Analyzing the role of ferroptosis in ribosome-related bone marrow failure disorders: From pathophysiology to potential pharmacological exploitation.

Within the last decade, the scientific community has witnessed the importance of ferroptosis as a novel cascade of molecular events leading to cellular decisions of death distinct from apoptosis and other known forms of cell death. Notably, such non- apoptotic and iron-dependent regulated cell death has been found to be intricately linked to several physiological processes as well as to the pathogenesis of various diseases. To this end, recent data support the notion that a potential molecular connection between ferroptosis and inherited bone marrow failure (IBMF) in individuals with ribosomopathies may exist. In this review, we suggest that in ribosome-related IBMFs the identified mutations in ribosomal proteins lead to changes in the ribosome composition of the hematopoietic progenitors, changes that seem to affect ribosomal function, thus enhancing the expression of some mRNAs subgroups while reducing the expression of others. These events lead to an imbalance inside the cell as some molecular pathways are promoted while others are inhibited. This disturbance is accompanied by ROS production and lipid peroxidation, while an additional finding in most of them is iron accumulation. Once lipid peroxidation and iron accumulation are the two main characteristics of ferroptosis, it is possible that this mechanism plays a key role in the manifestation of IBMF in this type of disease. If this molecular mechanism is further confirmed, new pharmacological targets such as ferroptosis inhibitors that are already exploited for the treatment of other diseases, could be utilized to improve the treatment of ribosomopathies.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

The RNA-Binding Function of Ribosomal Proteins and Ribosome Biogenesis Factors in Human Health and Disease.

The ribosome is a macromolecular complex composed of RNA and proteins that interact through an integrated and interconnected network to preserve its ancient core activities. In this review, we emphasize the pivotal role played by RNA-binding proteins as a driving force in the evolution of the current form of the ribosome, underscoring their importance in ensuring accurate protein synthesis. This category of proteins includes both ribosomal proteins and ribosome biogenesis factors. Impairment of their RNA-binding activity can also lead to ribosomopathies, which is a group of disorders characterized by defects in ribosome biogenesis that are detrimental to protein synthesis and cellular homeostasis. A comprehensive understanding of these intricate processes is essential for elucidating the mechanisms underlying the resulting diseases and advancing potential therapeutic interventions.

New Insights into Dyskerin-CypA Interaction: Implications for X-Linked Dyskeratosis Congenita and Beyond.

The highly conserved family of cyclophilins comprises multifunctional chaperones that interact with proteins and RNAs, facilitating the dynamic assembly of multimolecular complexes involved in various cellular processes. Cyclophilin A (CypA), the predominant member of this family, exhibits peptidyl-prolyl cis-trans isomerase activity. This enzymatic function aids with the folding and activation of protein structures and often serves as a molecular regulatory switch for large multimolecular complexes, ensuring appropriate inter- and intra-molecular interactions. Here, we investigated the involvement of CypA in the nucleus, where it plays a crucial role in supporting the assembly and trafficking of heterogeneous ribonucleoproteins (RNPs). We reveal that CypA is enriched in the nucleolus, where it colocalizes with the pseudouridine synthase dyskerin, the catalytic component of the multifunctional H/ACA RNPs involved in the modification of cellular RNAs and telomere stability. We show that dyskerin, whose mutations cause the X-linked dyskeratosis (X-DC) and the Hoyeraal-Hreidarsson congenital ribosomopathies, can directly interact with CypA. These findings, together with the remark that substitution of four dyskerin prolines are known to cause X-DC pathogenic mutations, lead us to indicate this protein as a CypA client. The data presented here suggest that this chaperone can modulate dyskerin activity influencing all its partecipated RNPs.

POLR1A variants underlie phenotypic heterogeneity in craniofacial, neural, and cardiac anomalies.

Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

Nucleolar Protein 56 Deficiency in Zebrafish Leads to Developmental Abnormalities and Anemia via p53 and JAK2-STAT3 Signaling.

Ribosomes are the vital molecular machine for protein translation in a cell. Defects in several nucleolar proteins have been observed in human ribosomopathies. In zebrafish, a deficiency in these ribosomal proteins often results in an anemic phenotype. It remains to be determined whether any other ribosome proteins are involved in regulating erythropoiesis. Here, we generated a nucleolar protein 56 (nop56)-/- zebrafish model and investigated its function. A nop56 deficiency induced severe morphological abnormalities and anemia. WISH analysis showed that the specification of the erythroid lineage in definitive hematopoiesis and the maturation of erythroid cells were impaired in the nop56 mutants. Additionally, transcriptome analysis revealed that the p53 signaling pathway was abnormally activated, and the injection of a p53 morpholino partially rescued the malformation, but not the anemia. Moreover, qPCR analysis showed that the JAK2-STAT3 signaling pathway was activated in the mutants, and the inhibition of JAK2 partially rescued the anemic phenotype. This study suggests that nop56 is a potential target for investigation in erythropoietic disorders, particularly those that may be associated with JAK-STAT activation.

Nucleolar stress: From development to cancer.

The nucleolus is a large nuclear membraneless organelle responsible for ribosome biogenesis. Ribosomes are cytoplasmic macromolecular complexes comprising RNA and proteins that link amino acids together to form new proteins. The biogenesis of ribosomes is an intricate multistep process that involves the transcription of ribosomal DNA (rDNA), the processing of ribosomal RNA (rRNA), and the assembly of rRNA with ribosomal proteins to form active ribosomes. Nearly all steps necessary for ribosome production and maturation occur in the nucleolus. Nucleolar shape, size, and number are directly linked to ribosome biogenesis. Errors in the steps of ribosomal biogenesis are sensed by the nucleolus causing global alterations in nucleolar function and morphology. This phenomenon, known as nucleolar stress, can lead to molecular changes such as stabilization of p53, which in turn activates cell cycle arrest or apoptosis. In this review, we discuss recent work on the association of nucleolar stress with degenerative diseases and developmental defects. In addition, we highlight the importance of de novo nucleotide biosynthesis for the enhanced nucleolar activity of cancer cells and discuss targeting nucleotide biosynthesis as a strategy to activate nucleolar stress to specifically target cancer cells.

Molecular etiology of defective nuclear and mitochondrial ribosome biogenesis: Clinical phenotypes and therapy.

Ribosomopathies are rare congenital disorders associated with defective ribosome biogenesis due to pathogenic variations in genes that encode proteins related to ribosome function and biogenesis. Defects in ribosome biogenesis result in a nucleolar stress response involving the TP53 tumor suppressor protein and impaired protein synthesis leading to a deregulated translational output. Despite the accepted notion that ribosomes are omnipresent and essential for all cells, most ribosomopathies show tissue-specific phenotypes affecting blood cells, hair, spleen, or skin. On the other hand, defects in mitochondrial ribosome biogenesis are associated with a range of clinical manifestations affecting more than one organ. Intriguingly, the deregulated ribosomal function is also a feature in several human malignancies with a selective upregulation or downregulation of specific ribosome components. Here, we highlight the clinical conditions associated with defective ribosome biogenesis in the nucleus and mitochondria with a description of the affected genes and the implicated pathways, along with a note on the treatment strategies currently available for these disorders.

Ribosomopathies and cancer: pharmacological implications.

INTRODUCTION: The ribosome is a ribonucleoprotein organelle responsible for protein synthesis, and its biogenesis is a highly coordinated process that involves many macromolecular components. Any acquired or inherited impairment in ribosome biogenesis or ribosomopathies is associated with the development of different cancers and rare genetic diseases. Interference with multiple steps of protein synthesis has been shown to promote tumor cell death. AREAS COVERED: We discuss the current insights about impaired ribosome biogenesis and their secondary consequences on protein synthesis, transcriptional and translational responses, proteotoxic stress, and other metabolic pathways associated with cancer and rare diseases. The modulation of different therapeutic chemical entities targeting cancer in in vitro and in vivo models have also been detailed. EXPERT OPINION: Despite the association between inherited mutations affecting ribosome biogenesis and cancer biology, the development of therapeutics targeting the essential cellular machinery has only started to emerge. New chemical entities should be designed to modulate different checkpoints (translating oncoproteins, dysregulation of specific ribosome-assembly machinery, ribosomal stress, and rewiring ribosomal functions). Although safe and effective therapies are lacking, consideration should be given to using existing drugs alone or in combination for long-term safety, with known risks for feasibility in clinical trials and synergistic effects.

Mutations in RPS19 may affect ribosome function and biogenesis in Diamond Blackfan anemia.

Ribosomes, the cellular organelles translating the genetic code to proteins, are assemblies of RNA chains and many proteins (RPs) arranged in precise fine-tuned interwoven structures. Mutated ribosomal genes cause ribosomopathies, including Diamond Blackfan anemia (DBA, a rare heterogeneous red-cell aplasia connected to ribosome malfunction) or failed biogenesis. Combined bioinformatical, structural, and predictive analyses of potential consequences of possibly expressed mutations in eS19, the protein product of the highly mutated RPS19, suggest that mutations in its exposed surface could alter its positioning during assembly and consequently prevent biogenesis, implying a natural selective strategy to avoid malfunctions in ribosome assembly. A search for RPS19 pseudogenes indicated > 90% sequence identity with the wild-type, hinting at its expression in cases of absent or truncated gene products.

Cell senescence and malignant transformation in the inherited bone marrow failure syndromes: Overlapping pathophysiology with therapeutic implications.

Fanconi anemia, telomeropathies and ribosomopathies are members of the inherited bone marrow failure syndromes, rare genetic disorders that lead to failure of hematopoiesis, developmental abnormalities, and cancer predisposition. While each disorder is caused by different genetic defects in seemingly disparate processes of DNA repair, telomere maintenance, or ribosome biogenesis, they appear to lead to a common pathway characterized by premature senescence of hematopoietic stem cells. Here we review the experimental data on senescence and inflammation underlying marrow failure and malignant transformation. We conclude with a critical assessment of current and future therapies targeting these pathways in inherited bone marrow failure syndromes patients.

Proteostasis Perturbations and Their Roles in Causing Sterile Inflammation and Autoinflammatory Diseases.

Proteostasis, a portmanteau of the words protein and homeostasis, refers to the ability of eukaryotic cells to maintain a stable proteome by acting on protein synthesis, quality control and/or degradation. Over the last two decades, an increasing number of disorders caused by proteostasis perturbations have been identified. Depending on their molecular etiology, such diseases may be classified into ribosomopathies, proteinopathies and proteasomopathies. Strikingly, most-if not all-of these syndromes exhibit an autoinflammatory component, implying a direct cause-and-effect relationship between proteostasis disruption and the initiation of innate immune responses. In this review, we provide a comprehensive overview of the molecular pathogenesis of these disorders and summarize current knowledge of the various mechanisms by which impaired proteostasis promotes autoinflammation. We particularly focus our discussion on the notion of how cells sense and integrate proteostasis perturbations as danger signals in the context of autoinflammatory diseases to provide insights into the complex and multiple facets of sterile inflammation.

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis. Loss of these DExD/H-box proteins, which we name Aramis, Athos, and Porthos, aberrantly stabilizes p53, arrests the cell cycle, and stalls germline stem cell (GSC) differentiation. Aramis controls cell-cycle progression by regulating translation of mRNAs that contain a terminal oligo pyrimidine (TOP) motif in their 5' UTRs. We find that TOP motifs confer sensitivity to ribosome levels that are mediated by La-related protein (Larp). One such TOP-containing mRNA codes for novel nucleolar protein 1 (Non1), a conserved p53 destabilizing protein. Upon a sufficient ribosome concentration, Non1 is expressed, and it promotes GSC cell-cycle progression via p53 degradation. Thus, a previously unappreciated TOP motif in Drosophila responds to reduced RiBi to co-regulate the translation of ribosomal proteins and a p53 repressor, coupling RiBi to GSC differentiation.

Invariable Ribosome Stoichiometry During Murine Erythroid Differentiation: Implications for Understanding Ribosomopathies.

Heterogeneity of the main ribosomal composition represents an emerging, yet debatable, mechanism of gene expression regulation with a purported role in ribosomopathies, a group of disorders caused by mutations in ribosomal protein genes (RPs). Ribosomopathies, mysteriously relate with tissue-specific symptoms (mainly anemia and cancer predisposition), despite the ubiquitous expression and necessity of the associated RPs. An outstanding question that may shed light into disease pathogenicity and provide potential pharmacological interventions, is whether and how the ribosomal composition is modified during, the highly affected by RP mutations, process of erythroid differentiation. To address this issue, we analyzed ribosome stoichiometry using an established model of erythroid differentiation, through sucrose gradient ultracentrifugation and quantitative proteomics. We found that differentiation associates with an extensive reprogramming of the overall ribosomal levels, characterized by an increase in monosomes and a decrease in polysomes. However, by calculating a stoichiometry score for each independent ribosomal protein, we found that the main ribosomal architecture remained invariable between immature and differentiated cells. In total, none of the 78 Ribosomal Proteins (RPs- 74 core RPs, Rack1, Fau and 2 paralogs) detected was statistically different between the samples. This data was further verified through antibody-mediated quantification of 6 representative RPs. Moreover, bioinformatic analysis of whole cell proteomic data derived out of 4 additional models of erythropoiesis revealed that RPs were co-regulated across these cell types, too. In conclusion, ribosomes maintain an invariant protein stoichiometry during differentiation, thus excluding ribosome heterogeneity from a potential mechanism of toxicity in ribosomopathies and other erythroid disorders.

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.

Translational control through ribosome heterogeneity and functional specialization.

The conceptual origins of ribosome specialization can be traced back to the earliest days of molecular biology. Yet, this field has only recently begun to gather momentum, with numerous studies identifying distinct heterogeneous ribosome populations across multiple species and model systems. It is proposed that some of these compositionally distinct ribosomes may be functionally specialized and able to regulate the translation of specific mRNAs. Identification and functional characterization of specialized ribosomes has the potential to elucidate a novel layer of gene expression control, at the level of translation, where the ribosome itself is a key regulatory player. In this review, we discuss different sources of ribosome heterogeneity, evidence for ribosome specialization, and also the future directions of this exciting field.

Enhanced p53 Levels Are Involved in the Reduced Mineralization Capacity of Osteoblasts Derived from Shwachman-Diamond Syndrome Subjects.

Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.

A Recurrent Variant in POLR1B, c.3007C>T; p.Arg1003Cys, Associated with Atresia of the External Canal and Microtia in Treacher Collins Syndrome Type 4.

Treacher Collins syndrome (TCS) is a heterogenous malformation syndrome characterized by a distinct facial appearance including downslanting palpebral fissures, malar hypoplasia, conductive hearing loss, and mandibular hypoplasia. Recently, a new causative gene, POLR1B, encoding DNA-directed RNA polymerase I subunit RPA2, was identified as a fourth type of TCS (TCS4). We describe another patient with TCS4 caused by a recurrent POLR1B variant, c.3007C>T; p.Arg1003Cys. Including our patient, all 4 patients with p.(Arg1003Cys) had atresia of the external auditory canal and microtia. All of the reported pathogenic variants in POLR1B were clustered at only 2 residues. Our patient highlights the genotype-phenotype correlation in TCS4 associated with POLR1B.

Angiogenin (ANG)-Ribonuclease Inhibitor (RNH1) System in Protein Synthesis and Disease.

Protein synthesis is a highly complex process executed by well-organized translation machinery. Ribosomes, tRNAs and mRNAs are the principal components of this machinery whereas RNA binding proteins and ribosome interacting partners act as accessory factors. Angiogenin (ANG)-Ribonuclease inhibitor (RNH1) system is one such accessory part of the translation machinery that came into focus afresh due to its unconventional role in the translation. ANG is conventionally known for its ability to induce blood vessel formation and RNH1 as a "sentry" to protect RNAs from extracellular RNases. However, recent studies suggest them to be important in translation regulation. During cell homeostasis, ANG in the nucleus promotes rRNA transcription. While under stress, ANG translocates to the cytosol and cleaves tRNA into fragments which inhibit ribosome biogenesis and protein synthesis. RNH1, which intimately interacts with ANG to inhibit its ribonucleolytic activity, can also bind to the 40S ribosomes and control translation by yet to be known mechanisms. Here, we review recent advancement in the knowledge of translation regulation by the ANG-RNH1 system. We also gather information about this system in cell homeostasis as well as in pathological conditions such as cancer and ribosomopathies. Additionally, we discuss the future research directions and therapeutic potential of this system.