Rethinking Nucleolar Evolution: New Insights into Structure and Function

The Nucleolus Aspects are Essential for its Role in Cellular Processes

The role of nucleolus in ribosome synthesis is well- established, whereas its major impartment in cellular processes remains less clear. This gap needs about deep insights on nucleolar structure and physiology to explore its influence on cellular physiology and in this aspect study of nucleolus evolution is crucial to defined complexity of ribosomal biogenesis and cellular physiology across diverse organisms (Figure 1). The bacteria and archaea ever not possess nucleolus, however, rely upon analogous nucleolus-like structure essentially for the accurate function in their life cycle. In present, it must be clarified about evolutionary adaptation during the life evolution which exhibiting selection pressures and alteration for cellular efficiency and so knowledge about nucleolar processes not only valuable to appreciate cellular functions but also significantly insights on nucleolar dysfunction which is linked with several human diseases as cancer and neurodegenerative disorders [9]. The disease can progressed through nucleolar function through unbalanced ribosome synthesis and alteration in cellular homeostasis. Thus deep insights about nucleolus physiology consists potential to uncover novel diagnostic and therapeutic strategies become possible through hope and inspiration in modern cellular and molecular biology.

There recent progress in imaging, molecular techniques and proteomic approaches have revealed previous unexplored vision and perspective approach into dynamics and functions of nucleolus through high-resolution polar microscopy, genome editing and proteomic analysis of nucleolar components [10]. These approaches have certainly improved nucleolus display in cellular biology. The complexity and mystery of the nucleolus can provide more inspiration to further research in the field of cellular and molecular biology.

Structural Complexity of the Nucleolus: Morphological and Functional Insights

There are key substructures has emerged as central to nucleolar function through transmission electron microscope (TEM). The fibrillar centre (FC) is critical area for ribosomal DNA (rDNA) storage. The transcription of nascent rDNA initially drives at the junction of FC and covering dense fibrillar component (DFC) with the help of RNA polymerase I complexes for pre-45S rRNA synthesis. The DFC exhibits role about processing of pre-45S rRNA into the mature 18S, 5.8S and 28S rRNA segments as key building blocks of eukaryotic ribosomes.

The DFC is also site for adaptive changes as methylation and pseudo-uridylation to organizing the rRNAs for their convergent integration into ribosomal domains. This DFC also activates the assembly of imported ribosomal proteins from the cytoplasm with the processed rRNAs to sustain the complete maturation of ribosomal subunits, where harmonized progression from rRNA processing to ribosome assembly occurs through interaction with granular component (GC) [11, 12].

The granular component (GC), which covers the DFC, is needful to complete the ribosome biogenesis, where maturation and combination of pre-ribosomal particles with nucleophosmin (B23) exhibiting key roles in this event. The size of the GC is directly related to the cell metabolism reflecting need to ribosome synthesis about cellular growth and cell division [13]. The regular integration between the nucleolar regions ascertain the normal production and maturation of functional ribosomes, which are essential for cellular homeostasis.

The recent researches identified nucleolar ‘vacuoles’ (NoVs) as membrane-less structures primarily observed in plants and some animals like Caenorhabditis elegans consists few granules and fibrils and association with FCs suggests a significant role in nucleolar dynamics [14]. Also, more recently identified nucleolar rim (NR) as distinct component signified by its special and more discrete proteomic composition is perhaps tether the nucleolus to chromatin and potentially impacting processes such as cell cycle regulation and ribosome biogenesis.

The recent studies aids in the complexity is physicochemical barcodes within the nucleolus that create unique micro-environments with varying features, which could uncover about additional nucleolar adaptations for diverse cellular functions [15]. The continuing research about emerging aspects about nucleolus structure can reveal vital role of each component in maintaining cellular function and supporting essential biological processes.

Communication and Interaction between Nucleolus and Nuclear Regions

The nucleolus not solely involved in ribosomal genesis, but also imparts an important role in the genome organization within the nucleus. This interacts with chromatin through nucleolar-associated domains (NADs) characterizes as domain of low transcriptional performance due to repressive transcription due to excessive histone modification consist causative H3K9me2 markers. This organization of repressive chromatin regions prevail such nucleolus, which easily divisible into euchromatic (active) and heterochromatic (inactive) regions [16]. The nucleolus is often observed as a centre for inactive chromatin and in such condition responding gene expression and preserving genomic fidelity.

The nucleolus operates as a central node that modulates localized pattern and activities of other nuclear domains as targeted depletion of specific genes led to nucleolar organization like localization of nuclear Cajal bodies, which are engaged in the maturation and modification of snRNAs and snoRNAs. These Cajal bodies are also needful for ribosome synthesis, telomere stability, transcriptional control, chromatin stress response and RNA breakdown [17] .

Nucleolar proteins regularly cycle between the nucleolus and the nucleoplasm, particularly in the cellular stress conditions and in case of such event nucleoplasmin (NPMI) translocated from the nucleolus to the nucleoplasm to reduce cellular stress involving the activation of the p53 signalling pathway as a critical stress response regulator. The damage in nucleolar structure in addition to impairing ribosome biogenesis but also cause abnormal nuclear architecture, defective signaling, and compromised cellular function [6].

The Nucleolar Proteins and their Functions

The diverse array of proteins in the nucleolus is crucial for revealing its functions and dynamics. The nucleolus contains both ribosomal proteins and enzymes involved in ribosome biogenesis, as well as a wide range of non-ribosomal proteins that contribute to processes like DNA repair, RNA processing, chromatin remodelling, transcription regulation and cell death [11, 18, 19, 20]. Comparative studies have shown that the composition and organization of the nucleolus can vary among species. For example, yeast nucleoli lack certain proteins found in higher Eukaryotes, reflecting evolutionary divergence in nucleolar function.

Both nucleophosmin (NPM1) and nucleolin play central roles in ribosome biogenesis, but their functions extend beyond this process. NPM1 regulates cell death pathways in response to cellular stress and DNA damage, specifically during apoptosis, where it is involved in both intrinsic and extrinsic apoptotic pathways. Mutations in NPM1, such as those found in acute myeloidleukaemia (AML), lead to its mislocalization, disrupting its ability to regulate apoptosis. Additionally, NPM1 acts as a chaperone of proteins involved in chromatin remodeling and DNA repair mechanisms and interacts with tumour suppressor factors like ARF [21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33].

Nucleolin is essential for cell growth and proliferation, regulating gene expression through interaction with nucleic acids involved in transcription and translation. Interestingly, nucleolin also facilitates viral replication by interacting with viral proteins, highlighting its role in viral pathogenesis [22]. Fibrillarin (FBL), a key player in rRNA methylation, is involved in the 2′-O-methylation of numerous pre-rRNA sites, a process crucial for ribosome biogenesis. As a core component of small nucleolar ribonucleoprotein (snoRNP) complexes, fibrillarin is vital for rRNA maturation and initiation of transcription by RNA polymerase I. Fibrillarin can undergo liquid–liquid phase separation (LLPS), facilitating the formation of membrane-less organelles like nucleolus [23]. This protein also interacts with p53 and ARF, suggesting a potential role in cancer progression [24]. Importantly, NPM1, nucleolin and fibrillarin all contain intrinsically disordered regions, which may regulate their localization and function within the nucleolus.

Recent studies have shown the presence of telomerase components in the nucleolus, suggesting that part of telomerase biosynthesis may occur there. This discovery has implications for cellular ageing and genomic instability, suggesting they influence telomere maintenance [20]. Moreover, some nucleolar proteins re-localize to the chromosomal periphery during mitosis, indicating a role in nucleolar disassembly and reassembly during cell division [19]. These proteins can be classified into two groups: those recruited early during pro-metaphase and those recruited later during post-metaphase. Understanding these recruitment dynamics helps clarify how the nucleolus disassembles and reassembles during the cell cycle.

Dynamic Nucleolar Organization Model as Liquid–Liquid Phase Separation

The nucleolus is a liquid-like substructure within cell assembled by LLPS pattern through modification of certain bio-molecules to arise distinct region which facilitates the formation of dense and dynamic liquid state not covered by membranes. The LLPS causes multivalent interactions within nucleus region involving intrinsically disordered regions (IDRs) of proteins and RNA jointly mediates the formation of dynamic membrane-less compartments [24, 25].

The nucleolus is organized into three functionally and physically distinct sub-compartments as the fibrillar centre (FC), dense fibrillar component (DFC) and granular component (GC)-whose spatial segregation is governed by LLPS (Figure 2). The FC is enriched in rDNA and gene transcription, while the surrounding DFC concentrates early RNA processing factors and small nucleolar RNPs that mediate initial transcription maturation. In parallel, the granular component (GC) forms through LLPS governed by nucleophosmin and related scaffold proteins which IDRs drive the assembly of liquid-like condensates that support the final steps of ribosome biogenesis [26].

The LLPS provides a versatile biophysical framework for regulating nuclear function by dynamically concentrating or excluding specific macromolecules in response to cellular cues beyond driving nucleolar structures [24]. Phase-separated condensates enhance reaction kinetics by increasing local concentrations of enzymes, substrates, and RNA, while simultaneously buffering stochastic fluctuations in gene expression. LLPS further enables rapid, reversible reorganization of nuclear architecture during stress, cell- cycle progression, and differentiation, thereby coordinating transcription, RNA processing, and ribo-nucleoprotein assembly in a context-dependent manner [25]. In addition, condensates can act as selective molecular filters, modulating the diffusion and residency time of regulatory factors to fine- tune signaling outputs and dysregulation of these material properties shifts condensates toward aberrant, less dynamic states, linking altered phase behavior to impaired genome maintenance, defective RNA metabolism, and the emergence of pathological protein aggregation observed in cancer and neurodegenerative disorders [27] (Table 1).

Stress granules represent prototypical bio-molecular condensates formed through liquid–liquid phase separation (LLPS) that transiently sequester un-translated mRNAs and RNA-binding proteins to preserve cellular homeostasis during environmental stress. The persistent stress or disease-associated mutations can shift these assemblies from dynamic, liquid-like states toward more rigid, gel- or solid-like phases, thereby impairing granule clearance and normal RNA metabolism. Such aberrant phase transitions promote the accumulation of insoluble protein aggregates and are increasingly implicated in the pathogenesis of neurodegenerative disorders, including amyotrophic lateral sclerosis and fronto-temporal dementia. Thus, LLPS not only enables adaptive stress responses but also constitutes a critical vulnerability point where altered condensate material properties can precipitate cellular dysfunction and disease [26].

The Evolutionary Origins of the Nucleolus

The nucleolus is the most prominent compartment of the eukaryotic nucleus and arises through self-organization around actively transcribed ribosomal DNA (rDNA) loci.

The distinctive architecture of nucleolus suggests that the nucleolus did not emerge as a static organelle but rather evolved as a dynamic, self-assembling ribonucleoprotein condensate, shaped by increasing cellular and genomic complexity in eukaryotes.

From an evolutionary perspective, the nucleolus likely originated from primitive ribosome biogenesis centers present in ancestral prokaryotic-like cells. In bacteria, rRNA transcription, processing, and ribosome assembly occur in spatially enriched regions termed nucleoid- associated ribosome assembly zones, although these lack stable compartmentalization [30]. Nevertheless, recent studies indicate that bacterial cells exhibit functional micro- domains enriched in ribosome biogenesis factors, suggesting that spatial organization of ribosomal production predates the emergence of the eukaryotic nucleus [34]. These early organizational tendencies may represent evolutionary precursors to the nucleolus, where clustering of rRNA synthesis and processing activities conferred kinetic and regulatory advantages (Figure 2).

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The transition from prokaryotic to eukaryotic cellular organization, marked by the appearance of the nuclear envelope and linear chromosomes, likely intensified selective pressure for spatial coordination of ribosome biogenesis. Expansion of genome size, increased gene regulatory complexity, and separation of transcription and translation created a need for specialized nuclear sub-domains that could efficiently integrate rRNA synthesis with processing and ribonucleoprotein assembly [35, 36]. The nucleolus, in this context, can be viewed as an emergent structure arising from the concentration of rDNA repeats and associated processing machinery within confined nuclear space.

Comparative genomics provides further insight into nucleolar evolution. Core components of ribosome biogenesis including RNA polymerase I subunits, small nucleolar RNAs (snoRNAs), ribosomal proteins, and processing enzymes are highly conserved across eukaryotes and share ancestry with bacterial ribosomal assembly factors [35]. However, eukaryotes possess an expanded repertoire of nucleolar proteins enriched in intrinsically disordered regions (IDRs) and low-complexity domains, which facilitate multivalent interactions and promote phase separation. This expansion suggests that the evolution of protein disorder was instrumental in enabling the formation of stable yet dynamic nucleolar compartments, supporting the hypothesis that biophysical self-assembly mechanisms were co-opted to generate higher-order nuclear organization.

The LLPS offers a parsimonious mechanism by which cellular systems can generate compartmentalization without membranes, relying instead on physicochemical properties of macromolecules. The emergence of LLPS-capable proteins may therefore have been a critical step in transforming dispersed ribosome biogenesis reactions into spatially coherent nucleolar condensates [26].

Phylogenetic analyses further indicate that structured nucleoli appear only in eukaryotes, whereas simpler ribosome assembly zones exist in archaea. Interestingly, some archaeal species possess nucleolus-like bodies enriched in rRNA processing factors, suggesting an intermediate organizational state [33]. These observations support a gradualist model in which increasing molecular crowding, genome expansion, and diversification of RNA- processing pathways favored the stabilization of ribosome biogenesis condensates into discrete nuclear bodies. Thus, the nucleolus likely evolved not as a novel invention but as an elaboration of pre-existing spatial clustering mechanisms that became progressively more refined.

The co-evolution of nucleolar structure and rDNA organization is also notable. Eukaryotic genomes typically contain hundreds of tandemly repeated rDNA units, which serve as nucleation platforms for nucleolar assembly. These repeats enable high transcriptional output while simultaneously providing a scaffold for recruitment of processing factors. Evolutionary amplification of rDNA arrays may therefore have reinforced nucleolar formation by enhancing local macromolecular concentration, creating positive feedback between gene organization and nuclear architecture [37]. In this sense, the nucleolus represents an example of how genome structure and cellular compartmentalization co-evolve to optimize essential biosynthetic processes.

Beyond ribosome production, the nucleolus has acquired additional regulatory roles during evolution, including involvement in cell-cycle control, stress sensing, and sequestration of regulatory proteins. These expanded functions likely emerged as secondary adaptations once nucleolar condensates became stable nuclear landmarks [38, 39]. The capacity of the nucleolus to act as a molecular reservoir or stress-responsive hub may reflect selective advantages conferred by pre-existing phase-separated architecture, which could be readily repurposed for regulatory integration. Such functional diversification underscores how biophysical properties can facilitate evolutionary innovation without requiring extensive structural redesign.

Importantly, the evolutionary persistence of the nucleolus across all extant eukaryotic lineages highlights its fundamental role in cellular fitness. Any perturbation of nucleolar integrity directly compromises ribosome biogenesis and protein synthesis, imposing strong selective constraints on its maintenance. At the same time, variability in nucleolar size, number, and organization among species indicates evolutionary plasticity, allowing adaptation to differing metabolic demands and developmental strategies. This balance between conservation and flexibility reflects the dual nature of the nucleolus as both an indispensable biosynthetic center and a tunable regulatory platform.

In summary, the nucleolus likely emerged through the evolutionary stabilization of ancestral ribosome biogenesis clusters, driven by genome expansion, increased regulatory complexity, and the appearance of proteins capable of multivalent, phase-separating interactions. Rather than being a static organelle defined by rigid boundaries, the nucleolus represents an emergent, self-organizing nuclear condensate whose architecture and functions have been progressively refined throughout eukaryotic evolution. This perspective not only clarifies the origins of nucleolar compartmentalization but also illustrates how fundamental biophysical principles can shape cellular organization over evolutionary timescales.