Regulation Of Ribosome Biogenesis Research Articles

Rapamycin induces morphological and physiological changes without increase in lipid content in Ustilago maydis.

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

Assessment of the nucleotide modifications in the high-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit.

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides’ positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

Diphthamide modification of eEF2 is required for gut tumor-like hyperplasia induced by oncogenic Ras.

Eukaryotic elongation factor 2 (eEF2) undergoes a unique post-translational modification called diphthamidation. Although eEF2 diphthamidation is highly conserved, its pathophysiological function is still largely unknown. To elucidate the function of diphthamidation in tumor, we examined the involvement of diphthamidation pathway enzyme Dph5 in tumor progression in Drosophila adult gut. Expression of oncogenic RasV12 in gut intestinal stem cells (ISCs) and enteroblasts (EBs) causes hypertrophy and disruption of gut epithelia, and shortened life span. Knockdown of Dph5 ameliorated these pathogenic phenotypes. Dph5 is required for gross translation activation and high dMyc protein level in RasV12 tumor-like hyperplasia. Transcriptome analysis revealed that Dph5 is involved in the regulation of ribosome biogenesis genes. These results suggest that diphthamidation is required for translation activation partly through the regulation of ribosome biogenesis in Ras-induced tumor-like hyperplasia model in Drosophila gut.

Subcellular mRNA Localization Regulates Ribosome Biogenesis In Migrating Cells

Translation of Ribosomal Protein-coding mRNAs (RP-mRNAs) constitutes a key step in ribosome biogenesis, but the mechanisms which modulate RP-mRNA translation in coordination with other cellular processes are poorly defined. Here we show that the subcellular localization of RP-mRNAs acts as a key regulator of their translation during cell migration. As cells migrate into their surroundings, RP-mRNAs localize to the actin-rich protrusive fronts of the cells. This localization is mediated by La-related protein 6 (LARP6), an RNA binding protein that is enriched in protrusions. Protrusions act as hotspots of translation for RP-mRNAs, resulting in enhancement of RP synthesis, ribosome biogenesis, and overall protein synthetic capacity of migratory cells, required for supporting efficient migration and proliferation. In human breast carcinomas, Epithelial to Mesenchymal Transition (EMT) upregulates LARP6 expression to enhance protein synthesis and support invasive growth. Our findings reveal LARP6 mediated mRNA localization as a key regulator of ribosome biogenesis during cell migration, and demonstrate a role for this process in cancer progression downstream of EMT.

MTORC1-Rps15 Axis Contributes to the Mechanisms Underlying Global Translation Reduction During Senescence of Mouse Embryonic Fibroblasts.

The reduction of protein translation is a common feature in senescent cells and aging organisms, yet the underlying mechanisms are not fully understood. Here we show that both global mRNA translation and mammalian/mechanistic target of rapamycin complex 1 (mTORC1) kinase activity are declined in a senescent model of mouse embryonic fibroblasts (MEFs). Furthermore, RNA-seq analyses from polysomal versus total mRNA fractions identify TOP-like mRNA of Rps15 whose translation is regulated by mTORC1 during MEF senescence. Overexpression of Rps15 delays MEF senescence, possibly through regulating ribosome maturation. Together, these findings indicate that the activation of mTORC1-Rps15 axis ameliorate senescence by regulating ribosome biogenesis, which may provide further insights into aging research.

Abstract LB-A12: A synthetic lethal drug screen identifies exploitable vulnerabilities in cohesin-deficient cells

Abstract The cohesin complex comprises four core subunits: RAD21, SMC3, SMC1A, and STAG2. Cohesin is required for many biological processes, including sister chromatid cohesion, DNA damage response, regulation of gene expression, and ribosomal biogenesis. Germline mutations of cohesin subunits or their regulators give rise to human developmental disorders, while somatic mutations in cohesin subunits are found in a wide range of human cancers, including acute myeloid leukemia (AML) (12%), Ewing sarcoma (15%) and glioblastoma (19%). In particular, cohesin subunit STAG2, has been identified as one of only 12 genes that are significantly mutated in more than 4 human cancers. Currently, there is no targeted therapeutic strategy for cancer patients with cohesin mutations. Synthetic lethality has emerged as a promising approach for targeted therapy. While synthetic lethal interactions of STAG2 have been demonstrated with its paralog, STAG1, and with the DNA damage repair pathway, synthetic lethal interactions with other pathways have not been explored. To identify synthetic lethal drug activity with cohesin mutations, we generated three isogenic cohesin-deficient MCF10A cell lines (RAD21+/-, SMC3+/-, and STAG2-/-) using CRISPR/Cas9. Cohesin-deficient MCF10A cell lines are sufficiently similar to their parental line concerning growth rate and chromosome stability. However, these cell lines exhibit alterations in nucleolar morphology and increased nuclear location of γH2AX and p53 (except in STAG2-/-). RNA-sequencing analysis of the cell lines showed that cohesin subunit mutation led to downregulation of genes involved in regulation of ribosome biogenesis and RNA processing. Furthermore, ribosomal stress mediated by Actinomycin D led to severe nucleolar fragmentation in cohesin-deficient MCF10A, but not MCF10A parental, suggesting that cohesin mutation might confer vulnerability to perturbation of ribosome biogenesis. Using MCF10A parental and isogenic cohesin-deficient MCF10A cell lines, we performed a quantitative high-throughput drug screen against 3,009 compounds of FDA-approved drugs, kinase, and epigenetic inhibitors. Several classes of inhibitors targeting mTOR signaling, Wnt signaling and chromatin modification were found to selectively inhibit the growth of cohesin-deficient MCF10A cells by 30% or more. WAY-600 (mTORC1/mTORC2 inhibitor) and I-BET-762 (Bromodomain inhibitor) were among the top hits. Mechanistic studies on how these drugs preferentially kill cohesin-deficient cells are under way. In summary, we have identified potential synthetic lethal compounds that may provide the basis for development of targeted therapies of cancers with cohesin deficiency or mutation. Citation Format: Chue Vin Chin, Jisha Antony, Amee J George, JinShu He, Kate Parson, Ross D Hannan, Julia A Horsfield. A synthetic lethal drug screen identifies exploitable vulnerabilities in cohesin-deficient cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A12. doi:10.1158/1535-7163.TARG-19-LB-A12

A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice

tRNA-derived small RNAs (tsRNAs) have been implicated in many cellular processes, yet the detailed mechanisms are not well defined. We previously found that the 3' end of Leu-CAG tRNA-derived small RNA (LeuCAG3'tsRNA) regulates ribosome biogenesis in humans by maintaining ribosomal protein S28 (RPS28) levels. The tsRNA binds to coding (CDS) and non-coding 3' UTR sequence in the RPS28 mRNA, altering its secondary structure and enhancing its translation. Here we report that the functional 3' UTR target site is present in primates while the CDS target site is present in many vertebrates. We establish that this tsRNA also regulates mouse Rps28 translation by interacting with the CDS target site. We further establish that the change in mRNA translation occurred at a post-initiation step in both species. Overall, our results suggest that LeuCAG3'tsRNA might maintain ribosome biogenesis through a conserved gene regulatory mechanism in vertebrates.

Dactinomycin in Acute Myeloid Leukemia with NPM1 Mutations

NPM1 mutations are frequent in acute myeloid leukemia (AML) and define a distinct entity according to the 2016 WHO classification. NPM1 mutations are generally associated with chemosensitivity and a favorable prognosis. However, outcome may vary according to co-mutational events, and still approximately 40% of patients relapse after achieving complete response. Wild-type NPM1 is mainly located in the nucleolus where it plays a key role in the regulation of ribosome biogenesis, protein synthesis and tumor suppression through TP53 activation. Mutated NPM1 loses its predominant nucleolar location and accumulates in cytoplasm contributing to leukemogenesis (Falini B, Blood 2011). Moreover, this mutational event leads to haploinsufficiency and cytoplasmic retention of wild type NPM1 creating a vulnerability to nucleolar stress. Indeed, complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug (Falini B, NEJM 2015). Here, we report our experience of off-label dactinomycin in untreated or relapsed/ refractory NPM1-mutated AML. Inclusion criteria for this retrospective study were: age ≥ 18 years-old, AML with NPM1 mutation, relapsed or refractory disease as well as treatment-naive patients unfit for intensive chemotherapy. Patients should also have completed one cycle of dactinomycin 12.5 µg/kg/day for 5 days every 28 days. From September 2015 to February 2019, 26 patients received dactinomycin. Median age was 62.5y, WBC count was > 50 giga/L in 8 patients (31%), 13 patients (50%) had FLT3-ITD mutation whereas 10 (38%) and 11 (42%) patients were classified as favorable or intermediate-I according to the ELN-2010 classification. There were 7 (27%) relapses post-allogeneic transplantation. Median number of dactinomycin cycle was 1 (1-8) and 7 patients (27%) received more than 3 cycles. Sorafenib was added in 6 patients with associated FLT3-ITD mutations whereas 2 others patients received ATRA in combination with dactinomycin. Dactinomycin was administered in different situations: front-line treatment in 4 unfit patients (16%); morphologic (n=16, 62%) or molecular relapses (n=4, 16%) following intensive chemotherapy, refractory disease (n=1, 13%) or post remission therapy in second complete response (CR) following salvage chemotherapy (n=1, 13%). Three out of 17 patients (18%) in morphologic relapse or refractory to chemotherapy reached complete remission after the first cycle of dactinomycin. The duration of response was 4 and 6 months in 2 patients whereas the third patient is still in CR 3 years after dactinomycin. One out of 4 patients in molecular relapses achieved a complete molecular remission with dactinomycin. None of the 4 patients unfit for intensive chemotherapy responded to dactinomycin as front-line therapy. The only patient treated in post-CR2 with dactinomycin achieved a complete molecular remission before allogeneic transplantation. Overall, 5 patients (19%) appeared to benefit from dactinomycin treatment. Grade 3-4 adverse events were thrombocytopenia (n=11, 42%), neutropenia (n=11, 42%), GI toxicity (n=6, 23%), mucositis (n=5, 19%), lung infection (n=5, 19%) and skin rash (n=2, 7.6%). Dactinomycin is an inexpensive and easily available drug that may induce significant responses in AML patients with NPM1 mutations with an acceptable safety profile. Prospective and controlled clinical trials are mandatory to clearly define the role of this agent in AML with NPM1 mutations. Disclosures Tavitian: Novartis: Membership on an entity's Board of Directors or advisory committees. Bertoli:Sanofi: Honoraria. Huguet:Incyte Biosciences: Honoraria; Servier: Honoraria; Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria. Bories:Abbvie: Consultancy. Recher:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Macrogenics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: NPM1 mutations are frequent in acute myeloid leukemia (AML) and define a distinct entity according to the 2016 WHO classification. Wild-type NPM1 is mainly located in the nucleolus where it plays a key role in the regulation of ribosome biogenesis, protein synthesis and tumor suppression through TP53 activation. Mutated NPM1 loses its predominant nucleolar location and accumulates in cytoplasm contributing to leukemogenesis (Falini B, Blood 2011). Moreover, this mutational event leads to haploinsufficiency and cytoplasmic retention of wild type NPM1 creating a vulnerability to nucleolar stress. Indeed, complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug (Falini B, NEJM 2015).

Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes

ABSTRACTIn addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5ʹ leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.

Phosphorylation of eukaryotic initiation factor 4E is dispensable for skeletal muscle hypertrophy.

The eukaryotic initiation factor 4E (eIF4E) is a major mRNA cap-binding protein that has a central role in translation initiation. Ser209 is the single phosphorylation site within eIF4E and modulates its activity in response to MAPK pathway activation. It has been reported that phosphorylation of eIF4E at Ser209 promotes translation of key mRNAs, such as cyclin D1, that regulate ribosome biogenesis. We hypothesized that phosphorylation at Ser209 is required for skeletal muscle growth in response to a hypertrophic stimulus by promoting ribosome biogenesis. To test this hypothesis, wild-type (WT) and eIF4E knocked-in (KI) mice were subjected to synergist ablation to induce muscle hypertrophy of the plantaris muscle as the result of mechanical overload; in the KI mouse, Ser209 of eIF4E was replaced with a nonphosphorylatable alanine. Contrary to our hypothesis, we observed no difference in the magnitude of hypertrophy between WT and KI groups in response to 14 days of mechanical overload induced by synergist ablation. Similarly, the increases in cyclin D1 protein levels, ribosome biogenesis, and translational capacity did not differ between WT and KI groups. Based on these findings, we conclude that phosphorylation of eIF4E at Ser209 is dispensable for skeletal muscle hypertrophy in response to mechanical overload.

P11.36 Ribosome hydroxylase Mina53 is required for Glioblastoma and is involved in regulation of translation rateand fidelity by regulating ribosomal biogenesis

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive types of tumors with a poor response to standard treatment and a median 5-year survival of less than 5%. Therefore, there is an urgent need for new treatments. Recently, a large number of genome-wide studies have shown that the epigenetic modifiers are frequently deregulated in cancer. Using a mouse GBM model, we performed in vitro and in vivo shRNA screens to identify epigenetic regulators required for the tumorigenic process in GBM. Among these regulators is a ribosome hydroxylase Mina53 which hydroxylates His-39 of ribosomal protein, RPL27a. We have found that the knock-down (KD) of Mina53 reduces the in vitro proliferation and colony forming ability of mouse glioma initiating cells (mGIC) and this is dependent on the catalytic activity of Mina. Knock-down of Mina resulted into a small but significant reduction in the global protein synthesis rate. A tandem affinity purification experiment to identify proteins associated with Mina revealed that it is associated mainly with ribosomal proteins, including its substrate RPL27a. Global proteomic analyses revealed that final amounts and de novo protein synthesis of many ribosomal proteins were reduced upon Mina depletion. Isolation and identification of different polysome fraction bound mRNAs using high-throughput sequencing found that mRNAs encoding many ribosomal proteins have lower number of ribosomes loaded on them in the Mina depleted samples compared to the control. Taken together, this study has found that Mina53 is required for glioblastoma and it regulates translation through regulation of ribosomal biogenesis

Loss of Peter Pan (PPAN) Affects Mitochondrial Homeostasis and Autophagic Flux.

Nucleolar stress is a cellular response to inhibition of ribosome biogenesis or nucleolar disruption leading to cell cycle arrest and/or apoptosis. Emerging evidence points to a tight connection between nucleolar stress and autophagy as a mechanism underlying various diseases such as neurodegeneration and treatment of cancer. Peter Pan (PPAN) functions as a key regulator of ribosome biogenesis. We previously showed that human PPAN localizes to nucleoli and mitochondria and that PPAN knockdown triggers a p53-independent nucleolar stress response culminating in mitochondrial apoptosis. Here, we demonstrate a novel role of PPAN in the regulation of mitochondrial homeostasis and autophagy. Our present study characterizes PPAN as a factor required for maintaining mitochondrial integrity and respiration-coupled ATP production. PPAN interacts with cardiolipin, a lipid of the inner mitochondrial membrane. Down-regulation of PPAN enhances autophagic flux in cancer cells. PPAN knockdown promotes recruitment of the E3-ubiquitin ligase Parkin to damaged mitochondria. Moreover, we provide evidence that PPAN knockdown decreases mitochondrial mass in Parkin-expressing cells. In summary, our study uncovers that PPAN knockdown is linked to mitochondrial damage and stimulates autophagy.

Mechanosensitive pathways controlling translation regulatory processes in skeletal muscle and implications for adaptation.

The ability of myofibers to sense and respond appropriately to mechanical signals is one of the primary determinants of the skeletal muscle phenotype. In response to a change in mechanical load, muscle cells alter their protein metabolism, primarily through the regulation of protein synthesis rate. Protein synthesis rates are determined by both translation efficiency and translational capacity within the muscle. Translational capacity is strongly determined by the ribosome content of the muscle; thus the regulation of ribosomal biogenesis by mechanical inputs has been an area of recent interest. Despite the clear association between mechanical signals and changes in protein metabolism, the molecular pathways that link these events are still not fully elucidated. This review focuses on recent studies looking at how mechanosignaling impacts translational events. The role of impaired mechanotransduction in aging is discussed, as is the connection between age-dependent signaling defects and compromised ribosomal biogenesis during mechanical overload. Finally, emerging evidence suggests that the nucleus can act as a mechanosensitive element and that this mode of mechanotransduction may have an important role in skeletal muscle physiology and adaptation.

Signal Transduction in Ribosome Biogenesis: A Recipe to Avoid Disaster.

Energetically speaking, ribosome biogenesis is by far the most costly process of the cell and, therefore, must be highly regulated in order to avoid unnecessary energy expenditure. Not only must ribosomal RNA (rRNA) synthesis, ribosomal protein (RP) transcription, translation, and nuclear import, as well as ribosome assembly, be tightly controlled, these events must be coordinated with other cellular events, such as cell division and differentiation. In addition, ribosome biogenesis must respond rapidly to environmental cues mediated by internal and cell surface receptors, or stress (oxidative stress, DNA damage, amino acid depletion, etc.). This review examines some of the well-studied pathways known to control ribosome biogenesis (PI3K-AKT-mTOR, RB-p53, MYC) and how they may interact with some of the less well studied pathways (eIF2α kinase and RNA editing/splicing) in higher eukaryotes to regulate ribosome biogenesis, assembly, and protein translation in a dynamic manner.

Regulation of Ribosome Biogenesis During Skeletal Muscle Hypertrophy

An increase in ribosomal capacity is a hallmark of the hypertrophying muscle. We review evidence demonstrating that transcription of ribosomal RNA genes is necessary for the increase in ribosomal capacity, and this is critical for muscle growth in human and animal models of hypertrophy.

Sphingolipid/Pkh1/2-TORC1/Sch9 Signaling Regulates Ribosome Biogenesis in Tunicamycin-Induced Stress Response in Yeast.

Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.

Low GAS5 Levels as a Predictor of Poor Survival in Patients with Lower-Grade Gliomas

Introduction Gliomas are infiltrative neoplasms of a highly invasive nature. Different stages of gliomas feature distinct genomic, genetic, and epigenetic changes. The long noncoding RNA Growth Arrest Specific Transcript 5 (GAS5) is an identified tumour suppressor involved in several cancers. However, the underlying roles of the GAS5 gene in lower-grade glioma (LGG) patients are not clear. Methods Via bioinformatic analysis based on TCGA-LGG and TCGA-GBM data, we explored the mechanisms of GAS5 expression in LGG (grades II and III) and high-grade glioma (glioblastoma multiforme, grade IV). The log-rank test and multivariate Cox analysis were performed to find the association between GAS5 and overall survival (OS) in LGG patients. Weighted gene coexpression network analysis (WGCNA) and RNA-Seq analysis were applied to find the key gene network associated with GAS5. Results We found that GAS5 expression was downregulated in both LGG and glioblastoma multiforme (GBM) compared with normal brain tissue. Low methylation in the GAS5 promoter region was detected in both LGG and GBM tissues. The amplification type was the predominant type of GAS5 gene alteration in both LGG and GBM. High GAS5 expression was more associated with long overall survival (OS) in LGG patients than in GBM patients. The multivariate survival analysis of GAS5 and clinical and molecular characteristics in LGG patients further confirmed the association between GAS5 and OS in LGG patients. We then developed a nomogram for clinical use. WGCNA and RNA-Seq analysis indicated that ribosomal biogenesis and translation initiation were the predominant events regulated by GAS5 in LGG patients. Conclusion Taken together, these results demonstrate that GAS5 expression is associated with OS in LGG patients and that its underlying roles involve the regulation of ribosomal biogenesis and translation initiation, which may aid in identifying a new target for the treatment of LGG.

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform.

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml -1 . Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

Regulation of Ribosome Biogenesis in Skeletal Muscle Hypertrophy.

The ribosome is the enzymatic macromolecular machine responsible for protein synthesis. The rates of protein synthesis are primarily dependent on translational efficiency and capacity. Ribosome biogenesis has emerged as an important regulator of skeletal muscle growth and maintenance by altering the translational capacity of the cell. Here, we provide evidence to support a central role for ribosome biogenesis in skeletal muscle growth during postnatal development and in response to resistance exercise training. Furthermore, we discuss the cellular signaling pathways regulating ribosome biogenesis, discuss how myonuclear accretion affects translational capacity, and explore future areas of investigation within the field.

Notchless defines a stage-specific requirement for ribosome biogenesis during lineage progression in adult skeletal myogenesis.

Cell fate decisions occur through the action of multiple factors, including signalling molecules and transcription factors. Recently, the regulation of translation has emerged as an important step for modulating cellular function and fate, as exemplified by ribosomes that play distinct roles in regulating cell behaviour. Notchless (Nle) is a conserved nuclear protein that is involved in a crucial step in ribosome biogenesis, and is required for the maintenance of adult haematopoietic and intestinal stem/progenitor cells. Here, we show that activated skeletal muscle satellite cells in conditional Nle mutant mice are arrested in proliferation; however, deletion of Nle in myofibres does not impair myogenesis. Furthermore, conditional deletion of Nle in satellite cells during homeostasis did not impact on their fate for up to 3 months. In contrast, loss of Nle function in primary myogenic cells blocked proliferation because of major defects in ribosome formation. Taken together, we show that muscle stem cells undergo a stage-specific regulation of ribosome biogenesis, thereby underscoring the importance of differential modulation of mRNA translation for controlling cell fate decisions.