Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1-4]). Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.

HEATR3 variants impair nuclear import of uL18 (RPL5) and drive Diamond-Blackfan anemia.

The congenital bone marrow failure syndrome Diamond-Blackfan anemia (DBA) is typically associated with variants in ribosomal protein (RP) genes impairing erythroid cell development. Here we report multiple individuals with biallelic HEATR3 variants exhibiting bone marrow failure, short stature, facial and acromelic dysmorphic features, and intellectual disability. These variants destabilize a protein whose yeast homolog is known to synchronize the nuclear import of RPs uL5 (RPL11) and uL18 (RPL5), which are both critical for producing ribosomal subunits and for stabilizing the p53 tumor suppressor when ribosome biogenesis is compromised. Expression of HEATR3 variants or repression of HEATR3 expression in primary cells, cell lines of various origins, and yeast models impairs growth, differentiation, pre-ribosomal RNA processing, and ribosomal subunit formation reminiscent of DBA models of large subunit RP gene variants. Consistent with a role of HEATR3 in RP import, HEATR3-depleted cells or patient-derived fibroblasts display reduced nuclear accumulation of uL18. Hematopoietic progenitor cells expressing HEATR3 variants or small-hairpin RNAs knocking down HEATR3 synthesis reveal abnormal acceleration of erythrocyte maturation coupled to severe proliferation defects that are independent of p53 activation. Our study uncovers a new pathophysiological mechanism leading to DBA driven by biallelic HEATR3 variants and the destabilization of a nuclear import protein important for ribosome biogenesis.

Functionally impaired RPL8 variants associated with Diamond-Blackfan anemia and a Diamond-Blackfan anemia-like phenotype.

Diamond-Blackfan anemia is a rare genetic disease characterized by erythroblastopenia and a large spectrum of developmental anomalies. The vast majority of the cases genetically described are linked to heterozygous pathogenic variants in more than 20 ribosomal protein genes. Here we report an atypical clinical case of DBA associated with a missense variant in RPL8, which encodes RPL8/uL2, a protein of the 60S large ribosomal subunit. RPL8 has been previously implicated as a candidate disease gene in one patient with DBA bearing another type of missense variant; however, evidence for pathogenicity was limited to computational tools. Using functional studies in lymphoblastoid cells as well as yeast models, we show that the RPL8 variants detected in these two patients encode functionally deficient proteins that affect ribosome production and are therefore likely pathogenic. We propose to include RPL8 in the list of DBA-associated genes.

Poly (A)-specific ribonuclease (PARN): More than just "mRNA stock clearing".

In eukaryotic cells, the balance between the synthesis and the degradation decides the steady-state levels of messenger RNAs (mRNA). The removal of adenosine residues from the poly(A) tail, called deadenylation, is the first and the most crucial step in the process of mRNA degradation. Poly (A)-specific ribonuclease (PARN) is one such enzyme that catalyses the process of deadenylation. Although PARN has been primarily known as the regulator of the mRNA stability, recent evidence clearly suggests several other functions of PARN, including a role in embryogenesis, oocyte maturation, cell-cycle progression, telomere biology, non-coding RNA maturation and ribosome biogenesis. Also, deregulated PARN activity is shown to be a hallmark of specific disease conditions. Pathogenic variants in the PARN gene have been observed in various cancers and inherited bone marrow failure syndromes. The focus in this review is to highlight the emerging functions of PARN, particularly in the context of human diseases.

The final step of 40S ribosomal subunit maturation is controlled by a dual key lock.

Preventing premature interaction of pre-ribosomes with the translation apparatus is essential for translational accuracy. Hence, the final maturation step releasing functional 40S ribosomal subunits, namely processing of the 18S ribosomal RNA 3' end, is safeguarded by the protein DIM2, which both interacts with the endoribonuclease NOB1 and masks the rRNA cleavage site. To elucidate the control mechanism that unlocks NOB1 activity, we performed cryo-electron microscopy analysis of late human pre-40S particles purified using a catalytically inactive form of the ATPase RIO1. These structures, together with in vivo and in vitro functional analyses, support a model in which ATP-loaded RIO1 cooperates with ribosomal protein RPS26/eS26 to displace DIM2 from the 18S rRNA 3' end, thereby triggering final cleavage by NOB1; release of ADP then leads to RIO1 dissociation from the 40S subunit. This dual key lock mechanism requiring RIO1 and RPS26 guarantees the precise timing of pre-40S particle conversion into translation-competent ribosomal subunits.

NHP2 deficiency impairs rRNA biogenesis and causes pulmonary fibrosis and Høyeraal-Hreidarsson syndrome.

Telomeres are nucleoprotein structures at the end of chromosomes. The telomerase complex, constituted of the catalytic subunit TERT, the RNA matrix hTR and several cofactors, including the H/ACA box ribonucleoproteins Dyskerin, NOP10, GAR1, NAF1 and NHP2, regulates telomere length. In humans, inherited defects in telomere length maintenance are responsible for a wide spectrum of clinical premature aging manifestations including pulmonary fibrosis (PF), dyskeratosis congenita (DC), bone marrow failure and predisposition to cancer. NHP2 mutations have been so far reported only in two patients with DC. Here, we report the first case of Høyeraal-Hreidarsson syndrome, the severe form of DC, caused by biallelic missense mutations in NHP2. Additionally, we identified three unrelated patients with PF carrying NHP2 heterozygous mutations. Strikingly, one of these patients acquired a somatic mutation in the promoter of TERT that likely conferred a selective advantage in a subset of blood cells. Finally, we demonstrate that a functional deficit of human NHP2 affects ribosomal RNA biogenesis. Together, our results broaden the functional consequences and clinical spectrum of NHP2 deficiency.

The human methyltransferase ZCCHC4 catalyses N6-methyladenosine modification of 28S ribosomal RNA.

RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.

Ribosomal protein gene RPL9 variants can differentially impair ribosome function and cellular metabolism.

Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5'UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5'UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5'UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.

RPL13 Variants Cause Spondyloepimetaphyseal Dysplasia with Severe Short Stature.

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.

Impaired telomere integrity and rRNA biogenesis in PARN-deficient patients and knock-out models.

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.

Maturation of pre-40S particles in yeast and humans.

The synthesis of ribosomal subunits in eukaryotes requires the interplay of numerous maturation and assembly factors (AFs) that intervene in the insertion of ribosomal proteins within pre-ribosomal particles, the ribosomal subunit precursors, as well as in pre-ribosomal RNA (rRNA) processing and folding. Here, we review the intricate nuclear and cytoplasmic maturation steps of pre-40S particles, the precursors to the small ribosomal subunits, in both yeast and human cells, with particular emphasis on the timing and mechanisms of AF association with and dissociation from pre-40S particles and the roles of these AFs in the maturation process. We highlight the particularly complex pre-rRNA processing pathway in human cells, compared to yeast, to generate the mature 18S rRNA. We discuss the information gained from the recently published cryo-electron microscopy atomic models of yeast and human pre-40S particles, as well as the checkpoint/quality control systems that seem to operate to probe functional sites within yeast cytoplasmic pre-40S particles. This article is categorized under: RNA Processing > rRNA Processing Translation > Ribosome Biogenesis.

The Genetic Landscape of Diamond-Blackfan Anemia.

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.

Pre-Ribosomal RNA Processing in Human Cells: From Mechanisms to Congenital Diseases.

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

Recurring mutations in RPL15 are linked to hydrops fetalis and treatment independence in Diamond-Blackfan anemia.

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.

RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes.

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.

Molecular approaches to diagnose Diamond-Blackfan anemia: The EuroDBA experience.

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia and inherited bone marrow failure syndrome that affects approximately seven individuals in every million live births. In addition to anemia, about 50% of all DBA patients suffer from various physical malformations of the face, hands, heart, or urogenital region. The disorder is almost exclusively driven by haploinsufficient mutations in one of several ribosomal protein (RP) genes, although for ∼30% of diagnosed patients no mutation is found in any of the known DBA-linked genes. Because DBA is such a rare disease with a particularly wide range of clinical phenotypes and molecular signatures, the development of collaborative efforts such as the ERARE-funded European DBA consortium (EuroDBA) has become imperative for DBA research. EuroDBA was founded in 2012 and brings together dedicated clinical and biological researchers of DBA from France, Italy, the Netherlands, Germany, Israel, Poland, and Turkey to achieve a number of goals including the consolidation of data in patient registries, establishment of minimal diagnostic criteria, and projects aimed at more fully describing the different mutations linked to DBA. This review will cover the history of the EuroDBA registries, the methods used by EuroDBA in the diagnosis of DBA, and how the consortium has successfully worked together towards the discovery of new DBA-linked genes and the better understanding their pathophysiological effects.

Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: Evidence from RPL11-deficient in vivo and in vitro models.

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.

Nucleolar sub-compartments in motion during rRNA synthesis inhibition: Contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant.

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.