Defects in ribosome biogenesis trigger stress response pathways, which perturb cell proliferation and differentiation in several genetic diseases. In Diamond–Blackfan anemia (DBA), a congenital erythroblastopenia, mutations in ribosomal protein genes often interfere with the processing of the internal transcribed spacer 1 (ITS1), the mechanism of which remains elusive in human cells. Using loss-of-function experiments and extensive RNA analysis, we have defined the precise position of the endonucleolytic cleavage E in the ITS1, which generates the 18S-E intermediate, the last precursor to the 18S rRNA. Unexpectedly, this cleavage is followed by 3′–5′ exonucleolytic trimming of the 18S-E precursor during nuclear export of the pre-40S particle, which sets a new mechanism for 18S rRNA formation clearly different from that established in yeast. In addition, cleavage at site E is also followed by 5′–3′ exonucleolytic trimming of the ITS1 by exonuclease XRN2. Perturbation of this step on knockdown of the large subunit ribosomal protein RPL26, which was recently associated to DBA, reveals the putative role of a highly conserved cis-acting sequence in ITS1 processing. These data cast new light on the original mechanism of ITS1 elimination in human cells and provide a mechanistic framework to further study the interplay of DBA-linked ribosomal proteins in this process.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.

Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocyctic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30–50% of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26 and RPL26 and GATA1 in about 60–65% of patients. Here we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1 (ITS1). In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.

The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN.

Pearson marrow pancreas syndrome in patients suspected to have Diamond-Blackfan anemia. Blood 17, 437-440. The authors apologize for this error.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis shows that ribosome biogenesis is abruptly interrupted by the drop of rDNA transcription and the collapse of ribosomal protein neo-synthesis. Its premature arrest by RNA polI inhibitor, CX-5461 targets the proliferation of immature erythroblasts. We also show that p53 is activated spontaneously or in response to CX-5461 concomitantly to ribosome biogenesis arrest, and drives a transcriptional program in which genes involved in cell cycle arrest, negative regulation of apoptosis and DNA damage response were upregulated. RNA polI transcriptional stress results in nucleolar disruption and activation of ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation are crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold of ribosome biogenesis down-regulation could be prematurely reached and together with pathological p53 activation prevents a normal expansion of erythroid progenitors.

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.