Insights into the nucleolar dysfunction and associated genetic disorders

Abstract

The nucleolus, a dynamic and membrane-less nuclear subcompartment, plays a central role in maintaining cellular homeostasis. Traditionally recognized as the site of ribosomal RNA (rRNA) synthesis and ribosome assembly, the nucleolus is now known to participate in diverse cellular processes, including cell-cycle regulation, DNA-damage response, and stress signaling. Structurally organized into three main components—the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC)—it undergoes rapid remodeling under physiological and pathological conditions.

Dysfunction of the nucleolus has been increasingly linked to a group of genetic disorders collectively termed ribosomopathies, characterized by defective ribosome biogenesis and function. Mutations affecting nucleolar proteins or rRNA-processing factors can disrupt ribosomal output, activate the p53 pathway, and result in tissue-specific abnormalities, hematological disorders, and heightened cancer susceptibility. Notable ribosomopathies include Diamond–Blackfan anemia, Treacher Collins syndrome, and Dyskeratosis congenita.

This review provides a comprehensive overview of the structural and functional aspects of the nucleolus, explores its role in genetic-disease mechanisms, and highlights current advances in understanding nucleolar stress and its downstream effects. We also discuss how emerging insights into nucleolar biology are revealing novel diagnostic and therapeutic strategies. Given the centrality of the nucleolus in cellular physiology, elucidating its dysfunction may significantly improve our understanding of a wide spectrum of human diseases.

Keywords

nucleolus mutations ribosome genetic disorders nucleolar stress p53

Introduction

The nucleolus is a multifunctional nuclear domain that plays a key role in coordinating ribosome biogenesis with cellular growth and stress responses. Beyond its classical function in ribosomal RNA synthesis and ribosome assembly, recent evidence highlights its involvement in genome stability, cell-cycle regulation, and the cellular response to stress. Dysregulation of nucleolar structure or function has been increasingly recognized as a hallmark of several human diseases, particularly genetic disorders that affect ribosome biogenesis and nucleolar integrity. This review focuses on the mechanisms of nucleolar dysfunction and their association with genetic diseases, providing an integrated overview of recent advances in this rapidly evolving field.

The nucleolus

The nucleolus, an intracellular structure present inside the nucleus, was first identified in the early nineteenth century.¹ It has the highest mass among all other intracellular structures.² The nucleolus can be notably visualized with the help of phase contrast microscopy. The underlying principle is the higher refractive index of the nucleolus compared to the peripheral nucleoplasm.

The nucleolus is structurally organized into three core compartments viz., the granular component (GC), the dense fibrillar component (DFC), and the fibrillar center (FC) as shown in Figure 1. The distribution and organization of these components vary with respect to the species, cell type, and functional state of the cell.⁴

Figure 1.

Nucleolar Structure: A schematic diagram showing the nucleolar structure inside the nucleus and three different regions, GC, FC, and DFC, of the nucleolus is shown in the image. Adapted from Tiku et al., 2018³.

The nucleolus is the region inside the nucleus where transcription and processing of rRNA occurs. The rDNA genes encoding the 18S, 5.8S, and 28S RNA species of ribosome are transcribed by RNA polymerase inside the nucleolus. These genes are present on the chromosome in high copy numbers in tandem arrays at the nucleolar organizer loci.^5,6 Thus, nucleolus is a genetically inferred element as recognized by the discovery of nucleolar organizer loci in the chromosome.⁷ The structural arrangement of the nucleolus is based upon these loci. Thus, these loci regulate the transcription of the RNA, their modification and assembly of ribosomal proteins.

Apart from the transcription of the ribosomal rRNA, the nucleolus is also involved in the pre-rRNA processing and assembly of ribosomal subunits. These functions are carried out spatially within the three compartments of the nucleolus. The nascent transcripts (pre-rRNA) are localized in the region between the FCs and the DFC. RNA Polymerase I (Pol I) machinery components, such as Upstream Binding Factor (UBF), are present in the fibrillar centers (FCs). Pre-rRNA processing factors, including small nucleolar RNAs (snoRNAs), nucleolar protein 58 (Nop58), fibrillarin, and ribonucleoproteins (RNPs), are enriched in the dense fibrillar component (DFC). Ultimately, the nucleolus major function is characterized by ribosome biogenesis.^8,9 Recent mass spec studies have demonstrated the involvement of nucleolus such as signal recognition particle (SRP) assembly, RNA editing, telomerase assembly, spliceosome, pre-transfer RNA (tRNA) maturation, and genome architecture maintenance. Since nucleolus performs different basic and important functions within the cells, therefore, any kind of nucleolar dysfunction may lead to different types of diseases. In this review, we will be discussing nuclear dysfunction-associated diseases with a major focus on genetic disorders.

Mechanism of nuclear dysfunction

The primary regulatory function linked to misregulated dynamics of nucleolar proteins is associated with the stress response and the release of ribosomal proteins (RPs) from nucleolus to the nucleoplasm.

Nucleolar stress

A continued transport of nucleolar proteins occurs between the nucleolus and the nucleoplasm. Some nucleolar proteins like NPM and nucleostemin regulate the functions of other nucleolar proteins that commute constantly between the inside and the outside of the nucleolus.^10,11 The disruption of nucleolar architecture and the inhibition of rRNA transcription initiates nucleolar stress which causes dysregulation in the influx and efflux of nucleolar proteins. This leads to the release of various nucleolar proteins from the nucleolus and entry of many non-nucleolar proteins in the nucleolus.¹² Quite a number of stress stimuli like nutrient deprivation, hypoxia, double stranded breaks in DNA (SSBs), and oxidative and thermal stress are known to cause nucleolar stress.^13,14 Nucleolar stress relays different stress-related signaling pathways in response to the abovementioned stress including the Mdm2-p53, NF-κB and HIF-1α pathways.¹⁵ In this section, we concisely throw light on the mechanisms that are intrinsic to the signaling pathways activated in response to nucleolar stress.

Mdm2-p53 pathway

One of the most well-studied pathways is Mdm2-p53 signaling in response to nucleolar stress.¹⁶ Mdm2/MDMX-mediated ubiquitination and proteolysis leads to a speedy turnover rate of p53 in quiescent cells.¹⁷ It has been previously reported that the breakdown of the nucleolus activates p53 upon receiving stress stimuli like DNA damage.^18,19 There is no consensus regarding the elaborate mechanisms underlying the activation of p53 due to nucleolar stress. Multiple hypotheses have been postulated to throw light upon the role of nucleolar stress in the stabilization of p53. One of the theories is that nucleolar stress leads to the interaction of Mdm2 and MDMX with ribosomal proteins in the nucleoplasm. This inactivates both Mdm2 and MDMX which stabilizes p53.^20,21 As of now many ribosomal proteins are known to play roles in Mdm2-p53 pathway. RPL5 and RPL11 are two significant proteins amongst them as they form 5S ribonucleoprotein particle complex with 5S rRNA to complement the Mdm2-p53 pathway.^22,23 Apart from the ribosomal proteins, it has also been found that the to-and-fro movement of p14^Arf may be involved in Mdm2-p53 pathway. Due to nucleolar stress, p14^Arf is released from the nucleolus which causes the activation of p53.^24,25 The inactivation of Mdm2 and consequent p53 accumulation due to stress stimuli can also occur by binding of ribosomal proteins to the Mdm2-p53 complex, as shown in Figure 2.^11,26 It is well known that nucleolar stress-mediated signaling and cell-fate decisions like apoptosis, growth arrest, and senescence are regulated by the Mdm2-p53 pathway.

Figure 2.

Schematic illustration showing activation of p53 molecule upon nucleolar stress, where nucleolar stress causes ribosomal protein (RP) interaction with MDM2, resulting in activation of p53.

NF-κB pathway

NF-κB transcription factors are known to have important functions in stress response, immune regulation and cellular homeostasis.²⁷ The member proteins of NF-κB family include cRel, RelB, p65(RelA), p52 (NF-κB2), and p50 (NF-κB1).²⁸ Apart from the extracellular cytokines, various intracellular stress signaling have been reported to initiate NF-κB signaling. One such is the interaction between NPM and NF-κB proteins revealed in an early study. They showed that NPM is a transcriptional co-activator of NF-κB that triggers the expression of MnSOD (Manganese Superoxide Dismutase).²⁹ NF-κB-inducing kinase (NIK) was shown to shuttle between the nucleoplasm and the nucleolus. NIK inside the nucleolus reduces NF-κB activation potential.³⁰ Literature also reveals that NF-κB proteins like RelA show nucleolar translocation. Many pro-apoptotic stimuli like UV-C radiation, serum withdrawal and aspirin cause sequestration of RelA inside the nucleolus which inhibits NF-κB transcriptional activity.³¹ RelA inside the nucleolus may get ubiquitinated and may also lead to improper localization of NPM when the cell is undergoing apoptosis.^32,33 The genetic and pharmacological triggers of nucleolar stress like activation of TIF-IA and actinomycin D treatment, respectively, can also lead to nuclear translocation and activation of NF-κB.^34–36 When treated with CX-5461 which gives a more potent nucleolar stress stimulus, there was no phosphorylation and activation of NF-κB. This showed that the activation of NF-κB does not depend upon inhibition of rRNA transcription.^36,37

HIF-1α

HIF-1α is a crucial transcription factor which responds to stress and shows nucleolar translocation. p14^ARF represses the transcriptional activity of HIF-1α by interacting with HIF-1α. This sequesters HIF-1α inside the nucleolus without affecting the activity of p53.³⁸ VHL is an important E3 ligase which catalyzes the ubiquitination and degradation of HIF-1α. Physiological conditions like hypoxia or normoxic acidosis leads to the nucleolar sequestration of VHL.³⁹ When VHL is sequestered inside the nucleolus, HIF-1α remains stable. This reverses hypoxia to normal oxygen conditions. One of the proteins of the nucleolus, SENP3 acts as a catalytic factor for de-SUMOylation of p300. This leads to increase in the transcriptional efficiency of HIF-1α which causes the release of SENP3 from nucleolus to nucleoplasm by induction of ROS.⁴⁰ It is known that hypoxia and ROS can cause disruption of RNA synthesis and condensation of nucleolus upon interaction of VHL with rDNA. It can be hypothesized that under these conditions, nucleolar stress plays a pivotal role in the activation of HIF-1α.^41,42 This suggests that the nucleolus contributes significantly to HIF-1α activation in response to cellular stress stimuli.

Stress signaling upon DNA damage

It is well known that nucleolus helps in monitoring the stability and integrity of genomic DNA. The chromatin region that undergoes intensive transcription in dividing cells corresponds to ribosomal DNA (rDNA).^43,44 rDNA is highly prone to DNA damages like double strand breaks (DSBs) because of robust transcription of its rRNA and its repetitive nature. These damages to DNA inhibit the transcriptional machinery of Pol I which ultimately cause nucleolar stress.⁴⁵ This leads to the localization of DNA repair proteins inside the nucleolus.⁴⁶ Ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia- and Rad3-related (ATR) assemble at nucleolar caps in response to DSBs in rDNA and initiate DNA repair.^47–49 DNA damage causing nucleolar stress also leads to the migration of a DNA check-point clamp, Rad9B into the nucleolus.⁵⁰ Hence, there occurs a complex interplay between DNA damage and nucleolar homeostasis,^51,52 which initiate DNA damage-related stress responses that operate via p53 independent signaling pathways.^53,54

Others

The arrangement of different proteins in the nucleolus has been often noticed, including those that control the cell cycle and those involved in metabolic and stress signaling pathways. Many cell-cycle proteins, like cyclin E, CDK2, CDKN1A/p21, and CDKN1B/p27, can show a presence in the nucleolus in various cell types.^55–58 Nucleolus plays a crucial role in cell-cycle regulation by promoting their ubiquitination.^58,59 The proteosomal machinery present in the nucleolus facilitates proteolysis of c-myc by causing its accumulation and ubiquitination.^60,61 Sirt6 and Sirt7 which are sirtuin proteins that regulate ribosome biogenesis also get sequestered in the nucleolus.^62–64 Sirt1 expression and function is also determined by nucleolar stress.⁶⁵ The nucleolus modulates metabolic regulators affecting the function of mitochondria during oxidative stress.⁶⁶

Ribosome biogenesis impairment

Ribosome biogenesis is an essential element in cell growth. Impairment in the ribosome biogenesis pathway can cause the cells to undergo oncogenic transformation. Many proto-oncogenes and tumor suppressors are involved in the process of ribosome biogenesis. This process is primarily regulated by PI3K-mTORC1 signaling pathway and C-MYC. Dividing cells carry out extensive protein synthesis which require ribosomes. Hence, ribosome biogenesis and protein synthesis are coupled to cell cycle.⁶⁷ The three RNA polymerases are involved in ribosome biogenesis which cause transcription of both rRNAs and mRNAs that encode 80 ribosomal proteins.⁶⁸ The translation machinery and the nuclear import-export apparatus are extensively required for ribosome biogenesis.^69,70 The primary location of ribosome biogenesis within the cell is the nucleolus. Clusters of tandem repeats of rRNA genes are arranged in this sub-nuclear compartment into what are called nucleolar organizing regions (NOR). RNA polymerase I (PolI) transcribes the rRNA genes to create the precursor 47S rRNA,⁷¹ which is then processed into mature rRNA species. The rRNA and RPs are then assembled to form the 40S and 60S ribosomal subunits. Notably, a number of human cancers and other malignancies exhibit dysregulation of distinct stages of the ribosome biogenesis process.

Since ribosome biogenesis is a highly energy- and resource-demanding process, its integrity is continuously monitored, and almost any kind of significant cellular stress will cause rRNA transcription to immediately stop.¹² The nucleolus experiences specific structural alterations in response to these stressors, such as exposure to various genotoxic agents like doxorubicin or inhibition of rRNA transcription by low concentrations of Actinomycin D (ActD). These changes include condensation and segregation into structures known as nucleolar caps, which are made up of nucleolar proteins and RNA.^72–74 Therefore, finding such structural changes could be interpreted as a sign of extreme nucleolar stress. Numerous forms of cellular stress, such as growth factor deprivation,⁷⁵ heat shock, and hypoxia,¹⁸ have been shown to activate p53 by preventing several ribosome biogenesis processes and causing nuclear stress. DNA damage has long been known to activate p53 via a number of ways.^76,77

Aberrant nucleolar dynamics

Disruptions to the nucleolus’s typical behavior, including as modifications to its size, shape, location, and component mobility, are known as aberrant nucleolar dynamics.⁷⁸ Many cell-associated factors can lead to aberrant nucleolar dynamics. Nucleolar stress, which results in abnormal nucleolar architecture and function, can be caused by disruption of ribosome synthesis in the nucleolus.⁷⁹ Protein shuttling and abnormal nucleolar dynamics can result from issues with the transcription of rRNA, the RNA that builds up ribosomes.⁸⁰ Aberrant dynamics can result from mutations in genes related to nucleolar structure or function.⁸¹ Nucleolar dynamics can also be disturbed by exposure to specific environmental stresses.⁸² Cell death or cell-cycle arrest may result from aberrant dynamics that set off stress-responsive pathways.⁷⁹ Numerous human illnesses, such as cancer, Diamond-Blackfan anemia, cardiomyopathy, and Hutchinson-Gilford progeria syndrome, are linked to abnormal nucleolar dynamics.⁸³ Nucleolar proteins may be released from the nucleolus or non-nucleolar proteins may be sequestered as a result of aberrant dynamics-induced aberrant shuttling and localization.⁸⁰ Nucleolar stress in cardiomyocytes can result in either an increase in cardioprotective proteins or, on the other hand, larger nucleoli and defective nucleolar organizer regions (AgNORs), which mimic tissue ageing.⁷⁹ In uncommon genetic disorders, de novo frameshift mutations in HMGB1 can result in abnormal phase separation and nucleolar dysfunction.⁸¹ Depletion of the nuclear structure-related protein lamin B2 alters nucleolar shape and nucleolin dynamics.⁸⁴

Nucleolar dysfunction and genetic disorders

A number of illnesses, such as early ageing, neurological diseases, and some types of cancer, are associated with nucleolar dysfunction or the compromised function of the nucleolus. This condition is frequently caused by abnormalities in ribosome synthesis. This review focuses on the genetic disorders associated with abnormalities in the function of the nucleolus. In this section following diseases will be discussed in detail:

(1) Werner Syndrome

The functional decline following a time of maturity is the definition of ageing, a complicated and inevitable process. Both ageing and maturity are dynamic processes; biological systems, such as cells and multicellular creatures, never reach a specific state of maturity. In contrast, although certain nearby pathways or tissues are already declining, development leads to conditions of maximum functional performance potential of subcellular signaling pathways or particular tissue functions. One can either look at centenarians who have a slower rate of morbidity or the opposite scenario, where children age as if time has stopped and have accelerated morbidity, in order to better understand the ageing process and the biological mechanisms that underlie it in humans. These juvenile conditions, known as progerias, are segmental in nature, exhibiting certain signs of ageing without reflecting the full range of age-related illnesses. Premature ageing, which is the term used to describe ageing that takes place before reaching maturity. There are still many unanswered problems because not all premature ageing disorders have been confirmed to exhibit a pathomechanism that also propels the “normal” ageing process, despite the fact that they are used as model diseases to research ageing.

Werner syndrome refers to a rapid, segmental ageing process that begins with the absence of the pubertal growth spurt. It is also known as an adult form of progeria. It manifests in the second and third decades as atherosclerosis, osteoporosis, cataracts, hair loss, and a decrease in subcutaneous fat.⁸⁵ Patients with Werner syndrome experience a wide range of age-related illnesses, however they do not exhibit age-related neurodegenerative conditions such as Parkinson’s or Alzheimer’s dementia. The majority of Werner syndrome cases are brought on by mutations in the WRN-helicase, which is implicated in several DNA metabolic processes, including telomere maintenance, DNA transcription, replication, and repair. The cellular pathways that are impacted span the entire range of ageing molecular markers.^86–88 Fibroblasts with Werner syndrome exhibit a markedly decreased replicative lifespan in culture, and after a few passages, they reach senescence.⁸⁹ One factor contributing to human ageing is the buildup of senescent cells⁹⁰; however, Werner syndrome and the pleiotropic WRN-helicase cases demonstrated that it is impossible to identify the crucial function whose malfunction results in accelerated ageing and age-related disorders.^87,88 As a result, the pleiotropic character of the ageing process in Werner’s syndrome has been highlighted in recent research. However, studying progeria offers a chance to examine the biological pathways that are important for the rate of human ageing. It should be easy to determine whether certain cellular pathways are engaged in or accountable for the accelerated ageing process because progerias are segmental in nature and are caused by recognized and characterized mutations. Recent studies have revealed that the WRN helicase localizes to the nucleolus, where it contributes to ribosomal DNA (rDNA) replication and repair. Loss of WRN function disrupts nucleolar integrity, leading to rDNA instability, impaired ribosomal biogenesis, and activation of nucleolar stress pathways. These findings indicate that nucleolar dysfunction plays a key role in the cellular senescence and genomic instability observed in Werner syndrome.

(2) Dyskeratosis congenita

Defects in telomere biology lead to dyskeratosis congenita (DC), a hereditary disease condition with bone marrow failure and cancer predisposition. All body systems are impacted by DC including the diagnostic triad of abnormal nails, reticular skin pigmentation, and oral leukoplakia. Other side effects include liver disease, pulmonary fibrosis, bone marrow failure, neurological and ocular abnormalities, and an elevated risk of cancer.^91,92

There are three possible inheritance patterns for DC: autosomal dominant (AD), autosomal recessive (AR), and X-linked recessive (XLR). Sporadic occurrences are also very common and are most likely caused by novel mutations in dominant genes. Till date, six telomere signaling genes—DKC1, TERC, TERT, TINF2, NOLA2, and NOLA3—have been shown to have mutations in DC patients.^91,93–97 One of these six genes is mutated in about half of DC patients, and the other half are waiting to find abnormalities in other telomere genes. The genes NOLA3 and NOLA2 encode for NOP10 and NHP2 proteins which are members of Nucleolar protein family. Mutations in these cause autosomal recessive DC. The dyskerin protein, encoded by the gene DKC1 which is the first DC gene, is involved in post-transcriptional pseudouridylation and forms a ribonucleoprotein (RNP) complex with NOP10 and NHP2.^96,97 As dyskerin, NOP10, and NHP2 are all nucleolar proteins essential for rRNA modification and ribosome assembly, mutations in these genes directly impair nucleolar function. This disruption results in defective ribosome biogenesis and triggers nucleolar stress, which contributes to the multisystemic manifestations of Dyskeratosis congenita. Therefore, DC exemplifies a classic nucleolar disorder in which telomere dysfunction and impaired nucleolar activity converge.

(3) Treacher Collins

Treacher-Collins syndrome (TCS) is an autosomal dominant condition of craniofacial morphogenesis. TCS, which is also referred to as mandibulofacial dysostosis and Franceschetti-Zwahlen-Klein syndrome, is thought to affect 1/50,000 live births.^98,99 Many head and neck-specific developmental abnormalities are hallmarks of Treacher Collins syndrome, a severe congenital condition of craniofacial development. One of the most prevalent characteristics of TCS is the hypoplasia of the facial bones, especially the zygomatic complex (81%), and mandible (78% of patients). A lack of lid lashes medial to the defect (69%) and downward slanting of the palpebral fissures (89%) with notching of the lower eyelids (69%) are examples of ocular abnormalities. Athesia of the external auditory canals and abnormalities of the middle ear ossicles are often linked to changes in the external ears’ size, shape, and location, which are further clinical characteristics of TCS.

The gene altered in TCS, TCOF1, was previously identified by genetic, physical, and transcript mapping approaches. It was discovered as the gene that encodes a low complexity, serine/alanine-rich, nucleolar phosphoprotein called Treacle.¹⁰⁰ More than 130 different mutations have since been found. The majority of the mutations described so far are family specific and occur throughout the gene; these include insertion, splicing, and non-sense mutations. The most frequent mutations are deletions, which vary in size from 1 to 40 nucleotides.¹⁰¹ Treacle shares the most structural similarities with Nopp140, which facilitates the import of ribosomal proteins from the cytoplasm and the export of pre-ribosomal ribonucleoprotein (pre-rRNP) from the nucleus. Treacle has been localized to the dense fibrillar component of the nucleus by immunofluorescence studies.¹⁰² These studies have also demonstrated that Treacle colocalizes with RNA pol1 and upstream binding factor in nucleolar organizing regions where it plays a role in the transcription of ribosomal DNA genes.¹⁰³ In the early phases of pre-RNA processing in the nucleolus, Treacle has been found to be a part of human Nop65p-associated pre-ribosomal ribonucleoprotein (pre-rRNP) complexes¹⁰⁴ that 2′-O-methylates pre-ribosomal RNA. According to these findings, Treacle is part of an RNP complex in the nucleolus and might be in charge of controlling particular phases of the ribosome biogenesis process.

Treacle has been demonstrated to have important roles in ribosome maturation, which in turn regulates neuroepithelial survival and neural crest cell proliferation, which is in line with its nucleolar localization.^101,103 The synthesis of the mature 28S subunit in neuroepithelial cells and neural crest cells indicates that haploinsufficiency of Tcof1 results in inadequate ribosome biogenesis. The high levels of tissue-specific death seen in the pathophysiology of TCS are caused by this deficiency, which is insufficient to meet the demands of these highly proliferative cell populations. This deficiency causes nucleolar stress to activate p53, and p53 stabilization in turn transcriptionally activates a number of pro-apoptotic effector genes, including Ccng1, Trp53inp1, Noxa, Perp, and Wig1, within the neuroepithelium.¹⁰⁵

(4) Progeria Syndrome

Progeria, also known as Hutchinson-Gilford Progeria (HGPS), is majorly characterized by premature ageing. The major phenotype of progeria involves retarded growth, a large skull, thin skin, less weight and an increase in the overall ageing process. Phenotype starts at an early stage of a child, starting from 2 years onwards. Individuals with progeria don’t survive beyond 12-14 years; there has been no treatment available currently.¹⁰⁶ Progeria is caused by a mutation in the nuclear envelope protein lamin A/C (LMNA). Mutation in LMNA causes the formation of a mutant form of lamin A known as progerin. Access accumulation of progerin surrounding the nuclear envelope causes different types of stresses within cells, which alters different signaling pathways as well. Among different stress within the cells, nucleolar stress emerged as one of the most important causes of the disease. Different reports showed that during the progeria syndrome, nucleolar size significantly increases, which results in increased production of ribosomes and different ribosomal proteins. Fibroblast isolated from progeria patients also exhibited increased protein synthesis due to increased nucleolar activity and ribosome biogenesis.³ At the molecular levels, progerin causes heterochromatin disorganization, which results in the loss of inactive ribosomal DNA loci that cause an abnormal increase in nucleolar activity and ribosomal biogenesis.¹⁰⁷ All of this evidence confirms that in progeria, elevated nucleolar activity (Nucleolar expansion) and increased protein synthesis are observed, which are considered the hallmarks of the disease, as shown in Figure 3. At the molecular level, because of nucleolar expansion and increased ribosomal biogenesis, ribosomal protein shuttling increases. An increased shuttling of ribosomal proteins enhances their interaction with Mdm2, which leads to the stabilization of p53. The elevated p53 levels subsequently upregulate the senescence marker p21, resulting in abnormal cellular ageing.¹⁰⁷

(5) Diamond–Blackfan Anemia (DBA)

Figure 3.

Schematic illustration showing nucleolar expansion in progeria syndrome, where an abnormal accumulation of progerin protein causes heterochromatin remodeling, resulting in increased production of ribosomal proteins and an increase in nucleolar size.

Diamond-Blackfan Anemia (DBA) is a rare genetic disorder that majorly affects bone marrow. DBA is majorly characterized by a reduced number or production of red blood cells (RBCs). RBCs are produced by bone marrow, and DBA mainly affects bone marrow, leading to a significant reduction in the levels of RBCs.¹⁰⁸ Phenotype starts at an early stage of a child, starting from 2 years onwards. Individuals with DBA exhibit weakness, development delay, fatigue, pale skin, and heart problems. Around 25% of patients with DBA die before the age of 50. Mutation in the ribosomal assembly proteins of the 40s and 60s leads to DBA. To date, 11 ribosomal genes including RPL5, RPL11, RPL35A, RPS10, RPS17, RPS19, RPS24, and RPS26 have been identified.^109,110 Mutation in any of these may lead to DBA. Around 25% of the DBA is caused by a mutation in RPS19, which is found in the ribosomal small unit. Mutation in these ribosomal genes leads to defects in the processing of pre-ribosomal RNS (rRNAs). Defects associated with ribosomal processing ultimately result in nucleolar stress. At the molecular level in different DBA model systems, increased levels of p53 have been observed. Interestingly, a reduction in the levels of p53 leads to reverse the phenotypes that are associated with increased levels of ribosomal proteins, indicating an important role of p53 in the pathophysiology of DBA. Similar to other nucleolar dysfunction-associated diseases in DBA, also at molecular levels, increased activity has been observed. Because of the mutation in the ribosomal proteins ribosomal assembly compromises that lead to production of liberated ribosomal proteins that interact with MDM2 leading to activation of p53 molecule.^111–113

(6) Cartilage hair hypoplasia

Cartilage hair hypoplasia (CHH) is a rare genetic disorder mainly characterized by short stature, short limbs, light color fragile hair, and other skeletal defects, including short fingers and toes. Some of the individuals with CHH also exhibited gastrointestinal defects. CHH is caused by a mutation in a gene known as the RMRP gene.¹¹⁴ RMRP is a long, non-coding RNA that plays a very important function in rRNA processing and ribosome biogenesis. Mutation in RMRP causes aberrant ribosomal biogenesis, resulting in nucleolar dysfunction or stress. Nucleolar stress caused by a mutation in RMRP causes activation of p53, suggesting that similar to other diseases in this also, phenotypes may be mediated through the p53 pathway. To understand the disease in more detail, RMRP knockout mice were generated; however, the homozygous knockout mice of RMRP were not viable, suggesting its important role during embryonic development^115–117

(7) Cockayne Syndrome

The Cockayne syndrome is a rare genetic disorder majorly characterized by slow growth, neurological defects, sensitivity to sunlight, thinning of the skin, bone abnormalities and dental defects. Mutations in genes ERCC6 or ERCC8, also known as Cockayne syndrome A protein (CSA), lead to Cockayne syndrome. These genes are majorly involved in DNA damage repair; mutation in these DNA damage repair genes leads to the accumulation of DNA-damaged cells, eventually leading to cell death.^116,118 DNA damage may occur through chemical exposure or UV light exposure. Based on the severity of the disease, it has been categorized into three types. In type I, symptoms start appearing during childhood, and death occurs during early adolescence. Type II is the most severe form of the disease, where symptoms start from birth itself, and they die before completing 10 years. Type III is the mildest form in which symptoms start appearing in adult life. CSA protein has been known to be involved in ribosome biogenesis and serves as a rate-limiting step in ribosomal biogenesis by regulating transcription by RNA polymerase. Mutation in ERCC6 causes impaired ribosomal biogenesis, which causes nucleolar stress. Around 90 % of Cockayne syndrome is caused by a mutation in the CSA genes. Previous studies have exhibited that CSA co-localizes with RNA pol-I inside the nucleolus, suggesting its direct connection to rRNA formation. CSA-deficient cells showed impaired RNA Pol-I activity, which leads to impairment in rRNA transcription. Defects in rRNA transcription also cause mitochondrial dysfunction. Mitochondrial dysfunction is strongly associated with ROS production. Increased ROS production further enhances DNA damage within the cells, which activates the p53 gene that further activates either the apoptotic pathway or the senescing pathway.^119,120

Conclusions and future directions

The nucleolus is the region inside the nucleus where transcription and processing of rRNA occurs. The rDNA genes encoding the 18S, 5.8S, and 28S RNA species of the ribosome are transcribed by RNA polymerase inside the nucleolus. Eventually, the nucleolus plays a very important role in the assembly and biogenesis of ribosomes. By regulating ribosome assembly and biogenesis, the nucleolus emerges as one of the key players in protein synthesis. Therefore, a mutation in any of the nucleolar genes or ribosomal genes causes nucleolar dysfunction, resulting in nucleolar stress. Most of the genetic diseases associated with nucleolar dysfunction overlap their phenotype. One of the most common phenotypes is accelerated ageing of the cells. During accelerated ageing, nucleolar expansion happens where nucleolar activity abnormally increases, leading to an increase in abnormal protein synthesis. Werner Syndrome, Dyskeratosis congenita, Treacher Collins, Progeria Syndrome, Diamond-Blackfan Anemia, Cartilage hair hypoplasia, and Cockayne Syndrome, these seven genetic disorders have been found to be associated with nucleolar dysfunction. The genes that have been identified in this disease are essential for ribosome maintenance, assembly and biogenesis. At the molecular levels in most of these genetic diseases, p53 levels were found to be increased. Nucleolar stress results in the activation of many different signaling pathways, such as MDM2-p53, HIF1-alpha, and Nf kappa b, but the most important and common signaling has emerged as an MDM2-p53. In this pathway MDM2 which is an E3 ubiquitin enzyme responsible to degrade p53 under normal circumstances. However, in the disease condition or during nucleolar stress, ribosomal proteins interact with MDM2, which inhibits MDM2-p53 interaction, leading to the accumulation of p53 inside the cell upon nucleolar stress (Figure 2). Since p53 accumulation has been observed in these genetic disorders suggesting p53 as therapeutic target to treat nucleolar dysfunction-associated disease. However, p53 plays an important role in DNA damage repair; therefore, inhibiting p53 directly may cause different problems. Inhibiting the upstream or downstream effector molecules of p53 may have therapeutic potential, but still, a lot of unanswered questions exist in the nucleolar dysfunction and associated disease areas. The molecular genetics approach to the loss of functions of different molecules may reveal some new therapeutic targets to treat nucleolar dysfunction-associated genetic disorders (Table 1).

Table 1.

List of genetic disorders associated with nucleolar dysfunction.

S.No.	Disease name	Genes	Disease phenotypes	Nucleolar defects	References
1	Werner syndrome	WRN	Atherosclerosis, osteoporosis, cataracts, hair loss, and a decrease in subcutaneous fat	The WRN gene is involved in maintaining genome integrity. Mutations cause defects in ribosome biogenesis, leading to nucleolar dysfunction	^86–90
2	Dyskeratosis congenita	DKC1, TERC, TERT, TINF2, NOLA2, NOLA3	Abnormal nails, reticular skin pigmentation, oral leukoplakia	It is caused by mutations in the genes involved in telomere maintenance and ribosome biogenesis. Mutation in any of these genes causes nucleolar stress	^96,97
3	Treacher Collins syndrome	TCOF1	Neck-specific developmental abnormalities are hallmarks of TCS, a severe congenital condition of craniofacial development	Haploinsufficiency of TCOF1 results in inadequate ribosome biogenesis. This deficiency causes nucleolar stress to activate p53	^104,105
4	Progeria syndrome	LMNA	Retarded growth, large skull, thin skin, low body weight, and premature death	Nucleolar size significantly increases, resulting in overproduction production of ribosomes and ribosomal proteins	^3,106,107
5	Diamond-Blackfan anemia	RPL5, RPL11, RPL35A, RPS10, RPS17, RPS19, RPS24, RPS26	Primarily affects bone marrow. Phenotypes include weakness, developmental delay, fatigue, pale skin, and heart problems	Defects in the processing of rRNAs cause nucleolar stress. Increased levels of p53 have also been observed in DBA.	^109–113
6	Cartilage hair hypoplasia	RMRP	Short stature, short limbs, light-colored fragile hair, and other skeletal defects	Mutation in RMRP causes aberrant ribosome biogenesis, resulting in nucleolar dysfunction or stress	^114–117
7	Cockayne syndrome	ERCC6, ERCC8	Slow growth, neurological defects, sensitivity to sunlight, thinning of the skin, bone abnormalities, and dental defects	ERCC (CSA) colocalizes with RNA Pol I inside the nucleolus. Mutation in ERCC causes impaired ribosome biogenesis, resulting in nucleolar stress	^{116,118–120}

Footnotes

Acknowledgments

The authors would like to thank the faculty and staff of Teerthanker Mahaveer Medical College & Research Centre for their support and cooperation during the course of this work.

ORCID iDs

Pothu Ushakiran

Vinod Kumar Singh

Author contributions

All authors contributed equally to the conception, literature review, analysis, and writing of the manuscript. All authors have read and approved the final version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.*

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