Feline leukaemia virus: half a century since its discovery

In the early 1960s, Professor William (Bill) F.H. Jarrett was presented with a timeGÇôspace cluster of cats with lymphoma identified by a local veterinary practitioner, Harry Pfaff, and carried out experiments to find if the condition might be caused by a virus, similar to lymphomas noted previously in poultry and mice. In 1964, the transmission of lymphoma in cats and the presence of virus-like particles that resembled GÇÿthe virus of murine leukaemiasGÇÖ in the induced tumours were reported in Nature. These seminal studies initiated research on feline leukaemia virus (FeLV) and launched the field of feline retrovirology. This review article considers the way in which some of the key early observations made by Bill Jarrett and his coworkers have developed in subsequent years and discusses progress that has been made in the field since FeLV was first discovered

Pathogenesis of the Erythroid Failure in Diamond Blackfan Anemia

Author's Manuscript 2011 February 1.Diamond Blackfan anaemia (DBA) is a severe congenital failure of erythropoiesis. Despite mutations in one of several ribosome protein genes, including RPS19, the cause of the erythroid specificity is still a mystery. We hypothesized that, because the chromatin of late erythroid cells becomes condensed and transcriptionally inactive prior to enucleation, the rapidly proliferating immature cells require very high ribosome synthetic rates. RNA biogenesis was measured in primary mouse fetal liver erythroid progenitor cells; during the first 24 h, cell number increased three to fourfold while, remarkably, RNA content increased sixfold, suggesting an accumulation of an excess of ribosomes during early erythropoiesis. Retrovirus infected siRNA RPS19 knockdown cells showed reduced proliferation but normal differentiation, and cell cycle analysis showed a G1/S phase delay. p53 protein was increased in the knockdown cells, and the mRNA level for p21, a transcriptional target of p53, was increased. Furthermore, we show that RPS19 knockdown decreased MYB protein, and Kit mRNA was reduced, as was the amount of cell surface KIT protein. Thus, in this small hairpin RNA murine model of DBA, RPS19 insufficient erythroid cells may proliferate poorly because of p53-mediated cell cycle arrest, and also because of decreased expression of the key erythroid signalling protein KIT.National Institutes of Health (U.S.) (Grant PO1 HL 32262)Amgen Inc

Many Ribosomal Protein Genes Are Cancer Genes in Zebrafish

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now

The Bowen–Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen–Conradi syndrome (BCS) is caused by a specific Nep1D86G mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific 14C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1ts mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome

Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen–Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase

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

The NIP7 protein is required for accurate pre-rRNA processing in human cells

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

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

The NIP7 protein is required for accurate pre-rRNA processing in human cells

Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells

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