEG-3 cells, especially on mitochondria, energy metabolism, and protein synthesis.

Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome

Dysregulated protein synthesis is a core pathogenic mechanism in Fragile X Syndrome (FX). The mGluR Theory of FX predicts that pathological synaptic changes arise from the excessive translation of mRNAs downstream of mGlu1/5 activation. Here, we use a combination of CA1 pyramidal neuron-specific TRAP-seq and proteomics to identify the overtranslating mRNAs supporting exaggerated mGlu1/5 -induced long-term synaptic depression (mGluR-LTD) in the FX mouse model (Fmr1−/y). Our results identify a significant increase in the translation of ribosomal proteins (RPs) upon mGlu1/5 stimulation that coincides with a reduced translation of long mRNAs encoding synaptic proteins. These changes are mimicked and occluded in Fmr1−/y neurons. Inhibiting RP translation significantly impairs mGluR-LTD and prevents the length-dependent shift in the translating population. Together, these results suggest that pathological changes in FX result from a length-dependent alteration in the translating population that is supported by excessive RP translation.

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.

Interdependency and Redundancy Add Complexity and Resilience to Biogenesis of Bacterial Ribosomes.

The pace and efficiency of ribosomal subunit production directly impact the fitness of bacteria. Biogenesis demands more than just the union of ribosomal components, including RNA and proteins, to form this functional ribonucleoprotein particle. Extra-ribosomal protein factors play a fundamental role in the efficiency and efficacy of ribosomal subunit biogenesis. A paucity of data on intermediate steps, multiple and overlapping pathways, and the puzzling number of functions that extra-ribosomal proteins appear to play in vivo make unraveling the formation of this macromolecular assemblage difficult. In this review, we outline with examples the multinodal landscape of factor-assisted mechanisms that influence ribosome synthesis in bacteria. We discuss in detail late-stage events that mediate correct ribosome formation and the transition to translation initiation and thereby ensure high-fidelity protein synthesis.

Extrinsic noise effects on ribosomal traffic during the translation process

Any cellular process at the microscopic level is governed by both extrinsic and intrinsic noise (IN) and same is true for mRNA translation as well. Even though the effect of stochasticity via the IN is well studied for mRNA translation, same cannot be said about extrinsic noise (EN). In this article, we incorporate EN in a model of mRNA translation and carry out stochastic simulations of the same. We then evaluate various statistics related to the residence time of the ribosome and the ribosomal traffic on the mRNA and subsequent protein production. We also study the effect of slow codons and ribosome dropoff. From our simulations, we show that noise in the translation initiation rate rather than the translation termination rate acts to significantly broaden the distribution of ribosome residence times on the mRNA lattice. This EN also has an effect on ribosomal current, density and protein production. Further, the presence of slow codons acts to increase the mean residence times. However, this increase also depends on the number and position of the slow codons on the lattice. We also show that the slow codons act to mask any effect from the EN themselves. Our results, therefore, have implications towards a better understanding of the role the individual components play during the translation process.

ZNF692, a novel nucleolar regulator in cancer

Nucleolar function and ribosome biogenesis are essential for protein synthesis and cellular growth. Therefore, aberrant increase in nucleolar size and number are hallmarks of many cancers and have been associated with poor prognosis; nucleolar size is one of the most frequently used parameters by pathologists to grade solid tumors. While basic components and general molecular functions of the nucleolus have been studied, fundamental information on the mechanisms and molecules involved in the increased nucleolar size and activity specifically in cancer cells is missing. In an unbiased screen designed to identify growth promoting pathways in MYC‐transformed cells, our lab identified a novel and critical nucleolar regulator, the Zinc finger protein 692 (ZNF692) (manuscript in preparation). Our data has shown that ZNF692 downregulation interferes with protein synthesis and nucleolar function, and overexpression promotes nucleolar activity and protein synthesis. Moreover, we found that ZNF692 expression is dramatically increased in nearly all solid tumors deposited in the TGCA and is correlated with poor patient outcome. On a molecular level, our lab has shown that ZNF692 might play a role in rRNA processing. Through this project, we have unexpectedly discovered that ZNF692 is likely to coordinate cellular and mitochondrial ribosome production to fulfill the needs for rapid cell proliferation. This project aims to define understudied mechanisms and key regulators of increased ribosome biogenesis in cancer cells and could become the basis for the development of novel and unexplored approaches to alter nucleolar activity specifically in solid tumors.

RNAP II produces capped 18S and 25S ribosomal RNAs resistant to 5\u2032-monophosphate dependent processive 5\u2032 to 3\u2032 exonuclease in polymerase switched Saccharomyces cerevisiae

BackgroundWe have previously found that, in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5′-end were resistant to 5′-monophosphate requiring 5′ → 3″ exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them.ResultsWe now show the production of such 18S and 25S rRNAs in Saccharomyces cerevisiae that have been permanently switched to RNAP II (due to deletion of part of RNAP I upstream activator alone, or in combination with deletion of one component of RNAP I itself). They contain more than one phosphate at their 5′-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to have been modified in the cytoplasm.ConclusionsOur data confirm the existence of such molecules and firmly establish RNAP II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis.

The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, reduces the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide-associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

Labeling of heterochronic ribosomes reveals C1ORF109 and SPATA5 control a late step in human ribosome assembly.

SUMMARYAlthough features of ribosome assembly are shared between species, our understanding of the diversity, complexity, dynamics, and regulation of ribosome production in multicellular organisms remains incomplete. To gain insights into ribosome biogenesis in human cells, we perform a genome-wide loss-of-function screen combined with differential labeling of pre-existing and newly assembled ribosomes. These efforts identify two functionally uncharacterized genes, C1orf109 and SPATA5. We provide evidence that these factors, together with CINP and SPATA5L1, control a late step of human pre-60S maturation in the cytoplasm. Loss of either C1orf109 or SPATA5 impairs global protein synthesis. These results link ribosome assembly with neurodevelopmental disorders associated with recessive SPATA5 mutations. Based on these findings, we propose that the expanded repertoire of ribosome biogenesis factors likely enables multicellular organisms to coordinate multiple steps of ribosome production in response to different developmental and environmental stimuli.

The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress.

Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that affect energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here, we demonstrate how a physiologically relevant ribosome population arises during high Na+, sorbitol, or pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na+ or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes reincorporation of the protein, thereby repairing the subunit after the Na+ stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input and without bypass of quality control mechanisms.

Transcriptomic and Metabolomic Analyses Provide Insights into the Growth and Development Advantages of Triploid Apostichopus japonicus

Polyploid breeding is widely used in aquaculture as an important area of new research. We have previously grown Apostichopus japonicus triploids with a growth advantage. The body length, body weight, and aestivation time of triploid and diploid A. japonicus were measured in this study, and the transcriptome and metabolome were used to examine the growth advantage of triploids A. japonicus. The results showed that the proportion of triploid A. japonicus with a body length of 6–12 cm and 12–18 cm was significantly higher than that of diploid A. japonicus, and triploid A. japonicus had a shorter aestivation time (39 days) than diploid (63 days). We discovered 3296 differentially expressed genes (DEGs); 13 DEGs (for example, cyclin-dependent kinase 2) related to growth advantage, immune regulation, and energy storage were screened as potential candidates. According to Gene Ontology (GO) enrichment analysis, DEGs were significantly enriched in the cytoplasm (cellular component), ATP binding process (molecular function), oxidation–reduction process (biological process), and other pathways. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment data, DEGs were significantly enriched in ribosome production and other areas. We discovered 414 significant differential metabolites (SDMs), with 11 important SDMs (for example, nocodazole) linked to a growth advantage. SDMs are significantly enriched in metabolic pathways, as well as other pathways, according to the KEGG enrichment results. According to a combined transcriptome and metabolome analysis, 6 DEGs have regulatory relationships with 11 SDMs, which act on 11 metabolic pathways together. Our results further enrich the biological data of triploid A. japonicus and provide useful resources for genetic improvement of this species.

Ribosome production factor 2 homolog promotes migration and invasion of colorectal cancer cells by inducing epithelial–mesenchymal transition via AKT/Gsk-3β signaling pathway

Colorectal cancer (CRC) is one of the most common malignancies of the gastrointestinal tract, and its prognosis is closely related to the degree of tumor invasion and metastasis. Ribosome production factor 2 homolog (RPF2) plays an important role in the process of ribosome biogenesis; however, its biological function in the progression of malignant tumors including CRC remains unknown. It was found that RPF2 expression was significantly higher in CRC tissues than in the adjacent normal tissue, using mRNA chip technology. This study aimed to explore the role of RPF2 in the invasion and migration of CRC cells and investigate its probable molecular mechanism. The results demonstrated that RPF2 is not only highly expressed in CRC tissues and cell lines but can also activate the AKT/GSK-3β signaling pathway through direct interaction with CARM1, promoting epithelial–mesenchymal transition, and consequently enhancing the migration and invasion abilities of CRC cells in vitro and in vivo. Thus, we speculated that RPF2 may become a novel therapeutic target for suppression of local invasion and distant metastasis of CRC.

Interaction of TOR and PKA Signaling in S. cerevisiae.

TOR and PKA signaling are the major growth-regulatory nutrient-sensing pathways in S. cerevisiae. A number of experimental findings demonstrated a close relationship between these pathways: Both are responsive to glucose availability. Both regulate ribosome production on the transcriptional level and repress autophagy and the cellular stress response. Sch9, a major downstream effector of TORC1 presumably shares its kinase consensus motif with PKA, and genetic rescue and synthetic defects between PKA and Sch9 have been known for a long time. Further, studies in the first decade of this century have suggested direct regulation of PKA by TORC1. Nonetheless, the contribution of a potential direct cross-talk vs. potential sharing of targets between the pathways has still not been completely resolved. What is more, other findings have in contrast highlighted an antagonistic relationship between the two pathways. In this review, I explore the association between TOR and PKA signaling, mainly by focusing on proteins that are commonly referred to as shared TOR and PKA targets. Most of these proteins are transcription factors which to a large part explain the major transcriptional responses elicited by TOR and PKA upon nutrient shifts. I examine the evidence that these proteins are indeed direct targets of both pathways and which aspects of their regulation are targeted by TOR and PKA. I further explore if they are phosphorylated on shared sites by PKA and Sch9 or when experimental findings point towards regulation via the PP2ASit4/PP2A branch downstream of TORC1. Finally, I critically review data suggesting direct cross-talk between the pathways and its potential mechanism.

Functionally impaired RPL8 variants associated with Diamond–Blackfan anemia and a Diamond–Blackfan anemia‐like phenotype

Diamond-Blackfan anemia is a rare genetic disease characterized by erythroblastopenia and a large spectrum of developmental anomalies. The vast majority of the cases genetically described are linked to heterozygous pathogenic variants in more than 20 ribosomal protein genes. Here we report an atypical clinical case of DBA associated with a missense variant in RPL8, which encodes RPL8/uL2, a protein of the 60S large ribosomal subunit. RPL8 has been previously implicated as a candidate disease gene in one patient with DBA bearing another type of missense variant; however, evidence for pathogenicity was limited to computational tools. Using functional studies in lymphoblastoid cells as well as yeast models, we show that the RPL8 variants detected in these two patients encode functionally deficient proteins that affect ribosome production and are therefore likely pathogenic. We propose to include RPL8 in the list of DBA-associated genes.

Trimethylguanosine synthase 1 is a novel regulator of pancreatic beta-cell mass and function

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in β-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in β-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human β-cells from type 2 diabetes donors. Using mice with conditional (βTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates β-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates β-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating β-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes.

High-grade ovarian cancer associated H/ACA snoRNAs promote cancer cell proliferation and survival.

ABSTRACTSmall nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, assessing the nature and extent of snoRNAs’ contribution to cancer biology has been largely limited by difficulties in detecting highly structured RNA. In this study, we used a dedicated midsize non-coding RNA (mncRNA) sensitive sequencing technique to accurately survey the snoRNA abundance in independently verified high-grade serous ovarian carcinoma (HGSC) and serous borderline tumour (SBT) tissues. The results identified SNORA81, SNORA19 and SNORA56 as an H/ACA snoRNA signature capable of discriminating between independent sets of HGSC, SBT and normal tissues. The expression of the signature SNORA81 correlates with the level of ribosomal RNA (rRNA) modification and its knockdown inhibits 28S rRNA pseudouridylation and accumulation leading to reduced cell proliferation and migration. Together our data indicate that specific subsets of H/ACA snoRNAs may promote tumour aggressiveness by inducing rRNA modification and synthesis.

Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors.

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.

PpGpp is a bacterial cell size regulator

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the adder principle, by unknown mechanisms.1,2 Growth is well known to be regulated by guanosine tetraphosphate (ppGpp), which controls diverse processes from ribosome production to metabolic enzyme activity and replication initiation and whose absence or excess can induce stress, filamentation, and small growth-arrested cells.3-6 These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth. Here, to untangle effects of ppGpp and nutrients, we gained control of cellular ppGpp by inducing the synthesis and hydrolysis enzymes RelA and Mesh1. We found that ppGpp not only exerts control over the growth rate but also over cell division and thus the steady state cell size. In response to changes in ppGpp level, the added size already establishes its new constant value while the growth rate still adjusts, aided by accelerated or delayed divisions. Moreover, the magnitude of the added size and resulting steady-state birth size correlate consistently with the ppGpp level, rather than with the growth rate, which results in cells of different size that grow equally fast. Our findings suggest that ppGpp serves as a key regulator that coordinates cell size and growth control.