The nucleolar detention pathway

Abstract

Molecular dynamics ensures that proteins and other factors reach their site of action in a timely and efficient manner. This is essential to the formation of molecular complexes, as they require an ever-changing framework of specific interactions to facilitate a model of self-assembly. Therefore, the absence or reduced availability of any key component would significantly impair complex formation and disrupt all downstream molecular networks. Recently, we identified a regulatory mechanism that modulates protein mobility through the inducible expression of a novel family of long noncoding RNA. In response to diverse environmental stimuli, the nucleolar detention pathway (NoDP) captures and immobilizes essential cellular factors within the nucleolus away from their effector molecules. The vast array of putative NoDP targets, including DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the delta catalytic subunit of DNA polymerase (POLD1), suggests that this may be a common and significant regulatory mechanism. Here, we discuss the implications of this new posttranslational strategy for regulating molecular networks.

The field of molecular dynamics was born nearly 40 years ago through the study of lateral mobility within a two-dimensional membrane. Early work focused on the analysis of cell surface particles and utilized fluorescent dyes to monitor mobility during recovery after photobleaching.^1-5 Advances in time-lapse imaging technology and the cloning of green fluorescent protein⁶ has allowed scientists to pass through the barrier of the cell membrane, with minimal invasiveness and map the dynamic properties of intracellular particles. Historically, most have assumed that some level of mobility was necessary for molecules to carry out their cellular role, i.e., DNA polymerase must traverse along the genome to facilitate DNA replication (Fig. 1A). However, the question of how proteins and RNA are present in the right place at the right time is not well understood.

Figure 1. Regulation of molecular networks by the nucleolar detention pathway. (A) Under normal growth conditions cellular proteins are highly mobile and capable of executing essential cellular functions such as: ubiquitination (VHL), proteasomal degradation (SUG1), DNA replication (POLD1) and methylation (DNMT1); (B) Activation of the nucleolar detention pathway immobilizes proteins in the nucleolus away from their downstream effectors inhibiting basic cellular functions; (C) Capture and immobilization of NoDS-containing proteins in the nucleolus is mediated by inducible noncoding RNAs that originate from stimulus-specific loci within the ribosomal intergenic spacer.

In both the cytoplasm^7-9 and nucleus,^10-12 biologically active molecules diffuse throughout their cellular compartments in a random, rapid and energy-independent manner.^11,13-16 Proper function of these factors requires the formation of complexes with other protein, RNA and/ or DNA molecules. This is believed to be accomplished through a stop-and-go scanning mechanism, whereby highly mobile particles randomly associate and dissociate from other molecules until transient, high-affinity and appropriate interactions can be found.^13,14 Therefore, it appears that the highly chaotic and dynamic environment within the cell is, ironically, indispensible to generating order and maintaining proper cellular function.

Protein Dynamics as a Site of Posttranslational Regulation

This necessity for functional mobility presents the cell with an interesting opportunity to provide another layer of posttranslational control. To date the focus of posttranslational regulation has been on protein modifications through the addition of chemical/peptide groups or alterations in conformation and stability.¹⁷ Currently, hundreds of phosphatases, kinases, proteases and other modifying enzymes have been identified to fine-tune molecular networks by shifting the affinity of proteins toward one binding partner or another. However, many of these alterations are unable to affect global changes on multiple molecular networks in response to significant environmental stimuli. Altering protein mobility could provide a more systemic, rapid and reversible approach to regulating vast cellular pathways.

The study of regulated protein dynamics has been limited to a handful of molecules. Analysis of the lamin B receptor and the yeast protein Septin has shown that the same molecule can possess radically divergent kinetic properties, depending on its cellular localization¹⁸ or the stage of the cell cycle.¹⁹ Nucleostemin, a regulator of cancer/stem cell proliferation, further demonstrated that mobility can be affected by the GTP binding state of a molecule.²⁰ The most dramatic display of altered protein dynamics was observed for the E3 ubiquitin ligases VHL and MDM2.²¹ Under normal physiological conditions, these highly dynamic molecules are diffused throughout the cytoplasm or nucleus, allowing them to locate their downstream effectors and target them for proteasomal degradation^22,23 (Fig. 1A). However, in response to diverse stimuli, such as acidosis, heat shock and transcriptional stress, a novel class of inducible long noncoding RNA expressed from distinct loci within the ribosomal intergenic spacer (IGS RNA) has been shown to capture and immobilize these molecules within the nucleolus,^21,24 away from their targets, rendering them functionally inert¹⁴
(Fig. 1B and C).

Subcellular Targeting vs. Subcellular Detention

Numerous factors have been shown to affect the subcellular distribution of proteins.^7,25-31 Those studying these phenomena have used ambiguous terms such as targeting, recruitment and sequestration, to denote changes in the subcellular localization of molecules in response to environmental and cellular stimuli. While on the surface, the nucleolar detention pathway (NoDP) appears to emulate these other forms of subcellular redistribution, the term nucleolar “detention” has been specifically chosen to convey two fundamental distinctions unique to this form of localization.

First, live cell photobleaching analysis has demonstrated that proteins detained by the NoDP are both localized and statically immobilized within the nucleolus.^21,24 In contrast, the more vague terms: targeting, recruitment and sequestration, generally overlook the concept of mobility and primarily focus on the assessment of steady-state localization by immunofluorescence microscopy of fixed cells. While the prevalent historical belief was that “targeted/recruited/sequestered” molecules were not dynamic and remained associated with their specific subcellular domains, recent studies have shown that most subcellular compartments are fluid structures composed of proteins that are rapidly entering and exiting the region.^7,11,32 Second, the purpose of nucleolar detention appears to differ from other forms of subcellular trafficking. In many cases, molecules are targeted to a particular region in order to perform a specific cellular function. Conversely, nucleolar detention functions by removing important factors from their active sites, thereby disrupting molecular networks through the temporary imprisonment of key cellular factors within the nucleolus (Fig. 1).

Nucleolar Detention Signal as a Molecular Marker

The identification of proteins targeted to the nucleolus has historically been problematic, as most localization signals (NoLS) generally contain a seemingly random series of charged arginine and lysine residues.³³ In contrast, the nucleolar detention signal (NoDS) is characterized by a position-independent consensus sequence, consisting of at least one arginine motif (RR^I/_L) and a minimum of two hydrophobic triplets Lh^L/_v (where h represents a hydrophobic residue).³⁴ While discreet, this motif is strongly predictive of NoDP activity. To date, localization and mobility studies have confirmed that 18 of 18 molecules tested are validated targets of nucleolar detention.^21,24,34-36 In addition, analysis of the literature has found several putative NoDS-containing proteins that undergo stimuli-induced “sequestration” within the nucleolus, DAXX,³⁷ SENP5,³⁸ PML,³⁹ TERT⁴⁰ and TIP5,⁴¹ though their mobility has yet to be reported. Bioinformatic analysis using this motif has yielded numerous additional NoDP candidates, suggesting that the number of putative targets will increase substantially.

In conclusion, the potentially staggering array of NoDP targets hints at the systemic nature of RNA-mediated regulation of protein dynamics. These molecules have diverse functions in ubiquitination, proteasomal degradation, protein folding, DNA replication and methylation (Fig.≈1), indicating that the NoDP may control all aspects of cellular life.^21,24,34,35 With the emergence of molecular dynamics, we recommend that photobleaching experiments become standard practice when studying the relocalization of molecules, especially within the nucleolus. Further examination of the nucleolar detentiome should highlight the significance of this novel form of posttranslational regulation and reveal other molecular networks under the control of the NoDP.