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Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44 - CD25 + double-negative stage. In vivo labeling with O -propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3' untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success., Competing Interests: Conflict of interest disclosure The authors declare no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Nephrogenesis is driven by complex signaling pathways that control cell growth and differentiation. The endoplasmic reticulum chaperone calreticulin (Calr) is well known for its function in calcium storage and in the folding of glycoproteins. Its role in kidney development is still not understood. We provide evidence for a pivotal role of Calr in nephrogenesis in this investigation. We show that Calr deficiency results in the disrupted formation of an intact nephrogenic zone and in retardation of nephrogenesis, as evidenced by the disturbance in the formation of comma-shaped and s-shaped bodies. Using proteomics and transcriptomics approaches, we demonstrated that in addition to an alteration in Wnt-signaling key proteins, embryonic kidneys from Calr -/- showed an overall impairment in expression of ribosomal proteins which reveals disturbances in protein synthesis and nephrogenesis. CRISPR/cas9 mediated knockout confirmed that Calr deficiency is associated with a deficiency of several ribosomal proteins and key proteins in ribosome biogenesis. Our data highlights a direct link between Calr expression and the ribosome biogenesis.

[URE3] is an amyloid-based prion of Ure2p, a negative regulator of poor nitrogen source catabolism in Saccharomyces cerevisiae. Overproduced Btn2p or its paralog Cur1p, in processes requiring Hsp42, cure the [URE3] prion. Btn2p cures by collecting Ure2p amyloid filaments at one place in the cell. We find that rpl4aΔ, rpl21aΔ, rpl21bΔ, rpl11bΔ, and rpl16bΔ (large ribosomal subunit proteins) or ubr2Δ (ubiquitin ligase targeting Rpn4p, an activator of proteasome genes) reduce curing by overproduced Btn2p or Cur1p. Impaired curing in ubr2Δ or rpl21bΔ is restored by an rpn4Δ mutation. No effect of rps14aΔ or rps30bΔ on curing was observed, indicating that 60S subunit deficiency specifically impairs curing. Levels of Hsp42p, Sis1p, or Btn3p are unchanged in rpl4aΔ, rpl21bΔ, or ubr2Δ mutants. Overproduction of Cur1p or Btn2p was enhanced in rpn4Δ and hsp42Δ mutants, lower in ubr2Δ strains, and restored to above wild-type levels in rpn4Δ ubr2Δ strains. As in the wild-type, Ure2N-GFP colocalizes with Btn2-RFP in rpl4aΔ, rpl21bΔ, or ubr2Δ strains, but not in hsp42Δ. Btn2p/Cur1p overproduction cures [URE3] variants with low seed number, but seed number is not increased in rpl4aΔ, rpl21bΔ or ubr2Δ mutants. Knockouts of genes required for the protein sorting function of Btn2p did not affect curing of [URE3], nor did inactivation of the Hsp104 prion-curing activity. Overactivity of the ubiquitin/proteasome system, resulting from 60S subunit deficiency or ubr2Δ, may impair Cur1p and Btn2p curing of [URE3] by degrading Cur1p, Btn2p or another component of these curing systems., (Published by Oxford University Press on behalf of Genetics Society of America 2021. This work is written by US Government employees and is in the public domain in the US.)

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3’UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3’UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.

Monocyte infiltration to the site of pathogenic invasion is critical for inflammatory response and host defence. However, this process demands precise regulation as uncontrolled migration of monocytes to the site delays resolution of inflammation and ultimately promotes chronic inflammation. C-C motif chemokine ligand 2 (CCL2) plays a key role in monocyte migration, and hence, its expression should be tightly regulated. Here, we report a post-transcriptional regulation of CCL2 involving the large ribosomal subunit protein L22 (RPL22) in LPS-activated, differentiated THP-1 cells. Early events following LPS treatment include transcriptional upregulation of RPL22 and its nuclear accumulation. The protein binds to the first 20 nt sequence of the 5'UTR of ccl2 mRNA. Simultaneous nuclear translocation of up-frameshift-1 protein and its interaction with RPL22 results in cytoplasmic degradation of the ccl2 mRNA at a later stage. Removal of RPL22 from cells results in increased expression of CCL2 in response to LPS causing disproportionate migration of monocytes. We propose that post-transcriptional regulation of CCL2 by RPL22 fine-tunes monocyte infiltration during a pathogenic insult and maintains homeostasis of the immune response critical to resolution of inflammation. DATABASES: Microarray data are available in NCBI GEO database (Accession No GSE126525)., (© 2020 Federation of European Biochemical Societies.)

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Previously, we identified a mechanism of inflammation control directed by ribosomal protein L13a and "GAIT" (Gamma Activated Inhibitor of Translation) elements in target mRNAs and showed that its elimination in myeloid cell-specific L13a knockout mice (L13a KO) increased atherosclerosis susceptibility and severity. Here, we investigated the mechanistic basis of this endogenous defense against atherosclerosis. We compared molecular and cellular aspects of atherosclerosis in high-fat diet (HFD)-fed L13a KO and intact (control) mice. HFD treatment of control mice induced release of L13a from 60S ribosome, formation of RNA-binding complex, and subsequent GAIT element-mediated translational silencing. Atherosclerotic plaques from HFD-treated KO mice showed increased infiltration of M1 type inflammatory macrophages. Macrophages from KO mice showed increased phagocytic activity and elevated expression of LDL receptor and pro-inflammatory mediators. NanoString analysis of the plaques from KO mice showed upregulation of a number of mRNAs encoding inflammatory proteins. Bioinformatics analysis suggests the presence of the potential GAIT elements in the 3'UTRs of several of these mRNAs. Macrophage induces L13a/GAIT-dependent translational silencing of inflammatory genes in response to HFD as an endogenous defense against atherosclerosis in ApoE -/- model., (© 2020 Federation of American Societies for Experimental Biology.)

Plant cytoplasmic ribosomal proteins not only participate in protein synthesis, but also have specific roles in developmental regulation. However, the high heterogeneity of plant ribosome makes our understanding of these proteins very limited. Here we reported that RPL14B, a component of the ribosome large subunit, is critical for fertilization in Arabidopsis. RPL14B is existed in a majority of organs and tissues. No homozygous rpl14b mutant is available, indicating that RPL14B is irreplaceable for sexual reproduction. Smaller-sized rpl14b pollens could germinate normally, but pollen tube competitiveness is grievously weakened. Beside, cell fate specification is impaired in female gametophytes from heterozygous rpl14b/RPL14B ovules, resulting in defect of micropylar pollen tube attraction. However, this defect could be restored by restricted expression of RPL14B in synergid cells. Successful fertilization requires normal pollen tube growth and precise pollen tube guidance. Thus our results show a novel role of RPL14B in fertilization and shed new light on regulatory mechanism of pollen tube growth and precise pollen tube guidance., Competing Interests: Declaration of Competing Interest There are no conflicts to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, 'ribosome pegging', to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The ribosome is not only a protein-making machine, but also a regulatory element in protein synthesis. This view is supported by our earlier data showing that Arabidopsis mitoribosomes altered due to the silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 differentially translate mitochondrial transcripts compared with the wild-type. Here, we used ribosome profiling to determine the contribution of transcriptional and translational control in the regulation of protein synthesis in rps10 mitochondria compared with the wild-type ones. Oxidative phosphorylation system proteins are preferentially synthesized in wild-type mitochondria but this feature is lost in the mutant. The rps10 mitoribosomes show slightly reduced translation efficiency of most respiration-related proteins and at the same time markedly more efficiently synthesize ribosomal proteins and MatR and TatC proteins. The mitoribosomes deficient in S10 protein protect shorter transcript fragments which exhibit a weaker 3-nt periodicity compared with the wild-type. The decrease in the triplet periodicity is particularly drastic for genes containing introns. Notably, splicing is considerably less effective in the mutant, indicating an unexpected link between the deficiency of S10 and mitochondrial splicing. Thus, a shortage of the mitoribosomal S10 protein has wide-ranging consequences on mitochondrial gene expression., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Control of ticks has been achieved primarily by the application of acaricides, which has drawbacks such as environmental contamination leading to the selection of pesticide-resistant ticks. The potential of dsRNA to suppress genes critical for tick survival due to its sequence specificity suggests that dsRNAs could be developed as tailor-made pesticides. In this study, the dsRNA of P0 gene from the tick, Rhipicephalus haemaphysaloides, was evaluated as a potential anti-tick agent. Effects of using different dsRNA delivery methods were tested by quantitative RT-PCR and tick bioassays to determine survival, feeding and reproduction. The results showed that P0 dsRNAs could be effectively delivered into ticks and silenced by incubating with liposomes. Incubation time was found to be the most important factor in dsRNA delivery and gene silencing compared with liposome types and dsRNA concentration. The effects of P0 dsRNA treatment on ticks were found to be significant on blood feeding, molting or reproduction. These data show that anti-tick agents based on dsRNAs could have potential use in tick control., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of rpl11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, Rpl11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of Rpl11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mutations resulting in constitutive activation of signaling pathways that regulate ribosome biogenesis are among the most common genetic events in acute myeloid leukemia (AML). However, whether ribosome biogenesis presents as a therapeutic target to treat AML remains unexplored. Perturbations in ribosome biogenesis trigger the 5S ribonucleoprotein particle (RNP)-Mdm2-p53 ribosomal stress pathway, and induction of this pathway has been shown to have therapeutic efficacy in Myc-driven lymphoma. In the current study we address the physiological and therapeutic role of the 5S RNP-Mdm2-p53 pathway in AML. By utilizing mice that have defective ribosome biogenesis due to downregulation of ribosomal protein S19 (Rps19), we demonstrate that induction of the 5S RNP-Mdm2-p53 pathway significantly delays the initiation of AML. However, even a severe Rps19 deficiency that normally results in acute bone marrow failure has no consistent efficacy on already established disease. Finally, by using mice that harbor a mutation in the Mdm2 gene disrupting its binding to 5S RNP, we show that loss of the 5S RNP-Mdm2-p53 pathway is dispensable for development of AML. Our study suggests that induction of the 5S RNP-Mdm2-p53 ribosomal stress pathway holds limited potential as a single-agent therapy in the treatment of AML.

Although ribosomal proteins (RP) are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein, emerging evidence suggests that individual RP can perform critical regulatory functions that control developmental processes. We showed previously that despite the ubiquitous expression of the RP ribosomal protein L22 (Rpl22), germline ablation of Rpl22 in mice causes a selective, p53-dependent block in the development of αβ, but not γδ, T cell progenitors. Nevertheless, the basis by which Rpl22 loss selectively induces p53 in αβ T cell progenitors remained unclear. We show in this study that Rpl22 regulates the development of αβ T cells by restraining endoplasmic reticulum (ER) stress responses. In the absence of Rpl22, ER stress is exacerbated in αβ, but not γδ, T cell progenitors. The exacerbated ER stress in Rpl22-deficient αβ T lineage progenitors is responsible for selective induction of p53 and their arrest, as pharmacological induction of stress is sufficient to induce p53 and replicate the selective block of αβ T cells, and attenuation of ER stress signaling by knockdown of protein kinase R-like ER kinase, an ER stress sensor, blunts p53 induction and rescues development of Rpl22-deficient αβ T cell progenitors. Rpl22 deficiency appears to exacerbate ER stress by interfering with the ability of ER stress signals to block new protein synthesis. Our finding that Rpl22 deficiency exacerbates ER stress responses and induces p53 in αβ T cell progenitors provides insight into how a ubiquitously expressed RP can perform regulatory functions that are selectively required by some cell lineages but not others., (Copyright © 2016 by The American Association of Immunologists, Inc.)

5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype., (Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and d-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.

Diamond-Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19-deficient cells- in a disease-specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease-specific treatments of DBA., (© 2015 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.)

Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous ribosomopathy and inherited bone marrow failure syndrome characterized by anemia, reticulocytopenia, and decreased erythroid precursors in the bone marrow with an increased risk of malignancy and, in approximately 50%, physical abnormalities., Methods: We retrospectively analyzed clinical data from 77 patients with DBA born in the Russian Federation from 1993 to 2014. In 74 families there was one clinically affected individual; in only three instances a multiplex family was identified. Genomic DNA from 57 DBA patients and their first-degree relatives was sequenced for mutations in RPS19, RPS10, RPS24, RPS26, RPS7, RPS17, RPL5, RPL11, RPL35a, and GATA1., Results: Severe anemia presented before 8 months of age in all 77 patients; before 2 months in 61 (78.2%); before 4 months in 71 (92.2%). Corticosteroid therapy was initiated after 1 year of age in the majority of patients. Most responded initially to steroids, while 5 responses were transient. Mutations in RP genes were detected in 35 of 57 patients studied: 15 in RPS19, 6 in RPL5, 3 in RPS7, 3 each in RPS10, RPS26, and RPL11 and 1 each in RPS24 and RPL35a; 24 of these mutations have not been previously reported. One patient had a balanced chromosomal translocation involving RPS19. No mutations in GATA1 were found., Conclusion: In our cohort from an ethnically diverse population the distribution of mutations among RP genes was approximately the same as was reported by others, although within genotypes most of the mutations had not been previously reported., (© 2015 Wiley Periodicals, Inc.)

Diamond-Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count. Mutations in ribosomal protein genes have been identified in approximately half of all DBA cases. Corticosteriod therapy and bone marrow transplantation are common treatment options for patients; however, significant risks and complications are associated with these treatment options. Therefore, novel therapeutic approaches are needed for treating DBA. Sotatercept (ACE-011, and its murine ortholog RAP-011) acts as an activin receptor type IIA ligand trap, increasing hemoglobin and hematocrit in pharmacologic models, in healthy volunteers, and in patients with β-thalassemia, by expanding late-stage erythroblasts through a mechanism distinct from erythropoietin. Here, we evaluated the effects of RAP-011 in zebrafish models of RPL11 ribosome deficiency. Treatment with RAP-011 dramatically restored hemoglobin levels caused by ribosome stress. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by sequestering lefty1 from erythroid cells. These findings identify lefty1 as a signaling component in the development of erythroid cells and rationalize the use of sotatercept in DBA patients., (© 2015 by The American Society of Hematology.)

The streptomycin counter-selection system is a useful tool for constructing unmarked in-frame gene deletions, which is a fundamental approach to study bacteria and their pathogenicity at the molecular level. A prerequisite for this system is acquiring a streptomycin-resistant strain due to rpsL mutations, which encodes the ribosomal protein S12. However, in this study no streptomycin resistance was found to be caused by rpsL mutations in all 127 clinical strains of Klebsiella pneumoniae isolated from liver abscess patients. By screening 107 spontaneous mutants of streptomycin resistance from a clinical strain of K. pneumoniae, nucleotide substitution or insertion located within the rpsL was detected in each of these strains. Thirteen different mutants with varied S12 proteins were obtained, including nine streptomycin-dependent mutants. The virulence of all four streptomycin-resistant mutants was further evaluated. Compared with the parental strain, the K42N, K42T and K87R mutants showed a reduction in growth rate, and the K42N and K42T mutants became susceptible to normal human serum. In the mice LD50 (the bacterial dose that caused 50% death) assay, the K42N and K42T mutants were ∼ 1,000-fold less lethal (∼ 2 × 10(5) CFU) and the K87R mutant was ∼ 50-fold less lethal (∼ 1 × 10(4) CFU) than the parental strain (∼ 2 × 10(2) CFU). A K42R mutant showed non-observable effects on the above assays, while this mutant exhibited a small cost (P < 0.01) in an in vitro growth competition experiment. In summary, most of the K. pneumoniae strains with streptomycin resistance caused by rpsL mutations are less virulent than their parental strain in the absence of streptomycin. The K42R mutant showed similar pathogenicity to its parental strain and should be one of the best choices when using rpsL as a counter-selection marker.

Background: Diamond-Blackfan anemia (DBA) is a class of human diseases linked to defective ribosome biogenesis that results in clinical phenotypes. Genetic mutations in ribosome protein (RP) genes lead to DBA phenotypes, including hematopoietic defects and physical deformities. However, little is known about the global regulatory network as well as key miRNAs and gene pathways in the zebrafish model of DBA., Results: In this study, we establish the DBA model in zebrafish using an RPS24 morpholino and found that RPS24 is required for both primitive hematopoiesis and definitive hematopoiesis processes that are partially mediated by the p53 pathway. Several deregulated genes and miRNAs were found to be related to hematopoiesis, vascular development and apoptosis in RPS24-deficient zebrafish via RNA-seq and miRNA-seq data analysis, and a comprehensive regulatory network was first constructed to identify the mechanisms of key miRNAs and gene pathways in the model. Interestingly, we found that the central node genes in the network were almost all targeted by significantly deregulated miRNAs. Furthermore, the enforced expression of miR-142-3p, a uniquely expressed miRNA, causes a significant decrease in primitive erythrocyte progenitor cells and HSCs., Conclusions: The present analyses demonstrate that the comprehensive regulatory network we constructed is useful for the functional prediction of new and important miRNAs in DBA and will provide insights into the pathogenesis of mutant rps24-mediated human DBA disease.

A point mutation [technical knockout(25t) (tko(25t))] in the Drosophila gene coding for mitoribosomal protein S12 generates a phenotype of developmental delay and bang sensitivity. tko(25t) has been intensively studied as an animal model for human mitochondrial diseases associated with deficiency of mitochondrial protein synthesis and consequent multiple respiratory chain defects. Transgenic expression in Drosophila of the alternative oxidase (AOX) derived from Ciona intestinalis has previously been shown to mitigate the toxicity of respiratory chain inhibitors and to rescue mutant and knockdown phenotypes associated with cytochrome oxidase deficiency. We therefore tested whether AOX expression could compensate the mutant phenotype of tko(25t) using the GeneSwitch system to activate expression at different times in development. The developmental delay of tko(25t) was not mitigated by expression of AOX throughout development. AOX expression for 1 d after eclosion, or continuously throughout development, had no effect on the bang sensitivity of tko(25t) adults, and continued expression in adults older than 30 d also produced no amelioration of the phenotype. In contrast, transgenic expression of the yeast alternative NADH dehydrogenase Ndi1 was synthetically semi-lethal with tko(25t) and was lethal when combined with both AOX and tko(25t). We conclude that AOX does not rescue tko(25t) and that the mutant phenotype is not solely due to limitations on electron flow in the respiratory chain, but rather to a more complex metabolic defect. The future therapeutic use of AOX in disorders of mitochondrial translation may thus be of limited value., (Copyright © 2014 Kemppainen et al.)

Long known as the center of ribosome synthesis, the nucleolus is connected to cell cycle regulation in more subtle ways. One is a surveillance system that reacts promptly when rRNA synthesis or processing is impaired, halting cell cycle progression. Conversely, the nucleolus also acts as a first-responder to growth-related stress signals. Here we review emerging concepts on how these "infraribosomal" links between the nucleolus and cell cycle progression operate in both forward and reverse gears. We offer perspectives on how new cancer therapeutic designs that target this infraribosomal mode of cell growth control may shape future clinical progress., (© FASEB.)

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA., (Copyright © 2014. Published by Elsevier B.V.)

Ribosomal biogenesis involves the processing of pre-ribosomal RNA. A deficiency of some ribosomal proteins (RPs) impairs processing and causes Diamond Blackfan anemia (DBA), which is associated with anemia, congenital malformations and cancer. p53 mediates many features of DBA, but the mechanism of p53 activation remains unclear. Another hallmark of DBA is the upregulation of adenosine deaminase (ADA), indicating changes in nucleotide metabolism. In RP-deficient zebrafish, we found activation of both nucleotide catabolism and biosynthesis, which is consistent with the need to break and replace the faulty ribosomal RNA. We also found upregulation of deoxynucleotide triphosphate (dNTP) synthesis - a typical response to replication stress and DNA damage. Both RP-deficient zebrafish and human hematopoietic cells showed activation of the ATR/ATM-CHK1/CHK2/p53 pathway. Other features of RP deficiency included an imbalanced dNTP pool, ATP depletion and AMPK activation. Replication stress and DNA damage in cultured cells in non-DBA models can be decreased by exogenous nucleosides. Therefore, we treated RP-deficient zebrafish embryos with exogenous nucleosides and observed decreased activation of p53 and AMPK, reduced apoptosis, and rescue of hematopoiesis. Our data suggest that the DNA damage response contributes to p53 activation in cellular and zebrafish models of DBA. Furthermore, the rescue of RP-deficient zebrafish with exogenous nucleosides suggests that nucleoside supplements could be beneficial in the treatment of DBA., (© 2014. Published by The Company of Biologists Ltd.)

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF., (© 2014 Society for Reproduction and Fertility.)

A temperature-sensitive mutation in rplB, designated rplB142, encodes a missense mutation at position 142 [His (CAT) to Leu (CTT)] of Bacillus subtilis ribosomal protein L2. The strain carrying the mutation grew more slowly than the wild-type, even at low temperatures, probably due to the formation of defective 70S ribosomes and the accumulation of incomplete 50S subunits (50S* subunits). Gel analysis indicated that amounts of L2 protein and also of L16 protein were reduced in ribosomes prepared from the rplB142 mutant 90 min after increasing the growth temperature to 45 °C. These results suggest that the assembly of the L16 protein into the 50S subunit requires the native L2 protein. The H142L mutation in the defective L2 protein affected sporulation as well as growth, even at the permissive temperature. A suppressor mutation that restored both growth and sporulation of the rplB142 mutant at low temperature was identified as a single base deletion located immediately upstream of the yaaA gene that resulted in an increase in its transcription. Furthermore, genetic analysis showed that enhanced synthesis of YaaA restores the functionality of L2 (H142L) by facilitating its assembly into 50S subunits., (© 2014 The Authors.)

Diamond-Blackfan anemia is an inherited genetic disease caused by mutations in ribosomal protein genes. The disease is characterized by bone marrow failure, congenital anomalies, and a severe erythroid defect. The activation of the TP53 pathway has been suggested to be critical for the pathophysiology of Diamond-Blackfan anemia. While this pathway plays a role in the morphological defects that associate with ribosomal protein loss-of-function in animal models, its role in the erythroid defects has not been clearly established. To understand the specificity of erythroid defects in Diamond-Blackfan anemia, we knocked down five RP genes (two Diamond-Blackfan anemia-associated and three non-Diamond-Blackfan anemia-associated) in zebrafish and analyzed the effects on the developmental and erythroid phenotypes in the presence and absence of Tp53. The co-inhibition of Tp53 activity rescued the morphological deformities but did not alleviate the erythroid aplasia indicating that ribosomal protein deficiency causes erythroid failure in a Tp53-independent manner. Interestingly, treatment with L-Leucine or L-Arginine, amino acids that augment mRNA translation via mTOR pathway, rescued the morphological defects and resulted in a substantial recovery of erythroid cells. Our results suggest that altered translation because of impaired ribosome function could be responsible for the morphological and erythroid defects in ribosomal protein-deficient zebrafish., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The v-erbA oncogene transforms chicken erythrocytic progenitors (T2EC) by blocking their differentiation and freezing them in a state of self-renewal. Transcriptomes of T2EC, expressing either v-erbA or a non-transforming form of v-erbA (S61G), were compared using serial analysis of gene expression and some, but not all, mRNA-encoding ribosomal proteins were seen to be affected by v-erbA. These results suggest that this oncogene could modulate the composition of ribosomes. In the present study, we demonstrate, using two-dimensional difference in gel electrophoresis, that v-erbA-expressing cells have a lower amount of RPL11 associated with the ribosomes. The presence of ribosomes devoid of RPL11 in v-erbA-expressing cells was further confirmed by immunoprecipitation. In order to assess the possible impact of these specialized ribosomes on the translational activity, we analyzed proteomes of either v-erbA or S61G-expressing cells using 2D/mass spectrometry, and identified nine proteins present in differing amounts within these cells. Among these proteins, we focused on HSP70 because of its involvement in erythroid differentiation. Our results indicate that, in v-erbA-expressing cells, hsp70 is not only transcribed but also translated more efficiently, as shown by polyribosome fractionation experiments. We demonstrate here, for the first time, the existence of ribosomes with different protein components, notably ribosomes devoid of RPL11, and a regulation of mRNA translation depending on v-erbA oncogene expression.

Objective: Unresolved inflammatory response of macrophages plays a pivotal role in the pathogenesis of atherosclerosis. Previously we showed that ribosomal protein L13a-dependent translational silencing suppresses the synthesis of a cohort of inflammatory proteins in monocytes and macrophages. We also found that genetic abrogation of L13a expression in macrophages significantly compromised the resolution of inflammation in a mouse model of lipopolysaccharide-induced endotoxemia. However, its function in the pathogenesis of atherosclerosis is not known. Here, we examine whether L13a in macrophage has a protective role against high-fat diet-induced atherosclerosis., Approach and Results: We bred the macrophage-specific L13a knockout mice L13a Flox(+/+) Cre(+/+) onto apolipoprotein E-deficient background and generated the experimental double knockout mice L13a Flox(+/+) Cre(+/+) apolipoprotein E deficient (apoE(-/-)). L13a Flox(+/+) Cre(-/-) mice on apolipoprotein E-deficient background were used as controls. Control and knockout mice were subjected to high-fat diet for 10 weeks. Evaluation of aortic sinus sections and entire aorta by en face showed significantly higher atherosclerosis in the knockout mice. Severity of atherosclerosis in knockout mice was accompanied by thinning of the smooth muscle cell layer in the media, larger macrophage area in the intimal plaque region and higher plasma levels of inflammatory cytokines. In addition, macrophages isolated from knockout mice had higher polyribosomal abundance of several target mRNAs, thus showing defect in translation control., Conclusions: Our data demonstrate that loss of L13a in macrophages increases susceptibility to atherosclerosis in apolipoprotein E-deficient mice, revealing an important role of L13a-dependent translational control as an endogenous protection mechanism against atherosclerosis.

The fate of cultivated primary hematopoietic stem cells (HSCs) with respect to genetic instability and telomere attrition has not yet been described in great detail. Thus, knowledge of the genetic constitution of HSCs is important when interpreting results of HSCs in culture. While establishing a cell culture model for myelodysplastic syndrome with a deletion in 5q by performing RPS14 knockdown, we found surprising data that may be of importance for any CD34+ cell culture experiments. We performed cytogenetic analyses and telomere length measurement on transduced CD34+ cells and untransduced control cells to observe the effects of long-term culturing. Initially, CD34+ cells had a normal median telomere length of about 12 kb and showed no signs of chromosomal instability. During follow-up, the median telomere length seemed to decrease and, simultaneously, increased chromosomal instability could be observed - in modified and control cells. One culture showed a clonal monosomy 7 - independent of prior RPS14 knockdown. During further culturing, it seemed that the telomeres re-elongated, and chromosomes stabilized, while TERT expression was not elevated. In summary, irrespective of our results of RPS14 knockdown in the long-term culture of CD34+ cells, it becomes clear that cell culture artefacts inducing telomere shortening and chromosomal instability have to be taken into account and regular cytogenetic analyses should always be performed., (© 2013 S. Karger AG, Basel.)

Isolated congenital asplenia is characterized by the absence of a spleen at birth without any other developmental defect. Isolated congenital asplenia is a rare and life-threatening disease that predisposes patients to severe bacterial infections. The first and main genetic etiology of isolated congenital asplenia was discovered in 2013. Mutations in the gene RPSA, which encodes ribosomal protein SA, cause more than half of the cases of isolated congenital asplenia. These disease-causing mutations lead to haploinsufficiency of RPSA. Haploinsufficiency of genes encoding other ribosomal proteins have been reported to cause other developmental defects in humans, and in model organisms like the fly or the mouse. About half of the patients with Diamond-Blackfan anemia, which is a well-characterized ribosomopathy, present developmental defects such as craniofacial defects, cardiac defects or thumb abnormalities. The mechanism of pathogenesis linking mutations in ribosomal proteins, which are highly and ubiquitously expressed, to specific developmental defects remains to be elucidated. One hypothesis is that the ribosome, and ribosomal proteins in particular, regulate the expression of specific genes during development., (© Société de Biologie, 2015.)

Yeast (Saccharomyces cerevisiae) RNA is an important source of 5'-ribonucleotides that is used in both the food and pharmaceutical industries. Efficient transcription of rDNA is very important to construct yeast strains with high RNA content. The gene RRN10, which encodes, a component of the upstream activation factor, is essential to promote high-level transcription of rDNA. In our previous study, we isolated SupE strain as a dominant suppressor, which showed the ability to restore the severe growth defects and reduced RNA content caused by disruption of the RRN10 gene. SupE strain has multiple mutations which we designated collectively as SUPE. Further analysis on SUPE mutation indicated that RPL40A was responsible for suppression of defect caused by rrn10 disruption. However, there were no base changes in this gene as compared with the parental Δrrn10 strain, thus suggesting that an additional copy of RPL40A suppress the defects caused by Δrrn10 disruption, and that, in SupE strain, these defects are suppressed by increased transcription of RPL40A whose copy is doubled. When multiple copies of RPL40A were combined with SUPE mutation on an RRN10⁺ background, the resultant SupE strain had significantly higher RNA content than wild-type strain. In addition, increased transcription of RPL40B also showed significant effect to restore the growth defect and reduced RNA content caused by Δrrn10 disruption. We propose a model to explain how SUPE mutation increases the transcription of ribosomal protein genes such as RPL40A and RPL40B in SupE strain, resulting in an increase in RNA content., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome that is characterized by pure red-cell aplasia and associated physical deformities. It has been proven that defects of ribosomal proteins can lead to this disease and that RPS19 is the most frequently mutated gene in DBA patients. Previous studies suggest that p53-dependent genes and pathways play important roles in RPS19-deficient embryos. However, whether there are other vital factors linked to DBA has not been fully clarified. In this study, we compared the whole genome RNA-Seq data of zebrafish embryos injected with RPS19 morpholino (RPS19 MO), RPS19 and p53 morpholino simultaneously (RPS19+p53 MO) and control morpholino (control). We found that genes enriched in the functions of hematological systems, nervous system development and skeletal and muscular disorders had significant differential expression in RPS19 MO embryos compared with controls. Co-inhibition of p53 partially alleviates the abnormalities for RPS19-deficient embryos. However, the hematopoietic genes, which were down-regulated significantly in RPS19 MO embryos, were not completely recovered by the co-inhibition of p53. Furthermore, we identified the genome-wide p53-dependent and -independent genes and pathways. These results indicate that not only p53 family members but also other factors have important impacts on RPS19-deficient embryos. The detection of potential pathogenic genes and pathways provides us a new paradigm for future research on DBA, which is a systematic and complex hereditary disease.

The Saccharomyces cerevisiae Pbp1 [poly(A)-binding protein (Pab1)-binding protein] is believed to be involved in RNA metabolism and regulation of translation, since Pbp1 regulates a length of poly(A) tail and is involved in stress granule (SG) formation. However, a physiological function of Pbp1 remains unclear, since the pbp1Δ mutation has no obvious effect on cell growth. In this study, we showed that PBP1 genetically interacts with CCR4 and KHD1, which encode a cytoplasmic deadenylase and an RNA-binding protein, respectively. Ccr4 and Khd1 modulate a signal from Rho1 in the cell wall integrity pathway by regulating the expression of RhoGEF and RhoGAP, and the double deletion of CCR4 and KHD1 confers a severe growth defect displaying cell lysis. We found that the pbp1Δ mutation suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. The pbp1Δ mutation also suppressed the growth defect caused by double deletion of POP2, encoding another cytoplasmic deadenylase, and KHD1. Deletion of the gene encoding previously known Pbp1-interacting factor Lsm12, Pbp4, or Mkt1 did not suppress the growth defect of the ccr4Δ khd1Δ mutant, suggesting that Pbp1 acts independently of these factors in this process. We then screened novel Pbp1-interacting factors and found that Pbp1 interacts with ribosomal proteins Rpl12a and Rpl12b. Similarly to the pbp1Δ mutation, the rpl12aΔ and rpl12bΔ mutations also suppressed the growth defect caused by the ccr4Δ khd1Δ mutation. Our results suggest that Pbp1 is involved in the Ccr4- and Khd1-mediated regulation of cell growth through the association with Rpl12a and Rpl12b.

The MDM2-p53 feedback loop is crucially important for restricting p53 level and activity during normal cell growth and proliferation, and is thus subjected to dynamic regulation in order for cells to activate p53 upon various stress signals. Several ribosomal proteins, such as RPL11, RPL5, RPL23, RPL26 or RPS7, have been shown to have a role in regulation of this feedback loop in response to ribosomal stress. Here, we identify another ribosomal protein S14, which is highly associated with 5q-syndrome, as a novel activator of p53 by inhibiting MDM2 activity. We found that RPS14, but not RPS19, binds to the central acidic domain of MDM2, similar to RPL5 and RPL23, and inhibits its E3 ubiquitin ligase activity toward p53. This RPS14-MDM2 binding was induced upon ribosomal stress caused by actinomycin D or mycophenolic acid. Overexpression of RPS14, but not RPS19, elevated p53 level and activity, leading to G1 or G2 arrest. Conversely, knockdown of RPS14 alleviated p53 induction by these two reagents. Interestingly, knockdown of either RPS14 or RPS19 caused a ribosomal stress that led to p53 activation, which was impaired by further knocking down the level of RPL11 or RPL5. Together, our results demonstrate that RPS14 and RPS19 have distinct roles in regulating the MDM2-p53 feedback loop in response to ribosomal stress.

Fission yeast cells express Rpl32-2 highly while Rpl32-1 lowly in log phase; in contrast, expression of Rpl32-1 raises and reaches a peak level while Rpl32-2 is downregulated to a low basic level when cells enter into stationary phase. Overexpression of Rpl32-1 inhibits cell growth while overexpression of Rpl32-2 does not. Deleting rpl32-2 impairs cell growth more severely than deleting rpl32-1 does. Cell growth impaired by deleting either paralog can be rescued completely by reintroducing rpl32-2, but only partly by rpl32-1. Overexpression of Rpl32-1 inhibits cell division, yielding 4c DNA and multiple septa, while overexpressed Rpl32-2 promotes it. Transcriptomics analysis proved that Rpl32 paralogs regulate expression of a subset of genes related with cell division and stress response in a distinctive way. This functional difference of the two paralogs is due to their difference of 95(th) amino acid residue. The significance of a competitive inhibition between Rpl32 paralogs on their expression is discussed.

In vitro studies suggest that haploinsufficiency is involved in the pathogenesis of myelodysplastic syndromes (MDS). In patients with del5q cytogenetic abnormality, RPS-14 and microRNAs (miRNAs) play a major role. In a multicenter phase II single-arm trial with lenalidomide in anemic primary del5q MDS patients with low- or int-1 risk IPSS, biological changes from baseline were investigated. Gene expression profiling of selected genes was performed (TaqMan® Low Density Array Fluidic card, Applied Biosystems PRISM® 7900HT) and normalized against the expression of the 18S housekeeping gene from a pool of healthy subjects. Thirty-two patients were evaluated at baseline and after 3 and 6 months of treatment. RPS-14, miR-145, and miR-146 were downregulated at baseline and significantly increased during treatment. Nuclear factor kappa B, IL-6, interferon regulatory factor-1, IFNγ-R2, IL-2, and many genes in the apoptotic pathways (TNF, IL-1B, and IL-10) were upregulated at baseline and significantly downregulated during lenalidomide treatment, while forkhead box P3, FAS, IFNγ, IL-12A, and IL-12B were downregulated at baseline and progressively upregulated during treatment. The crucial role of aberrant immunological pathways and haploinsufficiency in the pathogenesis of del5q MDS is confirmed in the present patient setting. Our results indicate that lenalidomide may act through defined immunological pathways in this condition.

Cellular ribosomal protein L29 (RPL29) is known to be important in protein synthesis, but its function during angiogenesis has never been described before. We have shown previously that mice lacking β3-integrins support enhanced tumour angiogenesis and, therefore, deletion of endothelial αvβ3 can provide a method for discovery of novel regulators of tumour angiogenesis. Here, we describe significant upregulation of RPL29 in β3-null endothelial cells at both the mRNA and protein level. Ex vivo, we show that VEGF-stimulated microvessel sprouting was reduced significantly in Rpl29-heterozygous and Rpl29-null aortic ring assays compared with wild-type controls. Moreover, we provide in vivo evidence that RPL29 can regulate tumour angiogenesis. Tumour blood vessel density in subcutaneously grown Lewis lung carcinomas was reduced significantly in Rpl29-mutant mice. Additionally, depletion of Rpl29 using RNA interference inhibited VEGF-induced aortic ring sprouting, suggesting that anti-RPL29 strategies might have anti-angiogenic potential. Overall, our results identify that loss or depletion of RPL29 can reduce angiogenesis in vivo and ex vivo.

Mutations in the human Shwachman-Bodian-Diamond syndrome (SBDS) gene cause defective ribosome assembly and are associated with exocrine pancreatic insufficiency, chronic neutropenia and skeletal defects. However, the mechanism underlying these phenotypes remains unclear. Here we show that knockdown of the zebrafish sbds ortholog fully recapitulates the spectrum of developmental abnormalities observed in the human syndrome, and further implicate impaired proliferation of ptf1a-expressing pancreatic progenitor cells as the basis for the observed pancreatic phenotype. It is thought that diseases of ribosome assembly share a p53-dependent mechanism. However, loss of p53 did not rescue the developmental defects associated with loss of zebrafish sbds. To clarify the molecular mechanisms underlying the observed organogenesis defects, we performed transcriptional profiling to identify candidate downstream mediators of the sbds phenotype. Among transcripts displaying differential expression, functional group analysis revealed marked enrichment of genes related to ribosome biogenesis, rRNA processing and translational initiation. Among these, ribosomal protein L3 (rpl3) and pescadillo (pes) were selected for additional analysis. Similar to knockdown of sbds, knockdown or mutation of either rpl3 or pes resulted in impaired expansion of pancreatic progenitor cells. The pancreatic phenotypes observed in rpl3- and pes-deficient embryos were also independent of p53. Together, these data suggest novel p53-independent roles for ribosomal biogenesis genes in zebrafish pancreas development.

Ribosomal proteins (RPs) play pivotal roles in developmental regulation. The loss or mutation of ribosomal protein L11 (RPL11) induces various developmental defects. However, few RPs have been functionally characterized in Apis cerana cerana. In this study, we isolated a single copy gene, AccRPL11, and characterized its connection to brain maturation. AccRPL11 expression was highly concentrated in the adult brain and was significantly induced by abiotic stresses such as pesticides and heavy metals. Immunofluorescence assays demonstrated that AccRPL11 was localized to the medulla, lobula and surrounding tissues of esophagus in the brain. The post-transcriptional knockdown of AccRPL11 gene expression resulted in a severe decrease in adult brain than in other tissues. The expression levels of other brain development-related genes, p38, ERK2, CacyBP and CREB, were also reduced. Immunofluorescence signal attenuation was also observed in AccRPL11-rich regions of the brain in dsAccRPL11-injected honeybees. Taken together, these results suggest that AccRPL11 may be functional in brain maturation in honeybee adults.

In Saccharomyces cerevisiae, 59 of the 78 ribosomal proteins are encoded by duplicated genes that, in most cases, encode identical or very similar protein products. However, different sets of ribosomal protein genes have been identified in screens for various phenotypes, including life span, budding pattern, and drug sensitivities. Due to potential suppressors of growth rate defects among this set of strains in the ORF deletion collection, we regenerated the entire set of haploid ribosomal protein gene deletion strains in a clean genetic background. The new strains were used to create double deletions lacking both paralogs, allowing us to define a set of 14 nonessential ribosomal proteins. Replicative life-span analysis of new strains corresponding to ORF deletion collection strains that likely carried suppressors of growth defects identified 11 new yeast replicative aging genes. Treatment of the collection of ribosomal protein gene deletion strains with tunicamycin revealed a significant correlation between slow growth and resistance to ER stress that was recapitulated by reducing translation of wild-type yeast with cycloheximide. Interestingly, enhanced tunicamycin resistance in ribosomal protein gene deletion mutants was independent of the unfolded protein response transcription factor Hac1. These data support a model in which reduced translation is protective against ER stress by a mechanism distinct from the canonical ER stress response pathway and further add to the diverse yet specific phenotypes associated with ribosomal protein gene deletions.

Diamond-Blackfan anemia is a congenital hypoproliferative macrocytic anemia and 5q- syndrome myelodysplastic syndrome is an acquired hypoproliferative macrocytic anemia. Their common erythroid phenotype reflects a shared pathophysiology-haploinsufficiency of one of many ribosomal proteins and somatic deletion of one allele of the ribosomal protein S14 gene, respectively. Although these abnormalities lead to defective ribosome biogenesis, why ribosomal protein hemizygosity results in anemia is not certain. Here, we characterize the hematopoietic phenotype of mice lacking one allele of the ribosomal protein S6 gene. The mice have an erythroid phenotype similar to both Diamond-Blackfan anemia and the 5q- syndrome and lenalidomide therapy improves their anemia., (Copyright © 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)