5′UTR Variants of Ribosomal Protein S19 Transcript Determine Translational Efficiency: Implications for Diamond-Blackfan Anemia and Tissue Variability

Background: Diamond-Blackfan anemia (DBA) is a lineage specific and congenital erythroblastopenia. The disease is associated with mutations in genes encoding ribosomal proteins resulting in perturbed ribosomal subunit biosynthesis. The RPS19 gene is mutated in approximately 25 % of DBA patients and a variety of coding mutations have been described, all presumably leading to haploinsufficiency. A subset of patients carries rare polymorphic sequence variants within the 59untranslated region (59UTR) of RPS19. The functional significance of these variants remains unclear. Methodology/Principal Findings: We analyzed the distribution of transcriptional start sites (TSS) for RPS19 mRNAs in testis and K562 cells. Twenty-nine novel RPS19 transcripts were identified with different 59UTR length. Quantification of expressed w.t. 59UTR variants revealed that a short 59UTR correlates with high levels of RPS19. The total levels of RPS19 transcripts showed a broad variation between tissues. We also expressed three polymorphic RPS19 59UTR variants identified in DBA patients. The sequence variants include two insertions (c.-147_-146insGCCA and c.-147_-146insAGCC) and one deletion (c.-144_-141delTTTC). The three 59UTR polymorphisms are associated with a 20–30 % reduction in RPS19 protein levels when compared to the wild-type (w.t.) 59UTR of corresponding length. Conclusions: The RPS19 gene uses a broad range of TSS and a short 59UTR is associated with increased levels of RPS19. Comparisons between tissues showed a broad variation in the total amount of RPS19 mRNA and in the distribution of TS

Loss of Ribosomal Protein L11 Affects Zebrafish Embryonic Development through a p53-Dependent Apoptotic Response

Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs) play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants) displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6–7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development

The Clinical Impact of Copy Number Variants in Inherited Bone Marrow Failure Syndromes

Inherited bone marrow failure syndromes (IBMFSs) comprise a genetically heterogeneous group of diseases with hematopoietic failure and a wide array of physical malformations. Copy number variants (CNVs) were reported in some IBMFSs. It is unclear what impact CNVs play in patients evaluated for a suspected diagnosis of IBMFS. Clinical and genetic data of 323 patients from the Canadian Inherited Marrow Failure Registry from 2001 to 2014, who had a documented genetic work-up, were analyzed. Cases with pathogenic CNVs (at least 1 kilobasepairs) were compared to cases with other mutations. Genotype-phenotype correlations were performed to assess the impact of CNVs. Pathogenic nucleotide-level mutations were found in 157 of 303 tested patients (51.8%). Genome-wide CNV analysis by single nucleotide polymorphism arrays or comparative genomic hybridization arrays revealed pathogenic CNVs in 11 of 67 patients tested (16.4%). In four of these patients, identification of CNV was crucial for establishing the correct diagnosis as their clinical presentation was ambiguous. Eight additional patients were identified to harbor pathogenic CNVs by other methods. Of the 19 patients with pathogenic CNVs, four had compound-heterozygosity of a CNV with a nucleotide-level mutation. Pathogenic CNVs were associated with more extensive non-hematological organ system involvement

Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway

Chordomas are radio- and chemo-resistant tumours and metastasise in as many as 40% of patients. The aim of this study was to identify potential molecular targets for the treatment of chordoma. In view of the reported association of chordoma and tuberous sclerosis complex syndrome, and the available therapeutic agents against molecules in the PI3K/AKT/TSC1/TSC2/mTOR pathway, a tissue microarray of 50 chordoma cases was analysed for expression of active molecules involved in this signalling pathway by immunohistochemistry and a selected number by western blot analysis. Chordomas were positive for p-AKT (92%), p-TSC2 (96%), p-mTOR (27%), total mTOR (75%), p-p70S6K (62%), p-RPS6 (22%), p-4E-BP1 (96%) and eIF-4E (98%). Phosphatase and tensin homologue deleted on chromosome 10 expression was lost in 16% of cases. Mutations failed to be identified in PI3KCA and RHEB1 in the 23 cases for which genomic DNA was available. Fluorescence in situ hybridisation analysis for mTOR and RPS6 loci showed that 11 of 33 and 21 of 44 tumours had loss of one copy of the respective genes, results which correlated with the loss of the relevant total proteins. Fluorescence in situ hybridisation analysis for loci containing TSC1 and TSC2 revealed that all cases analysed harboured two copies of the respective genes. On the basis of p-mTOR and or p-p70S6K expression there is evidence indicating that 65% of the chordomas studied may be responsive to mTOR inhibitors, rapamycin or its analogues, and that patients may benefit from combined therapy including drugs that inhibit AKT

Eukaryotic Cells Producing Ribosomes Deficient in Rpl1 Are Hypersensitive to Defects in the Ubiquitin-Proteasome System

It has recently become clear that the misassembly of ribosomes in eukaryotic cells can have deleterious effects that go far beyond a simple shortage of ribosomes. In this work we find that cells deficient in ribosomal protein L1 (Rpl1; Rpl10a in mammals) produce ribosomes lacking Rpl1 that are exported to the cytoplasm and that can be incorporated into polyribosomes. The presence of such defective ribosomes leads to slow growth and appears to render the cells hypersensitive to lesions in the ubiquitin-proteasome system. Several genes that were reasonable candidates for degradation of 60S subunits lacking Rpl1 fail to do so, suggesting that key players in the surveillance of ribosomal subunits remain to be found. Interestingly, in spite of rendering the cells hypersensitive to the proteasome inhibitor MG132, shortage of Rpl1 partially suppresses the stress-invoked temporary repression of ribosome synthesis caused by MG132.United States. National Institutes of Health (GM25532)United States. National Institutes of Health (ARRAGM25532-S1)United States. National Institutes of Health (GM085177)United States. National Institutes of Health (CAI-3330)Natural Sciences and Engineering Research Council of Canada (NSERC

Drosophila Ribosomal Protein Mutants Control Tissue Growth Non-Autonomously via Effects on the Prothoracic Gland and Ecdysone

The ribosome is critical for all aspects of cell growth due to its essential role in protein synthesis. Paradoxically, many Ribosomal proteins (Rps) act as tumour suppressors in Drosophila and vertebrates. To examine how reductions in Rps could lead to tissue overgrowth, we took advantage of the observation that an RpS6 mutant dominantly suppresses the small rough eye phenotype in a cyclin E hypomorphic mutant (cycEJP). We demonstrated that the suppression of cycEJP by the RpS6 mutant is not a consequence of restoring CycE protein levels or activity in the eye imaginal tissue. Rather, the use of UAS-RpS6 RNAi transgenics revealed that the suppression of cycEJP is exerted via a mechanism extrinsic to the eye, whereby reduced Rp levels in the prothoracic gland decreases the activity of ecdysone, the steroid hormone, delaying developmental timing and hence allowing time for tissue and organ overgrowth. These data provide for the first time a rationale to explain the counter-intuitive organ overgrowth phenotypes observed for certain members of the Minute class of Drosophila Rp mutants. They also demonstrate how Rp mutants can affect growth and development cell non-autonomously

Truncating ribosomal protein S19 mutations and variable clinical expression in Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a rare constitutional erythroblastopenia characterized by a specific defect in erythroid differentiation. Recently, mutations in the gene encoding ribosomal protein (RP) S19 were found in a subset of patients with the disease. To characterize further RPS19 mutations and to investigate genotype-phenotype relationships, we screened this gene for mutations in patients with DBA by direct sequencing and Southern-blot analysis. Four novel mutations were identified. A G120A nonsense mutation resulting in a stop at codon 33, a C302T nonsense mutation introducing a premature stop at codon 84, and a 327 delG which results in a frame shift at codon 103. A fourth and more complex mutation (TT157-158AA, 160insCT) resulting in a Leu45Gln and a frame shift from codon 47 was found in three affected family members with variable phenotypes. The different clinical expression for identical mutations suggest the presence of other modulating factors for the disease. The mutations presented here further support the role of RPS19 in erythropoietic differentiation and proliferation