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Diamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. Murine double minute 2 (MDM2), the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients.

Nucleoli are well defined for their function in ribosome biogenesis, but only a small fraction of the nucleolar proteome has been characterized. Here, we report that the proto-oncogene, c-Jun, is targeted to the nucleolus. Using live cell imaging in myogenic cells, we document that the c-Jun basic domain contains a unique, evolutionarily conserved motif that determines nucleolar targeting. Fos family Jun dimer partners, such as Fra2, while nuclear, do not co-localize with c-Jun in the nucleolus. A point mutation in c-Jun that mimics Fra2 (M260E) in its Nucleolar Localization sequence (NoLS) results in loss of c-Jun nucleolar targeting while still preserving nuclear localization. Fra2 can sequester c-Jun in the nucleoplasm, indicating that the stoichiometric ratio of heterodimeric partners regulates c-Jun nucleolar targeting. Finally, nucleolar localization of c-Jun modulates nucleolar architecture and ribosomal RNA accumulation. These studies highlight a novel role for Jun family proteins in the nucleolus, having potential implications for a diverse array of AP-1-regulated cellular processes.

We aimed to identify whether Rho GTPase activating proteins (RhoGAPs) were downregulated in cervical cancers and might be targeted to reduce the growth of cervical cancer using the GEO database and immunohistochemical analysis to identified changes in transcription and protein levels. We analyzed their proliferation, clone formation ability, and their growth as subcutaneous tumors in mice. To detect ARHGAP30 localization in cells, immunofluorescence assays were conducted. Mass spectrometry combined with immunoprecipitation experiments were used to identify binding proteins. Protein interactions were validated with co‐immunoprecipitation assays. Western‐blot and q‐PCR were applied to analyze candidate binding proteins that were associated with ribosome biogenesis. Puromycin incorporation assay was used to detect the global protein synthesis rate. We identified that ARHGAP30 was the only downregulated RhoGAP and was related to the survival of cervical cancer patients. Overexpression of ARHGAP30 in cervical cancer cells inhibited cell proliferation and migration. ARHGAP30 immunoprecipitated proteins were enriched in the ribosome biogenesis process. ARHGAP30 was located in the nucleous and interacted with nucleolin (NCL). Overexpression of ARHGAP30 inhibited rRNA synthesis and global protein synthesis. ARHGAP30 overexpression induced the ubiquitination of NCL and decreased its protein level in Hela cells. The function of ARHGAP30 on cervical cancer cell ribosome biogenesis and proliferation was independent of its RhoGAP activity as assessed with a RhoGAP‐deficient plasmid of ARHGAP30R55A. Overall, the findings revealed that ARHGAP30 was frequently downregulated and associated with shorter survival of cervical cancer patients. ARHGAP30 may suppress growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of NCL., ARHGAP30 suppressed growth of cervical cancer by reducing ribosome biogenesis and protein synthesis through promoting ubiquitination of nucleolin.

18 p.-9 fig., Premise: Light and gravity are fundamental cues for plant development. Our understanding of the effects of light stimuli on plants in space, without gravity, is key to providing conditions for plants to acclimate to the environment. Here we tested the hypothesis that the alterations caused by the absence of gravity in root meristematic cells can be counteracted by light., Methods: Seedlings of wild‐type Arabidopsis thaliana and two mutants of the essential nucleolar protein nucleolin (nuc1, nuc2) were grown in simulated microgravity,either under a white light photoperiod or under continuous darkness. Key variables of cell proliferation (cell cycle regulation), cell growth (ribosome biogenesis),and auxin transport were measured in the root meristem using in situ cellular markers and transcriptomic methods and compared with those of a 1 g control., Results: The incorporation of a photoperiod regime was sufficient to attenuate or suppress the effects caused by gravitational stress at the cellular level in the root meristem. In all cases, values for variables recorded from samples receiving light stimuli in simulated microgravity were closer to values from the controls than values from samples grown in darkness. Differential sensitivities were obtained for the two nucleolin mutants., Conclusions: Light signals may totally or partially replace gravity signals, significantly improving plant growth and development in microgravity. Despite that, molecular alterations are still compatible with the expected acclimation mechanisms, which need to be better understood. The differential sensitivity of nuc1 and nuc2 mutants to gravitational stress points to new strategies to produce more resilient plants to travel with humans in new extraterrestrial endeavors., This work was funded by the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation, Grants#ESP2015‐64323‐R and #RTI2018‐099309‐B‐I00 (co‐funded by EU‐ERDF) to F.J.M., and Bonus Recherche from the UPVD to J.S.V. The use of the facilities of microgravity simulation was provided by the ESA‐CORA‐Ground Based Facilities Program, contract Ref. #4000105761 to F.J.M. and R.H. A.M. was recipient of a contract of the Program for Young Researchers Training of the Agencia Estatal de Investigación of the Spanish Ministry of Science an Innovation Ref. #BES‐2013‐063933.

Fusarium oxysporum is the causal agent of the devastating Fusarium wilt by invading and colonizing the vascular system in various plants, resulting in substantial economic losses worldwide. Target of rapamycin (TOR) is a central regulator that controls intracellular metabolism, cell growth, and stress responses in eukaryotes, but little is known about TOR signalling in F. oxysporum. In this study, we identified conserved FoTOR signalling pathway components including FoTORC1 and FoTORC2. Pharmacological assays showed that F. oxysporum is hypersensitive to rapamycin in the presence of FoFKBP12 while the deletion mutant strain ΔFofkbp12 is insensitive to rapamycin. Transcriptomic data indicated that FoTOR signalling controls multiple metabolic processes including ribosome biogenesis and cell wall‐degrading enzymes (CWDEs). Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of F. oxysporum. Importantly, transcript levels of genes associated with ribosome biogenesis and CWDEs were dramatically downregulated in the ΔFotip4 mutant strain. Electrophoretic mobility shift assays showed that FoTIP4 can bind to the promoters of ribosome biogenesis‐ and CWDE‐related genes to positively regulate the expression of these genes. These results suggest that FoTOR signalling plays central roles in regulating hyphal growth and pathogenicity of F. oxysporum and provide new insights into FoTOR1 as a target for controlling and preventing Fusarium wilt in plants., Genetic analysis revealed that FoTOR1 interacting protein 4 (FoTIP4) acts as a new component of FoTOR signalling to regulate hyphal growth and pathogenicity of Fusarium oxysporum.

BCCIP was isolated based on its interactions with tumor suppressors BRCA2 and p21. Knockdown or knockout of BCCIP causes embryonic lethality in mice. BCCIP deficient cells exhibit impaired cell proliferation and chromosome instability. BCCIP also plays a key role in biogenesis of ribosome 60S subunits. BCCIP is conserved from yeast to humans, but it has no discernible sequence similarity to proteins of known structures. Here we report two crystal structures of an N-terminal truncated human BCCIPβ, consisting of residues 61-314. Structurally BCCIP is similar to GCN5-related acetyltransferases (GNATs) but contains different sequence motifs. Moreover, both acetyl-CoA and substrate-binding grooves are altered in BCCIP. A large 19-residue flap over the putative CoA binding site adopts either an open or closed conformation in BCCIP. The substrate binding groove is significantly reduced in size and is positively charged despite the acidic isoelectric point of BCCIP. BCCIP has potential binding sites for partner proteins and may have enzymatic activity.

The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

Ribosome assembly factor URB1 is essential for ribosome biogenesis. However, its latent role in cancer remains unclear. Analysis of The Cancer Genome Atlas database and clinical tissue microarray staining showed that URB1 expression was upregulated in colorectal cancer (CRC) and prominently related to clinicopathological characteristics. Silencing of URB1 hampered human CRC cell proliferation and growth in vitro and in vivo. Microarray screening, ingenuity pathway analysis, and JASPAR assessment indicated that activating transcription factor 4 (ATF4) and X‐box binding protein 1 (XBP1) are potential downstream targets of URB1 and could transcriptionally interact through direct binding. Silencing of URB1 significantly decreased ATF4 and cyclin A2 (CCNA2) expression in vivo and in vitro. Restoration of ATF4 effectively reversed the malignant proliferation phenotype of URB1‐silenced CRC cells. Dual‐luciferase reporter and ChIP assays indicated that XBP1 transcriptionally activated ATF4 by binding with its promoter region. X‐box binding protein 1 colocalized with ATF4 in the nuclei of RKO cells, and ATF4 mRNA expression was positively regulated by XBP1. This study shows that URB1 contributes to oncogenesis and CRC growth through XBP1‐mediated transcriptional activation of ATF4. Therefore, URB1 could be a potential therapeutic target for CRC., The classic ribosome assembly factor URB1 is overexpressed and related to unfavorable clinicopathological characteristics in colorectal cancer (CRC). URB1 silencing significantly hampered human CRC cell proliferation and decreased ATF4, CCNA2, and XBP1 expression in vivo and in vitro. Briefly, our study shows that URB1 contributes to oncogenesis and growth of CRC through XBP1‐medaited transcriptional activation of ATF4 and URB1 could be a potential therapeutic target for CRC.

Ribosomal DNA (rDNA) transcription is a limiting step in ribosome biogenesis, crucial for protein synthesis and cell growth-especially at the early stages of embryonic development-and is regulated in a mammalian target of rapamycin (mTOR)-dependent manner. Our previous report demonstrated that treatment with mTOR inhibitors during artificial embryonic activation improved the development of embryos derived from somatic cell nuclear transfer (SCNT). We hypothesize that inhibition of ribosome biogenesis in somatic cells facilitates reactivation of embryonic nucleolar establishment and ribosome biogenesis in SCNT embryos. Herein, we show that mTOR inhibitors suppressed ribosome biogenesis in somatic cells, and more importantly, improved development potential of SCNT embryos (blastocyst rate, 34% vs 24%). SCNT embryos derived from drug-treated somatic cells exhibited higher levels of 47S, 18S, and 5S rRNAs, upstream binding factor (UBF) mRNA, ribosomal protein S6; they also improved the rebuilding of the nucleolar ultrastructure. In addition, treatment of donor cells with the RNA polymerase I (Pol I) inhibitor cx5461 caused similar effects on SCNT embryos. These results indicated that transient inhibition of rDNA transcription in donor cells facilitated the establishment of functional nucleoli and improved preimplantation development of SCNT embryos.

Ribosomes are ribonucleoprotein particles that are essential for protein biosynthesis in all forms of life. During ribosome biogenesis, transcription, folding, modification, and processing of rRNA are coupled to the assembly of proteins. Various assembly factors are required to synchronize all different processes that occur during ribosome biogenesis. Herein, the RNA chaperone and RNA strand annealing activity of rRNA modification enzyme ribosome small subunit methyltransferase C (RsmC), which modifies guanine to 2-methylguanosine (m2 G) at position 1207 of 16S rRNA (Escherichia coli nucleotide numbering) located at helix 34 (h34), are reported. A 25-fold increase in the h34 RNA strand annealing rates is observed in the presence of RsmC. Single-molecule FRET experiments confirmed the ability of protein RsmC to denature a non-native structure formed by one of the two h34 strands and to form a native-like duplex. This observed RNA chaperone activity of protein RsmC might play a vital role in the rapid generation of functional ribosomes.

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.

Lipid metabolism is essential for stemness maintenance, self‐renewal, and differentiation of stem cells, however, the regulatory function of cholesterol metabolism in erythroid differentiation is poorly studied. In the present study, a critical role for cholesterol homeostasis in terminal erythropoiesis is uncovered. The master transcriptional factor GATA1 binds to Sterol‐regulatory element binding protein 2 (SREBP2) to downregulate cholesterol biosynthesis, leading to a gradual reduction in intracellular cholesterol levels. It is further shown that reduced cholesterol functions to block erythroid proliferation via the cholesterol/mTORC1/ribosome biogenesis axis, which coordinates cell cycle exit in the late stages of erythroid differentiation. The interaction of GATA1 and SREBP2 also provides a feedback loop for regulating globin expression through the transcriptional control of NFE2 by SREBP2. Importantly, it is shown that disrupting intracellular cholesterol hemostasis resulted in defect of terminal erythroid differentiation in vivo. These findings demonstrate that fine‐tuning of cholesterol homeostasis emerges as a key mechanism for regulating erythropoiesis., GATA binding protein 1 (GATA1) binds Sterol‐regulatory element binding protein 2 (SREBP2) to downregulate cholesterol biosynthesis, leading to a gradual reduction in cholesterol levels. Lower cholesterol inhibits mTORC1 and ribosome biogenesis and activates p53, thereby coordinating termination of cell cycling in the later stage of erythroid differentiation. Disrupting cholesterol homeostasis interferes with terminal erythropoiesis in vitro and in vivo.

The maternal nucleolus plays an indispensable role in zygotic genome activation (ZGA) and early embryonic development in mice. During oocyte-to-embryo transition, the nucleolus is subject to substantial transformation. Despite the primary role of the nucleolus is ribosome biogenesis, accumulating evidence has uncovered its functions in various other cell processes. However, the regulation of nucleolar maturation and ribosome biogenesis and the molecules involved remain unclear during early embryonic development. In this study, we observed that nucleolar protein 2 (NOP2) is restrictedly localized within the nucleolus, first detected in the late two-cell embryos, and increases to a peak level at the eight-cell stage in mice. RNAi-mediated NOP2 depletion leads to a developmental arrest during the morula-to-blastocyst transition. RNA-seq analyses reveal that 208 genes are differentially expressed, including multiple lineage-specific genes and several genes encoding ribosome proteins. Indeed, we observe a failure of the first lineage specification with reduced TEA domain transcription factor 4(TEAD4) (trophectoderm-specific), tir na nog (NANOG), and kruppel-like factor 4 (KLF4) (inner cell mass-specific). Importantly, by Transmission Electron Microscopy (TEM), we noted a decrease in the ratio of the nucleolus size and an increase in the ratio of the size of the nucleolus precursor body, suggesting the nucleolar maturation is disrupted. Moreover, both qPCR and Fluorescence In Situ Hybridization (FISH) data showcase a significant decrease in the abundance of ribosome RNAs. Similarly, NOP2 depletion causes reduced developmental potential and decreased rRNA level in bovine early embryos, suggesting a functional conservation of NOP2 in mammals. Taken together, these results suggest that NOP2 is required for mammalian preimplantation development, presumably by regulating nucleolar maturation and ribosome biogenesis.

Detoxification enzymes are necessary for insects to metabolize toxic substances and maintain physiological activities. Cytochromes P450 (CYPs), glutathione S-transferases (GSTs), and carboxylesterase (CarEs) are the main detoxification enzymes in insects. In addition, UDP-glucosyltransferase and ATP-binding cassette transporter also participate in the process of material metabolism. This study collected proteins related to detoxification in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). And we performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on these proteins to understand their biological function. We constructed the protein-protein interaction network for the silkworm's detoxification enzymes and analyzed the network's topological properties. We found that BGIBMGA014046-TA, BGIBMGA003221-TA, BGIBMGA011092-TA, BGIBMGA000074-TA, and LOC732976 are the essential proteins in the network. These proteins are primarily involved in the process of ribosome biogenesis and may be related to protein synthesis. We integrated GO, KEGG, and network analysis and found that ribosome-associated protein and GSTs played a vital role in the detoxification process.

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a non-enzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize and store large quantities of polyP. In this study, we applied a proven screening strategy to evaluate the polyphosphorylation status of over 200 candidate targets in the budding yeastS. cerevisiae.We report 8 new polyphosphorylated proteins that interact genetically and physically with a previously identified network of targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising additional questions regarding the location of polyphosphorylationin vivo.

The human fungal pathogen Candida albicans switches its morphology from yeast to hyphal forms. The morphological transition may render C. albicans virulent. Several signaling cascades, including those of the cyclic AMP-protein kinase A and mitogen-activated protein kinase pathways, are responsible for morphogenesis. In this study, we observed a reduction in gene transcription of ribosomal proteins during true hyphae formation. Moreover, morphogenesis-dependent decrease in ribosomal protein gene transcription was confirmed in constitutive yeast or filamentous growing strains. We consistently observed that polysome and monosome levels were decreased by hyphal stimuli through TORC1 and Sch9 kinases. Taken together, these results provide several lines of evidence to show that the Tor1-Sch9 kinase cascade, which stimulates transcription of ribosomal protein genes, exists in C. albicans. Thus, the present study revealed a novel link between ribosome biogenesis and morphogenesis in C. albicans that is mediated by Tor1 and Sch9.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

Multitoxin Bt‐crops expressing insecticidal toxins with different modes of action, for example, Cry and Vip, are expected to improve resistance management in target pests. While Cry1A resistance has been relatively well characterized in some insect species, this is not the case for Vip3A, for which no mechanism of resistance has yet been identified. Here we applied HT‐SuperSAGE to analyze the transcriptome of the gut tissue of tobacco budworm Heliothis virescens (F.) laboratory‐selected for Vip3Aa resistance. From a total of 1 324 252 sequence reads, 5 895 126‐bp tags were obtained representing 17 751 nonsingleton unique transcripts (UniTags) from genetically similar Vip3Aa‐resistant (Vip–Sel) and susceptible control (Vip–Unsel) strains. Differential expression was significant (≥2.5 fold or ≤0.4; P < 0.05) for 1989 sequences (11.2% of total UniTags), where 420 represented overexpressed (OE) and 1569 underexpressed (UE) genes in Vip–Sel. BLASTN searches mapped 419 UniTags to H. virescens sequence contigs, of which, 416 (106 OE and 310 UE) were unambiguously annotated to proteins in NCBI nonredundant protein databases. Gene Ontology distributed 345 of annotated UniTags in 14 functional categories with metabolism (including serine‐type hydrolases) and translation/ribosome biogenesis being the most prevalent. A UniTag homologous to a particular member of the REsponse to PAThogen (REPAT) family was found among most overexpressed, while UniTags related to the putative Vip3Aa‐binding ribosomal protein S2 (RpS2) were underexpressed. qRT‐PCR of a subset of UniTags validated the HT‐SuperSAGE data. This study is the first providing lepidopteran gut transcriptome associated with Vip3Aa resistance and a foundation for future attempts to elucidate the resistance mechanism.

Elevated ribosome biogenesis in oncogene‐driven cancers is commonly targeted by DNA‐damaging cytotoxic drugs. Our previous first‐in‐human trial of CX‐5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single‐agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX‐5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP‐dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I‐directed therapies., The elevated metabolic activity and activation of cAMP‐dependent pro‐survival mechanisms that lead to resistance of hematological cancers towards rRNA synthesis inhibition also poses a potentially targetable vulnerability.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function. Significance Statement AtRsgA is an assembly factor necessary for maturation of the small subunit of the chloroplast ribosome. Depletion of AtRsgA leads to dwarfed, chlorotic plants, a decrease of mature 16S rRNA and smaller, but more numerous, chloroplasts. Large-scale transcriptomic and proteomic analysis revealed that chloroplast-encoded and -targeted proteins were less abundant, while the corresponding transcripts were increased in the mutant. We analyze the transcriptional responses of several retrograde signaling pathways to suggest the mechanism underlying this compensatory response.

Nucleolar GTP-binding protein 1 (NOG1) is a highly conserved GTPase first reported in Trypanosoma as required for ribosome biogenesis. We characterized NbNOG1, a Nicotiana benthamiana NOG1 ortholog sharing more than 45% amino acid identity with Trypanosoma, yeast, and human NOG1. N. benthamiana plants silenced for NbNOG1 were stunted and produced sterile flowers. NbNOG1 is functionally interchangeable with yeast NOG1 (ScNOG1), rescuing yeast lethality caused by loss of ScNOG1. Finally, NbNOG1 silencing caused over-accumulation of pre-rRNA processing intermediates, and concomitant loss of mature rRNAs. Collectively, these data support a role for NbNOG1 in ribosomal RNA processing.

Ribosome biogenesis is a fundamental process in eukaryotic cells. Although Urb2 protein has been implicated in ribosome biogenesis in yeast, the Urb2 domain is loosely conserved between plants and yeast, and the function of Urb2 protein in plants remains unknown. Here, we isolated a maize mutant, designated as urb2, with defects in kernel development and vegetative growth. Positional cloning and transgenic analysis revealed that urb2 encodes an Urb2 domain-containing protein. Compared with the wild-type (WT), the urb2 mutant showed decreased ratios of 60S/40S and 80S/40S and increased ratios of polyribosomes. The pre-rRNA intermediates of 35/33S rRNA, P-A3 and 18S-A3 were significantly accumulated in the urb2 mutant. Transcriptome profiling of the urb2 mutant indicated that ZmUrb2 affects the expression of a number of ribosome-related genes. We further demonstrated that natural variations in ZmUrb2 are significantly associated with maize kernel length. The overall results indicate that, by affecting pre-rRNA processing, the Urb2 protein is required for ribosome biogenesis in maize.

Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF-Tu, EF-G and RF3, play well-known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), conversely, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit respectively. These findings have important implications for ribosome biogenesis in bacteria. Because the GTPase activity of each of these proteins depends on interactions with both ribosomal subunits, some portion of 30S and 50S assembly must occur in the context of the 70S ribosome. In this review, we introduce the trGTPases of bacteria, describe the new functional data on LepA and BipA, and discuss the how these findings shape our current view of ribosome biogenesis in bacteria.

Proper regulation of ribosome biosynthesis is mandatory for cellular adaptation, growth and proliferation. Ribosome biogenesis is the most energetically demanding cellular process, which requires tight control. Abnormalities in ribosome production have severe consequences, including developmental defects in plants and genetic diseases (ribosomopathies) in humans. One of the processes occurring during eukaryotic ribosome biogenesis is processing of the ribosomal RNA precursor molecule (pre-rRNA), synthesized by RNA polymerase I, into mature rRNAs. It must not only be accurate but must also be precisely coordinated with other phenomena leading to the synthesis of functional ribosomes: RNA modification, RNA folding, assembly with ribosomal proteins and nucleocytoplasmic RNP export. A multitude of ribosome biogenesis factors ensure that these events take place in a correct temporal order. Among them are endo- and exoribonucleases involved in pre-rRNA processing. Here, we thoroughly present a wide spectrum of ribonucleases participating in rRNA maturation, focusing on their biochemical properties, regulatory mechanisms and substrate specificity. We also discuss cooperation between various ribonucleolytic activities in particular stages of pre-rRNA processing, delineating major similarities and differences between three representative groups of eukaryotes: yeast, plants and humans.

Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm. Here, we cloned almost the entire set (∼180) of ribosome biogenesis factors from the thermophilic fungus Chaetomium thermophilum in order to perform an in-depth analysis of their protein-protein interaction network as well as exploring the suitability of these thermostable proteins for structural studies. First, we performed a systematic screen, testing about 80 factors for crystallization and structure determination. Next, we performed a yeast 2-hybrid analysis and tested about 32,000 binary combinations, which identified more than 1000 protein-protein contacts between the thermophilic ribosome assembly factors. To exemplary verify several of these interactions, we performed biochemical reconstitution with the focus on the interaction network between 90S pre-ribosome factors forming the ctUTP-A and ctUTP-B modules, and the Brix-domain containing assembly factors of the pre-60S subunit. Our work provides a rich resource for biochemical reconstitution and structural analyses of the conserved ribosome assembly machinery from a eukaryotic thermophile.

Secretory defects cause transcriptional repression of ribosome biogenesis in Saccharomyces cerevisiae. However, the molecular mechanism underlying secretory defect-induced transcriptional repression of ribosome biogenesis remains to be fully elucidated. In this study, we demonstrated that the Arp2/3 complex was required for reduction of ribosome protein gene expression in response to defective secretion by addition of tunicamycin. Two cmd1 mutants, cmd1-228 and cmd1-239 that cause mislocalization of calmodulin and defective mitotic spindle formation, respectively, failed to interact with Arc35, a component of the Arp2/3 complex. These mutants also caused defects in the reduction of ribosome protein gene expression induced by secretory blockade. A mutation in TUB4 (tub4-1), whose product has an essential function in microtubule organization, showed a similar response. In addition, we showed that the response to a secretory defect required SUN protein Mps3, which was localized at the nuclear envelope and involved in spindle pole body assembly. These results suggest that the Arp2/3 complex is required to transmit signals resulting from secretory blockade, and that the spindle pole body functions as a transit point from cytoplasm to Mps3 at the nuclear envelope. Copyright © 2016 John Wiley & Sons, Ltd.

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.

Diamond–Blackfan anaemia (DBA) is a congenital disorder that presents in the first year of life as severe anaemia, and in several patients is coupled with developmental defects. DBA is a ribosomopathy because almost all known mutations or deletions occur in genes encoding ribosomal proteins (RPs) that impair ribosome biogenesis. However, atypical examples of patients carry mutations in the erythroid specific transcription factor GATA1. DBA is a rare disease that displays a high level of heterogeneity with respect to the affected RP and disease penetrance. The reduced availability of ribosomes affects several cellular processes, including stabilisation of the p53 tumour suppressor, and impaired messenger ribonucleic acid (mRNA) translation. Registration of the genetic and phenotypic characteristics of DBA patients worldwide is needed to understand the relation between mutations, patient symptoms and cellular processes that underlie this pathophysiology. Key Concepts Diamond–Blackfan anaemia (DBA) is caused by haploinsufficiency of one of several ribosomal proteins in at least two-thirds of all patients; the disease causing mutation is unknown in approximately 30% of patients. The mechanisms underlying the ribosomopathy DBA are multiple, and are incompletely understood. Particularly, the roles of transcript specific translation and iron homeostasis are underestimated. Defective mRNA translation in ribosomopathies such as DBA should be investigated in cells that are affected in the disease, because an mRNA translation defect depends on the cell's specific transcriptome. DBA type I and DBA type II should be used to distinguish between patients carrying RP mutations versus those carrying mutations in genes that specifically affect erythropoiesis including GATA1. The ribosomopathy Diamond–Blackfan anaemia should be renamed to Diamond–Blackfan syndrome to emphasise that it is a systemic disease of which severe anaemia at young age is the most common, but not the exclusive symptom. The heterogeneity with which DBA presents, even when family members carry the same mutation, needs to be investigated to find a proper treatment for severe DBA. Keywords: ribosomopathy; ribosome biogenesis; ribosomal proteins; TP53; mRNA translation; Gata1

The pathogenic fungus Cryptococcus neoformans must adapt to glucose-limited conditions in the lung and glucose replete conditions upon dissemination to the brain. We report that glucose controls ribosome biogenesis and translation by modulating mRNA decay through a balance of PKA and Hog1 signalling. Glucose signalling through PKA stabilized ribosomal protein (RP) mRNAs whereas glucose starvation destabilized RP transcripts through Hog1. Glucose starvation-induced oxidative stress response genes, and treatment of glucose-fed cells with reactive oxygen species (ROS) generating compounds repressed RP transcripts, both of which were dependent on Hog1. Stabilization of RP transcripts led to retention of polysomes in a hog1Δ mutant, whereas stabilization of RP transcripts by cyclic AMP did not affect translation repression, suggesting that Hog1 alone signals translation repression. In sum, this work describes a novel antagonism between PKA and Hog1 controlling ribosome biogenesis via mRNA stability in response to glucose availability in this important human pathogen.

Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016.

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.

Most bacteria encode numerous chromosomal toxin-antitoxin (TA) systems that are proposed to contribute to stress tolerance, as they are able to shift the cells to a dormant state. Toxins act on a variety of targets with the majority attacking the translational apparatus. Intriguingly, the toxicity mechanisms of even closely related toxins may differ essentially. Here, we report on a new type of TA toxin that inhibits ribosome biogenesis. GraT of the GraTA system has previously been described in Pseudomonas putida as an unusually moderate toxin at optimal growth temperatures. However, GraT causes a severe growth defect at lower temperatures. Here, we demonstrate that GraT causes the accumulation of free ribosomal subunits. Mapping the rRNA 5' ends reveals incomplete processing of the free subunits and quantification of modified nucleosides shows an underrepresentation of late subunit assembly specific modifications. This indicates that GraT inhibits ribosome subunit assembly. Interestingly, GraT effects can be alleviated by modification of the chaperone DnaK, a known facilitator of late stages in ribosome biogenesis. We show that GraT directly interacts with DnaK and suggest two possible models for the role of this interaction in GraT toxicity.

The nucleolus is a nuclear organelle involved in ribosome biogenesis. In most eukaryotes this structure disperses during prophase through anaphase and reorganizes at telophase by a process known as nucleologenesis. This process involves new transcription of ribosomal DNA at the nucleolar organizer region and the formation of prenucleolar bodies fusing to it. In Giardia lamblia, for a long time considered the only anucleolated eukaryote, a very small nucleolus has been recently described. In order to evaluate whether nucleologenesis is also present in Giardia, we analyzed the distribution of nucleolar material during telophase using different light and electron microscopy techniques including silver staining for the nucleolar organizer. Results indicate that in G. lamblia, nucleolar elements persist mainly as an intranuclear peripheral organelle during all stages of division, including telophase, however, no prenucleolar bodies are detected in the nucleoplasm. Therefore, in the parasite, nucleolar material is present throughout cell division including telophase and formation of prenucleolar bodies may not be required for nucleologenesis.