Ribosomal Protein Rps3 Research Articles

The Neurobeachin-like 2 protein (NBEAL2) controls the homeostatic level of the ribosomal protein RPS6 in mast cells.

The Beige and Chediak-Higashi (BEACH) domain-containing, Neurobeachin-like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss-of-function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK-cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2-/- mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning-based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co-immunoprecipitation (co-IP) experiments, western blotting, enzyme-linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK-phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor-independent survival and in a pro-inflammatory MC-phenotype.

Potential biomarkers uncovered by bioinformatics analysis in sotorasib resistant-pancreatic ductal adenocarcinoma.

Mutant KRAS-induced tumorigenesis is prevalent in lung, colon, and pancreatic ductal adenocarcinomas. For the past 3 decades, KRAS mutants seem undruggable due to their high-affinity GTP-binding pocket and smooth surface. Structure-based drug design helped in the design and development of first-in-class KRAS G12C inhibitor sotorasib (AMG 510) which was then approved by the FDA. Recent reports state that AMG 510 is becoming resistant in non-small-cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC), and lung adenocarcinoma patients, and the crucial drivers involved in this resistance mechanism are unknown. In recent years, RNA-sequencing (RNA-seq) data analysis has become a functional tool for profiling gene expression. The present study was designed to find the crucial biomarkers involved in the sotorasib (AMG 510) resistance in KRAS G12C-mutant MIA-PaCa2 cell pancreatic ductal adenocarcinoma cells. Initially, the GSE dataset was retrieved from NCBI GEO, pre-processed, and then subjected to differentially expressed gene (DEG) analysis using the limma package. Then the identified DEGs were subjected to protein-protein interaction (PPI) using the STRING database, followed by cluster analysis and hub gene analysis, which resulted in the identification of probable markers. Furthermore, the enrichment and survival analysis revealed that the small unit ribosomal protein (RP) RPS3 is the crucial biomarker of the AMG 510 resistance in KRAS G12C-mutant MIA-PaCa2 cell pancreatic ductal adenocarcinoma cells. Finally, we conclude that RPS3 is a crucial biomarker in sotorasib resistance which evades apoptosis by MDM2/4 interaction. We also suggest that the combinatorial treatment of sotorasib and RNA polymerase I machinery inhibitors could be a possible strategy to overcome resistance and should be studied in in vitro and in vivo settings in near future.

The First Mitochondrial Genome of Ciborinia camelliae and Its Position in the Sclerotiniaceae Family.

Ciborinia camelliae is the causal agent of camellia flower blight (CFB). It is a hemibiotrophic pathogen, inoperculate Discomycete of the family Sclerotiniaceae. It shows host and organ specificity infecting only flowers of species belonging to the genus Camellia, causing serious damage to the ornamental component of the plant. In this work, the first mitochondrial genome of Ciborinia camellia is reported. The mitogenome was obtained by combining Illumina short read and Nanopore long read technology. To resolve repetitive elements, specific primers were designed and used for Sanger sequencing. The manually curated mitochondrial DNA (mtDNA) of the Italian strain DSM 112729 is a circular sequence of 114,660 bp, with 29.6% of GC content. It contains two ribosomal RNA genes, 33 transfer RNAs, one RNase P gene, and 62 protein-coding genes. The latter include one gene coding for a ribosomal protein (rps3) and the 14 typical proteins involved in the oxidative metabolism. Moreover, a partial mtDNA assembled from a contig list was obtained from the deposited genome assembly of a New Zealand strain of C. camelliae. The present study contributes to understanding the mitogenome arrangement and the evolution of this phytopathogenic fungus in comparison to other Sclerotiniaceae species and confirms the usefulness of mitochondrial analysis to define phylogenetic positioning of this newly sequenced species.

SATB1 Regulates Chromatin Organization and HSP70 Expression in Early Erythropoiesis and Is Downregulated in Models of Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare genetic disease predominantly caused by mutations carried within one of at least 20 ribosomal genes. DBA is characterized by red blood cell aplasia and normal myeloid and megakaryocyte progenitors, indicating that early uncommitted progenitors are relatively unaffected by the mutations. In DBA, the formation of BFU-E colonies and subsequent erythroblasts are severely restricted and indicate a defect in one of the earliest stages of erythroid expansion.To identify critical molecular mechanisms that may regulate early erythropoiesis, we used shRNAs against the ribosomal protein RPS19 (the most commonly mutated gene in DBA) in cord blood derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and performed bulk RNA-seq. After 3 days in an erythroid culture media, the transcriptomes in CD71+ erythroid progenitors were examined. We found that the special AT binding protein 1 (SATB1) was downregulated in RPS19-insufficient HSPCs compared to healthy cord blood HSPCs.SATB1 is modestly expressed in hematopoietic stem cells but is induced during lymphoid expansion and has been previously reported to suppress myeloid/erythroid progenitor (MEP) expansion. Our results showed that maintaining SATB1 expression is required for optimal expansion of MEP progenitors and that the premature loss of SATB1 in DBA contributes to the anemia phenotype.SATB1 binds to 3 specific regions upstream of the 5'UTR of the HSP70 genes and induces the formation of 2 chromatin loops. An enhancer element associates with the proximal promoters of the two HSP70 genes and facilitates the induction of HSP70. In DBA, HSP70 is not induced and contributes to DBA pathogenesis.HSPA1A is induced 4.3-fold while HSPA1B is induced 3.1-fold. Increased expression of the master erythroid transcription factor GATA1 during erythropoiesis occurs in two phases. The first induction precedes a more dramatic induction that accompanies later stages of erythroid differentiation. The absence of SATB1 or HSP70 reduced the earlier GATA1 induction that accompany MEP expansion by 46.1% and 49.3% respectively. The number of MEPs in SATB1 knockdown HSPCs was reduced, resulting in a 24.5% reduction in CD235+ erythroid and 20.8% reduction in CD41+ megakaryocytes.While SATB1-independent effects of RPS19-insufficiency contribute more significantly to erythroid defects in DBA, we have uncovered that SATB1 contributes to regulation of the earliest stages of erythropoiesis by facilitating the induction of HSP70 and subsequent stabilization of an early induction of GATA1. DisclosuresNo relevant conflicts of interest to declare.

Comparison of mitochondrial genomes from multi-, Bi-, and uninucleate Rhizoctonia

Six circular mitochondrial genomes of multi-, bi-, and uninucleate Rhizoctonia isolates were assembled and found that all the genomes contain 14 conserved protein-coding genes, one ribosomal protein (rps3), and 23 tRNA in the same order. The mitogenome sizes of uninucleate isolates were relatively smaller than binucleate and multinucleate stains. The size variations between uninucleate and multinucleate isolates were from both intergenic and intronic regions, whereas the differences between uninucleate and binucleate isolates were predominantly from intergenic regions. The phylogenetic analysis revealed that Rhizoctonia strains of the same nucleate types had a closer relationship.

Arginine Methylation Regulates Ribosome CAR Function.

The ribosome CAR interaction surface is hypothesized to provide a layer of translation regulation through hydrogen-bonding to the +1 mRNA codon that is next to enter the ribosome A site during translocation. The CAR surface consists of three residues, 16S/18S rRNA C1054, A1196 (E. coli 16S numbering), and R146 of yeast ribosomal protein Rps3. R146 can be methylated by the Sfm1 methyltransferase which is downregulated in stressed cells. Through molecular dynamics analysis, we show here that methylation of R146 compromises the integrity of CAR by reducing the cation-pi stacking of the R146 guanidinium group with A1196, leading to reduced CAR hydrogen-bonding with the +1 codon. We propose that ribosomes assembled under stressed conditions have unmethylated R146, resulting in elevated CAR/+1 codon interactions, which tunes translation levels in response to the altered cellular context.

Multilocus genotyping of ‘Candidatus Phytoplasma aurantifolia’ associated with witches’ broom disease in Citrus spp.

Witches’ broom disease of lime (WBDL), associated with ‘Candidatus Phytoplasma aurantifolia,’ is a serious disease of acid lime in Southern Iran. In order to characterize ‘Ca. P. aurantifolia’ strains, a multilocus sequence analysis protocol was developed based on different genomic loci, including 16S rRNA, 16S-23S rRNA intergenic spacer (IS), ribosomal protein genes rpl22 and rps3, cell division protein, and group II intron reverse transcriptase/mature gene, in 46 phytoplasma strains. Analyses of virtual RFLP, phylogenetics, and DNA sequence identity of 16S rRNA of Iranian phytoplasma strains associated with symptomatic citrus plants indicated that the Iranian strains were classified in ribosomal subgroup 16SrII-B. In total, three genotypes were recognized based on 16Sr virtual RFLP profiles. Phylogenetic analysis based on the rp and ftsH gene sequences showed clustering of all the strains as one group. Comparative sequence and phylogenetic analysis of group II intron genes indicated the highest differentiation of ‘Ca. P. aurantifolia’–related strains in four genotypes. The polymorphism of group II intron reverse transcriptase/mature genes fragmentation provides finer differentiation of closely related ‘Ca P. aurantifolia’ strains and reveals higher genetic diversity than previously reported.

GCN sensitive protein translation in yeast.

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons-the ramp region-of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

A Ribosome Interaction Surface Sensitive to mRNA GCN Periodicity.

A longstanding challenge is to understand how ribosomes parse mRNA open reading frames (ORFs). Significantly, GCN codons are over-represented in the initial codons of ORFs of prokaryote and eukaryote mRNAs. We describe a ribosome rRNA-protein surface that interacts with an mRNA GCN codon when next in line for the ribosome A-site. The interaction surface is comprised of the edges of two stacked rRNA bases: the Watson–Crick edge of 16S/18S rRNA C1054 and the adjacent Hoogsteen edge of A1196 (Escherichia coli 16S rRNA numbering). Also part of the interaction surface, the planar guanidinium group of a conserved Arginine (R146 of yeast ribosomal protein Rps3) is stacked adjacent to A1196. On its other side, the interaction surface is anchored to the ribosome A-site through base stacking of C1054 with the wobble anticodon base of the A-site tRNA. Using molecular dynamics simulations of a 495-residue subsystem of translocating ribosomes, we observed base pairing of C1054 to nucleotide G at position 1 of the next-in-line codon, consistent with previous cryo-EM observations, and hydrogen bonding of A1196 and R146 to C at position 2. Hydrogen bonding to both of these codon positions is significantly weakened when C at position 2 is changed to G, A or U. These sequence-sensitive mRNA-ribosome interactions at the C1054-A1196-R146 (CAR) surface potentially contribute to the GCN-mediated regulation of protein translation.

MOLECULAR CHARACTERIZATION OF ‘Candidatus PHYTOPLASMA MALI’ STRAINS FROM BULGARIA AND POLAND

During 2015, samples from 22 apple trees showing proliferation symptoms were collected in southwest Bulgaria and Central and South Poland and tested for phytoplasma presence. ‘Candidatus Phytoplasma mali’ was identified in 18 samples based on results of restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene amplified in nested PCR using primer pair P1/P7 followed by R16F2n/R16R2 and F1/B6 primer pairs. The nitroreductase and rhodonase like genes and ribosomal protein genes rpl22 and rps3 were then analyzed using PCR-RFLP technique to study the genetic variability of the phytoplasma strains. Two restriction profiles, P-I or P-II, were obtained for fragments of 16S rDNA plus 16S-23S spacer region digested with HpaII enzyme. Restriction fragment length polymorphism analysis of nitroreductase and rhodonase like genes using digestion with HincII endonuclease revealed that the all ‘Ca. P. mali’ strains belonged to subtype AP-15. Analysis of rpl22 and rps3 ribosomal protein genes digested with AluI enzyme resulted in classification of detected phytoplasma strains to rpX-A subgroup.

Maternal mitochondrial inheritance in two Fusarium pathogens of prickly ash (Zanthoxylum bungeanum) in northern China

ABSTRACTMitochondrial inheritance in Fusarium zanthoxyli and F. continuum, two canker-inducing pathogens of prickly ash (Zanthoxylum bungeanum) in northern China, was investigated by genotyping ascospore progeny obtained from laboratory crosses. Polymorphic regions of the mitochondrial genomes (mitogenomes) that contained indels and single-nucleotide polymorphisms (SNPs) were identified via comparative analyses of the complete mitogenomes of the parents used in the intraspecific crosses. A reciprocal genetic cross of F. zanthoxyli NRRL 66714 × NRRL 66285, and a separate cross of F. continuum ♀ NRRL 66286 × ♂ NRRL 66218, revealed that mitochondria were only inherited from the maternal parent. In addition, the reciprocal cross demonstrated that mitochondrial inheritance is not linked to mating type. Gene order in the circular mitogenomes of the prickly ash pathogens was identical to that previously reported for other fusaria and members of the Hypocreales, except that the TRNL tRNAs were duplicated in F. zanthoxyli NRRL 66714. The genomes contained 14 polypeptide-encoding genes involved in oxidative respiration, one intron-encoded ribosomal protein (rps3) gene, two ribosomal RNA (rRNA) genes, and 26–28 tRNA genes. The F. zanthoxyli mitogenomes were 80.9 and 98.7 kb in length, whereas those of F. continuum were considerably shorter and nearly identical in length at 63.4 kb. The significant differences in mitogenome length were primarily due to variable numbers of introns and open reading frames (ORFs) encoding hypothetical proteins.

Chromatin Organization By SATB1 Regulates HSP70 Induction in Early Erythropoiesis and Lost in Diamond Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome. The disease usually presents within the first year of life and is associated with anemia, congenital abnormalities, and an increased risk of developing cancer. The most prevalent mutations are found in the ribosomal protein RPS19, accounting for over 25% of cases but approximately 70% of DBA patients carry mutations in ribosomal genes. Downregulation of GATA1 has been attributed to the disease, but the upstream mechanisms leading to aberrant erythropoiesis is only beginning to be elucidated.SATB1 is highly upregulated in lymphocytes (especially thymocytes) and steadily downregulated in all myeloid ineages during differentiation. However the more modest expression of SATB1 in early progenitors is required for self-renewal of HSCs and globin switching in early erythropoiesis. Using transcriptomics, we identified SATB1 expression is prematurely lost in RPS19-insufficient erythropoiesis and that over a third (16/42) of RPS19-sensitive early erythroid genes were rescued upon SATB1 re-expression. One of the most deregulated transcripts encode the chaperone HSP70.GATA1 is an essential master regulator in erythroid differentiation and high GATA1 protein levels are required for efficient erythropoiesis. In DBA, GATA1 protein expression is drastically diminished. Two mechanisms contribute to this. At the translational level, GATA1 transcripts fail to be translated adequately due to ribosomal inefficiency at binding short, unstructured 5'UTRs. At the protein level,HSP70 loss leads to reduced GATA1 protein stability. During healthy erythropoiesis HSP70 genes are drastically upregulated (3.21, 4.18 and 1.37- fold) and this is lost in RPS19 insufficiency. Here we report that upregulation of HSP70 in healthy erythropoiesis requires SATB1. SATB1 binds to 3 sites within a 40kb region surrounding the HSP70 gene loci and recruits a distal predicted enhancer element to the proximal promoters via formation of two chromatin loops. During RPS19-insufficiency this chromatin organization is lost, but SATB1 re-expression restores chromatin looping and HSP70 expression, stabilizing GATA1 and promoting efficient erythropoiesis. As with HSP70, it is likely that permissive chromatin organization is essential for other RPS19-sensitive erythroid genes rescued by SATB1 re-expression. DisclosuresNo relevant conflicts of interest to declare.

Interactions between the mRNA and Rps3/uS3 at the entry tunnel of the ribosomal small subunit are important for no-go decay.

No-go Decay (NGD) is a process that has evolved to deal with stalled ribosomes resulting from structural blocks or aberrant mRNAs. The process is distinguished by an endonucleolytic cleavage prior to degradation of the transcript. While many of the details of the pathway have been described, the identity of the endonuclease remains unknown. Here we identify residues of the small subunit ribosomal protein Rps3 that are important for NGD by affecting the cleavage reaction. Mutation of residues within the ribosomal entry tunnel that contact the incoming mRNA leads to significantly reduced accumulation of cleavage products, independent of the type of stall sequence, and renders cells sensitive to damaging agents thought to trigger NGD. These phenotypes are distinct from those seen in combination with other NGD factors, suggesting a separate role for Rps3 in NGD. Conversely, ribosomal proteins ubiquitination is not affected by rps3 mutations, indicating that upstream ribosome quality control (RQC) events are not dependent on these residues. Together, these results suggest that Rps3 is important for quality control on the ribosome and strongly supports the notion that the ribosome itself plays a central role in the endonucleolytic cleavage reaction during NGD.

Annotation and analysis of the mitochondrial genome of Coniothyrium glycines, causal agent of red leaf blotch of soybean, reveals an abundance of homing endonucleases.

Coniothyrium glycines, the causal agent of soybean red leaf blotch, is a USDA APHIS-listed Plant Pathogen Select Agent and potential threat to US agriculture. Sequencing of the C. glycines mt genome revealed a circular 98,533-bp molecule with a mean GC content of 29.01%. It contains twelve of the mitochondrial genes typically involved in oxidative phosphorylation (atp6, cob, cox1-3, nad1-6, and nad4L), one for a ribosomal protein (rps3), four for hypothetical proteins, one for each of the small and large subunit ribosomal RNAs (rns and rnl) and a set of 30 tRNAs. Genes were encoded on both DNA strands with cox1 and cox2 occurring as adjacent genes having no intergenic spacers. Likewise, nad2 and nad3 are adjacent with no intergenic spacers and nad5 is immediately followed by nad4L with an overlap of one base. Thirty-two introns, comprising 54.1% of the total mt genome, were identified within eight protein-coding genes and the rnl. Eighteen of the introns contained putative intronic ORFs with either LAGLIDADG or GIY-YIG homing endonuclease motifs, and an additional eleven introns showed evidence of truncated or degenerate endonuclease motifs. One intron possessed a degenerate N-acetyl-transferase domain. C. glycines shares some conservation of gene order with other members of the Pleosporales, most notably nad6-rnl-atp6 and associated conserved tRNA clusters. Phylogenetic analysis of the twelve shared protein coding genes agrees with commonly accepted fungal taxonomy. C. glycines represents the second largest mt genome from a member of the Pleosporales sequenced to date. This research provides the first genomic information on C. glycines, which may provide targets for rapid diagnostic assays and population studies.

The metaplastic effects of NMDA receptors blockade on reactivation of instrumental memories in rats.

Metaplasticity, defined as the plasticity of synaptic plasticity, could affect learning and memory at different neural levels. It was hypothesized that metaplasticity changes on glutamate receptors may affect memory destabilization, promoting or preventing reconsolidation. We investigated the metaplastic effect of NMDA channel blocker MK-801 on sucrose instrumental memory reconsolidation in a behavioural rat model associated to the assessment of molecular markers of metaplasticity, memory retrieval, destabilization and reconsolidation. Following instrumental conditioning and forced abstinence, rats were intraperitoneally treated with MK-801 or vehicle 24 h before the exposure to memory retrieval or not-retrieval. Separate groups were tested for in-vivo extinction of responding (24 h and 7 d after reactivation) or ex-vivo assessment of transcription factor Zif268 and ribosomal protein rpS6 phosphorylation in nucleus accumbens (NAc) and amygdala (Amy). MK-801 significantly inhibited instrumental responding at extinction test, suggesting reconsolidation blockade of instrumental memory. The decrease of Zif268 and phosphorylated-rpS6 levels in NAc and Amy in MK-801/Retrieval vs. Vehicle/Retrieval group supported the behavioural findings. An increase of GluN2B, GluA1 and mGluR5 in NAc, and GluN2B in Amy, 24 h after MK-801 indicated the trigger of associated metaplastic changes. Our findings show that metaplastic changes induced by NMDA receptors blockade affected sucrose instrumental memory retrieval as shown by both behavioural and molecular changes. We hypothesize that these findings however suggested a switch to extinction rather than a reconsolidation.

Phosphorylation of Ribosomal Protein RPS6 Integrates Light Signals and Circadian Clock Signals.

The translation of mRNA into protein is tightly regulated by the light environment as well as by the circadian clock. Although changes in translational efficiency have been well documented at the level of mRNA-ribosome loading, the underlying mechanisms are unclear. The reversible phosphorylation of RIBOSOMAL PROTEIN OF THE SMALL SUBUNIT 6 (RPS6) has been known for 40 years, but the biochemical significance of this event remains unclear to this day. Here, we confirm using a clock-deficient strain of Arabidopsis thaliana that RPS6 phosphorylation (RPS6-P) is controlled by the diel light-dark cycle with a peak during the day. Strikingly, when wild-type, clock-enabled, seedlings that have been entrained to a light-dark cycle are placed under free-running conditions, the circadian clock drives a cycle of RPS6-P with an opposite phase, peaking during the subjective night. We show that in wild-type seedlings under a light-dark cycle, the incoherent light and clock signals are integrated by the plant to cause an oscillation in RPS6-P with a reduced amplitude with a peak during the day. Sucrose can stimulate RPS6-P, as seen when sucrose in the medium masks the light response of etiolated seedlings. However, the diel cycles of RPS6-P are observed in the presence of 1% sucrose and in its absence. Sucrose at a high concentration of 3% appears to interfere with the robust integration of light and clock signals at the level of RPS6-P. Finally, we addressed whether RPS6-P occurs uniformly in polysomes, non-polysomal ribosomes and their subunits, and non-ribosomal protein. It is the polysomal RPS6 whose phosphorylation is most highly stimulated by light and repressed by darkness. These data exemplify a striking case of contrasting biochemical regulation between clock signals and light signals. Although the physiological significance of RPS6-P remains unknown, our data provide a mechanistic basis for the future understanding of this enigmatic event.

Acquired ribosomopathies in leukemia and solid tumors.

A mutation in the gene encoding the small subunit-associated ribosomal protein RPS19, leading to RPS19 haploinsufficiency, is one of the ribosomal protein gene defects responsible for the rare inherited bone marrow failure syndrome Diamond Blackfan anemia (DBA). Additional inherited and acquired defects in ribosomal proteins (RPs) continue to be identified and are the basis for a new class of diseases called the ribosomopathies. Acquired RPS14 haploinsufficiency has been found to be causative of the bone marrow failure found in 5q- myelodysplastic syndromes. Both under- and overexpression of RPs have also been implicated in several malignancies. This review will describe the somatic ribosomopathies that have been found to be associated with a variety of solid tumors as well as leukemia and will review cancers in which over- or underexpression of these proteins seem to be associated with outcome.

Autophagic flux is required for the synthesis of triacylglycerols and ribosomal protein turnover in Chlamydomonas.

Autophagy is an intracellular catabolic process that allows cells to recycle unneeded or damaged material to maintain cellular homeostasis. This highly dynamic process is characterized by the formation of double-membrane vesicles called autophagosomes, which engulf and deliver the cargo to the vacuole. Flow of material through the autophagy pathway and its degradation in the vacuole is known as autophagic flux, and reflects the autophagic degradation activity. A number of assays have been developed to determine autophagic flux in yeasts, mammals, and plants, but it has not been examined yet in algae. Here we analyzed autophagic flux in the model green alga Chlamydomonas reinhardtii. By monitoring specific autophagy markers such as ATG8 lipidation and using immunofluorescence and electron microscopy techniques, we show that concanamycin A, a vacuolar ATPase inhibitor, blocks autophagic flux in Chlamydomonas. Our results revealed that vacuolar lytic function is needed for the synthesis of triacylglycerols and the formation of lipid bodies in nitrogen- or phosphate-starved cells. Moreover, we found that concanamycin A treatment prevented the degradation of ribosomal proteins RPS6 and RPL37 under nitrogen or phosphate deprivation. These results indicate that autophagy might play an important role in the regulation of lipid metabolism and the recycling of ribosomal proteins under nutrient limitation in Chlamydomonas.

Nuclear import of dimerized ribosomal protein Rps3 in complex with its chaperone Yar1.

After their cytoplasmic synthesis, ribosomal proteins need to be transported into the nucleus, where they assemble with ribosomal RNA into pre-ribosomal particles. Due to their physicochemical properties, they need protection from aggregation on this path. Newly synthesized ribosomal protein Rps3 forms a dimer that is associated with one molecule of its specific chaperone Yar1. Here we report that redundant pathways contribute to the nuclear import of Rps3, with the classical importin α/β pathway (Kap60/Kap95 in yeast) constituting a main import route. The Kap60/Kap95 heterodimer mediates efficient nuclear import of Rps3 by recognition of an N-terminal monopartite nuclear localization signal (NLS). This Rps3-NLS is located directly adjacent to the Yar1-binding site and, upon binding of Kap60 to Rps3, Yar1 is displaced from the ribosomal protein in vitro. While Yar1 does not directly interact with Kap60 in vitro, affinity purifications of Yar1 and Rps3, however, revealed that Kap60 is present in the Rps3/Yar1 complex in vivo. Indeed we could reconstitute such a protein complex containing Rps3 and both Yar1 and Kap60 in vitro. Our data suggest that binding of Yar1 to one N-domain and binding of Kap60 to the second N-domain of dimerized Rps3 orchestrates import and protection of the ribosomal protein.

Abstract 2457: Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma

Abstract Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia and congenital anomalies, notably skeletal defects. DBA is known in the majority of cases to be caused by haploinsufficiency of one of the small or large ribosomal protein (rp) subunits. In addition, there is a cancer predisposition in DBA. Indeed, somatic and germline rp mutations have been associated with a number of malignancies. In DBA patients one of the common malignancies is osteogenic sarcoma (OS). It is proposed that selective pressure caused by faulty translation may lead to clonal expansion of cells with acquired interdicting mutations leading to cancer. Purpose: To quantitate the incidence of cancer in patients with DBA and to examine osteoblast formation in vitro using a mouse embryonic stem (mES) cell model haploinsufficient for ribosomal protein Rps19 to identify putative bone defects that may promote tumorigenesis. Methods: The DBA Registry of North America (DBAR), the largest established patient cohort with prospective follow-up since 1991, was interrogated to ascertain the incidence of cancer in DBA. For in vitro studies, mES cells were differentiated towards osteoblasts using 1,25-dihydroxy-vitamin D3, dexamethasone, ascorbic acid and β-glycerophosphate for 10 days. qRT-PCR and histologic stains (alizarin red, alcian blue and alkaline phosphatase) were used to assess osteoblast production in wild type and Rps19+/- cultures. Results: The DBAR reported the first quantitative assessment of cancer incidence in DBA. The significantly elevated observed to expected ratios were 287, 36, 33 and 28 respectively, for myelodysplastic syndrome (MDS), colon carcinoma, OS and acute myeloid leukemia (AML). As of Nov 2015 (N = 751), with a median patient age of 20 years, 11 patients with gastrointestinal carcinoma, 5 with OS, 2 with AML and 8 with MDS are reported to the DBAR. The incidence of gastrointestinal carcinoma and OS is highly significant in this young population. We elected to study OS due to the skeletal defects and poor growth encountered in DBA patients. Using mES cells induced to differentiation towards osteoblasts, we observed reduced mineralization and therefore, bone formation, by alizarin red staining in Rps19+/- cells. Furthermore, rp haplo-insufficient cultures displayed a significant increase in alcian blue staining, a marker for chondrogenesis; these results suggest that regulation of osteochondrogenic potential was altered. In support of this phenotype, qRT-PCR analyses revealed that Rps19 haploinsufficiency induced abnormal expression of the master transcription factors and p53 involved in osteoblast and chondrocyte development. Conclusions: The most common cancers in DBA are AML, OS and adenocarcinoma of the colon. With regards to OS, we are now creating a conditional Rps19 CRISPR murine model, in order to clarify the contribution of both rp and p53 in the etiology of OS. Citation Format: Lionel Blanc, Brian Dulmovits, Julien Papoin, Jessica Kang, Eva Atsidaftos, Jeffrey M. Lipton, Adrianna Vlachos. Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2457.