Cellular function : Nucleolar malfunction can lead to disease – including cancer

The health of cells is maintained, in part, by two types of movement of their nucleoli, a team of scientists has found.

This dual motion within surrounding fluid, it reports, adds to our understanding of what contributes to healthy cellular function and points to how its disruption could affect human health.

“Nucleolar malfunction can lead to disease, including cancer,” explains Alexandra Zidovska, an assistant professor in New York University’s Department of Physics and the senior author of the study, which appears in the journal eLife.

“Thus, understanding the processes responsible for the maintenance of nucleolar shape and motion might help in the creation of new diagnostics and therapies for certain human afflictions.”

Recent discoveries have shown that some cellular compartments don’t have membranes, which were previously seen as necessary to hold a cell together.

Researchers have since sought to understand the forces that maintain the integrity of these building blocks of life absent these membranes.

What has been observed is the nature of this behavior.

Specifically, these compartments act as liquid droplets made of a material that does not mix with the fluid around them – similar to oil and water.

This process, known as liquid-liquid phase separation, has now been established as one of the key cellular organizing principles.

In their study, the researchers focused on the best known example of such cellular liquid droplet: the nucleolus, which resides inside the cell nucleus and is vital to cell’s protein synthesis.

“While the liquid-like nature of the nucleolus has been studied before, its relationship with the surrounding liquid is not known,” explains Zidovska, who co-authored the study with Christina Caragine, an NYU doctoral student, and Shannon Haley, an undergraduate in NYU’s College of Arts and Science at the time of the work and now a doctoral student at the University of California at Berkeley.

“This relationship is particularly intriguing considering the surrounding liquid – the nucleoplasm – contains the entire human genome.”

Yet, unclear is how the two fluids interact with each other.

To better understand this dynamic, the scientists examined the motion and fusion of human nucleoli in live human cells, while monitoring their shape, size, and smoothness of their surface.

The method for studying the fusion of the nucleolar droplets was created by the team in 2018 and reported in the journal Physical Review Letters.

Their latest study showed two types of nucleolar pair movements or “dances”: an unexpected correlated motion prior to their fusion and separate independent motion.

Moreover, they found that the smoothness of the nucleolar interface is susceptible to both changes in gene expression and the packing state of the genome, which surrounds the nucleoli.

“Nucleolus, the biggest droplet found inside the cell nucleus, serves a very important role in human aging, stress response, and general protein synthesis while existing in this special state,” observes Zidovska.

“Because nucleoli are surrounded by fluid that contains our genome, their movement stirs genes around them.

Consequently, because the genome in the surrounding fluid and nucleoli exist in a sensitive balance, a change in one can influence the other. Disrupting this state can potentially lead to disease.”

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The Multifunctional Nucleolus

The nucleolus is a highly dynamic, multifunctional, subnuclear organelle [3., 4., 7.]. Its most established role is as the hub of ribosome biogenesis, the process by which rRNA is transcribed, cleaved, and assembled into ribosomes (Figure 1).

This process is the most energy consuming in the cell and as such, is tightly linked to metabolic and proliferative activity.

The starting point, and arguably the rate-limiting step, is transcription of ribosomal DNA (rDNA) by the RNA polymerase I (PolI) preinitiation complex (PIC).

This complex includes upstream binding factor (UBF), which acts as derepressor and coactivator; SL1 proteins, which confer promotor specificity; and TIF-IA (gene name RRN3), which is essential as it tethers PolI to the rDNA promoter [8].

The output of rDNA transcription is the 47S preribosomal RNA transcript, which is cotranscriptionally matured through nucleolytic processing and assembly with ribosomal proteins and cofactors, to eventually generate the 40S and 60S ribosomal subunits [9].

In addition to its traditional role in ribosome biogenesis, the nucleolus plays a critical role in processes such as signal recognition particle assembly [10], pre-tRNA maturation [11], telomerase assembly [12], ribonucleoprotein biogenesis [13], and even organization of the epigenome [14., 15.].

Another key role for nucleoli is in the coordination of the cellular stress response (Figure 1) [3., 16.].

If cellular homeostasis is disrupted, for example, by nutrient deprivation, exposure to cytotoxic agents, viral infection, or oncogene inactivation, PolI-driven transcription is rapidly downregulated and a cascade of signalling events is triggered that influences cell physiology.

This process is broadly termed nucleolar stress and can take many forms, dependent on cell context and the nature of the insult [14., 17.]. Outcomes associated with nucleolar stress include differentiation, cell cycle arrest, autophagy, DNA repair, senescence, and apoptosis [4., 16., 18., 19.].

The paradigm of nucleolar stress response is inhibition of rDNA transcription leading to stabilization of p53 [20., 21.].

While this is undoubtedly an important mechanism by which the organelle regulates cellular homeostasis, there are multiple lines of evidence indicating nucleoli can control apoptosis, senescence, and autophagy in a p53-independent manner [19., 22., 23., 24.].

New research has revealed activation of the NF-κB pathway as an alternative key mechanism by which nucleolar stress regulates cell growth and death.

Nucleolar Stress and Activation of the NF-κB Pathway

NF-κB is a family of highly conserved, inducible transcription factors that play a critical role not only in innate and adaptive immunity, but also in stress response and the maintenance of cellular homeostasis [2., 25.].

The family comprise five members, namely, RelA (p65), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2). All members have a Rel homology domain which allows dimerization, translocation to the nucleus, and DNA binding, while only RelA, RelB, and C-Rel have the ability to drive transcription.

The most abundant form of NF-κB, and the primary mediator of NF-κB-dependent stress response, is the RelA:P50 heterodimer. In resting cells, this complex is held in the cytoplasm by the inhibitor of NF-κB (IκB) protein, IκBα [26., 27.].

When the cell is exposed to a wide array of stimuli, for example, inflammatory cytokines, bacterial pathogens, cytotoxic agents, DNA damaging agents, nutrient deprivation, hypoxia, and physical insult, IκBα is phosphorylated and targeted for degradation by the 26S proteasome.

This unmasks the nuclear localization signal on RelA, allowing NF-κB dimers to translocate to the nucleus and influence expression of over 150 target genes [28]. These genes control a broad range of physiological processes including proliferation, differentiation, senescence, cell cycle progression, and apoptosis.A number of upstream pathways have been described that lead to degradation of IκB. The most established of these is the canonical pathway.

This is induced by classic NF-κB stimuli [such as tumour necrosis factor alpha (TNFα)] and is characterized by rapid phosphorylation of IκBα by the inhibitor of κB kinase (IKK) complex [27]. By contrast, stress stimuli generally induce IκB phosphorylation/degradation with a delayed and slow kinetic. Specific mechanisms have been proposed for this delayed response [29., 30., 31., 32., 33.].

However, how multiple heterogeneous stresses converge on the NF-κB pathway has been far from clear. Intuitively, given the parallels between stresses that disrupt ribosome biogenesis and those that activate NF-κB (Table 1), nucleolar disruption could provide a unifying mechanism. Indeed, a number of nucleolar proteins are known to regulate NF-κB signalling. Furthermore, an atypical form of nucleolar stress has recently been shown to lie upstream of NF-κB pathway activation.

 Nucleolar Proteins Regulate NF-κB Signalling

The nucleolar proteome contains over 4500 proteins that shuttle dynamically between this and other cellular compartments, depending on cell context [34]. Several studies have shown the importance of such nucleolar proteins in the regulation of NF-κB signalling, especially in response to stress.

For example, casein kinase type 2 (CK2), which is bound to TIF-IA in the PolI complex [35], has previously been shown to phosphorylate IκBα in response to UV-C, potentiating NF-κB pathway activation [30., 36.].

Similarly, phosphorylation of eIF2α in response to endoplasmic reticulum stress can both inhibit PolI activity [37] and activate NF-κB [31., 32.]. The nucleolar protein p14^ARF, which sequesters MDM2 in the nucleolus to regulate p53 stability [38], interacts with RelA and inhibits NF-κB-driven transcription [39]. NIK (NF-κB-inducing kinase), which acts upstream of the IKK complex to stimulate NF-κB signalling [40], is a nucleolar shuttling protein [41], as is NF-κB repressing factor (NKRF), which modulates rRNA processing in response to heat stress and represses NF-κB-driven transcription [42., 43.].

Finally, ribosomal proteins themselves, when in ribosome-free form, can regulate NF-κB signalling. The ribosomal protein L3 prevents the degradation of IκB upon 5-fluorouracil treatment, thus repressing NF-κB activity [44., 45.], while S3 promotes activity by interacting with NF-κB complexes in the nucleus [46]. Hence, there are a number of signalling pathways by which stress-mediated nucleolar disruption could alter NF-κB nuclear translocation and transcriptional activity.

 Specific Nucleolar Disruption and Activation of the NF-κB Pathway

The first direct evidence that disruption of nucleolar homeostasis may activate NF-κB signalling came from studies showing that depletion of the multifunctional nucleolar protein, nucleophosmin (NPM, B23), induces degradation of IκB and nuclear translocation of RelA in colon cancer cells [47].

Depletion of the small nucleolar RNA host gene 15 (SNHG15) has also been shown to influence NF-κB signalling in a renal cell carcinoma model [48]. More recently, small interfering (si)RNA depletion of the PolI complex components UBF, TIF-IA, or RPA194 (a component of PolI) was shown to induce degradation of IκBα, S536 phosphorylation of RelA (a marker for activation), nuclear translocation of RelA, increased NF-κB transcriptional activity, and increased transcription of NF-κB target genes in multiple cell types [1].

Interestingly, this effect was not mimicked by actinomycin D, CX5461, or BMH-21 which block PolI transcription initiation (CX5461 and BMH-21) and elongation (Actinomycin D) [49., 50.]. Hence, unlike p53 nucleolar stress response, the link between nucleolar disruption and NF-κB activation is independent of rDNA transcription [21].

 TIF-IA–NF-κB Nucleolar Stress Response

Recent studies, aimed at further exploration of nucleoli–NF-κB crosstalk, uncovered a novel pathway by which nucleolar function is altered by stress and revealed this atypical nucleolar stress pathway lies upstream of NF-κB signalling [1]. This new pathway is centred around the PIC component TIF-IA.TIF-IA is the master regulator of PolI-driven transcription [35., 51.].

Not only is it essential for anchoring PolI to the rDNA promotor, it is also the component of the PIC that transduces environmental signals to the PolI transcriptional machinery. Upon exposure of cells to exogenous stresses, TIF-IA is targeted by a complex network of kinases and phosphatases including mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and protein phosphatase 2A (PP2A), which activate or inactivate the protein to fine-tune the transcriptional output [51., 52., 53., 54.].

The consequences of TIF-IA inactivation are context dependent but in general, its loss leads to cell cycle arrest and apoptosis, confirming the importance of this protein as a critical regulator of cellular homeostasis [55., 56., 57.].

Multiple stress stimuli of NF-kB, including aspirin, UV-C, and the second messenger ceramide, not only alter the phosphorylation status of TIF-IA, but also induce degradation of the protein [1]. This effect is not observed in response to TNFα or the DNA damaging agent camptothecin, indicating specificity. The mechanism underlying stress-mediated TIF-IA degradation is complex as it involves both proteasomal and lysosomal pathways. It is independent of MDM2, the E3 ligase reported to be responsible for basal TIF-IA turnover [58], suggesting it is distinct. Indeed, stress-mediated TIF-IA degradation is dependent on dephosphorylation of TIF-IA at serine 44 (S44) and the PolI complex–associated factors UBF and p14ARF (Figure 2, Key Figure).

Cyclin-dependent kinase 4 (CDK4) is a key cell cycle protein that is inhibited in response to many stress stimuli [59]. It is also known to phosphorylate UBF to regulate rDNA transcription [60]. It was found that small-molecule inhibitors of CDK4 induce TIF-IA degradation by an identical mechanism to that utilized by stress agents, that is, dependent on UBF, P14ARF, and dephosphorylation of TIF-IA at S44 [1]. Based on these data, it was concluded that stress-mediated CDK4 inhibition lies upstream of TIF-IA degradation (Figure 2).

On exploration of the downstream consequences of TIF-IA degradation, it was noted that this precedes degradation of IκBα and nuclear translocation of RelA/NF-κB, suggesting a potential link. Indeed, blocking degradation of TIF-IA, using specific siRNAs and dominant negative UBF and TIF-IA mutants, blocked the effects of specific stresses and CDK4 inhibitors on the NF-κB pathway (Figure 2) [1].

These data revealed a novel TIF-IA–NF-κB nucleolar stress axis.This novel nucleolar stress axis has now been observed in multiple mammalian cell types and in human colorectal tumours treated ex vivo with the chemopreventive agent aspirin, suggesting broad and in vivo relevance (see later) [1].

Although the signalling networks that link TIF-IA degradation to NF-κB pathway activation are not yet clear, it has been proposed that release of NF-κB regulatory proteins from the nucleolus to the nucleoplasm/cytoplasm upon TIF-IA degradation is responsible. Further research in this area will help us fully understand how both nucleoli and NF-κB regulate cellular homeostasis under normal and stress conditions.

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This research was supported by grants from the National Institutes of Health (R00-GM104152) and the National Science Foundation (CAREER PHY-1554880, CMMI-1762506).

Christina M Caragine, Shannon C Haley, Alexandra Zidovska. Nucleolar dynamics and interactions with nucleoplasm in living cells. eLife, 2019; 8 DOI: 10.7554/eLife.47533