Genome organization in and around the nucleolus

The nucleolus is the largest compartment of the cell nucleus and is where ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. In addition to rRNA gene clusters that build the core of this subnuclear structure, nucleoli are associated with condensed chromatin. Although the higher order structures of rRNA genes and nucleolus-associated chromatin have been studied for decades, detailed molecular insights into the constituents and organization of the nucleolar genome are only beginning to emerge. Here, we summarize current views on the structural organization of nucleolar DNA and on the targeting and anchoring of chromatin domains to this subnuclear compartment.     

The nucleolus： reviewing oldies to have new understandings

The nucleolus is the most prominent compartment in the nucleus and known as the site for ribosome biogenesis ineucaryotes.In contrast,there is no such equivalent structure for ribosome synthesis in procaryotes.This raises twoconcerns that how does the nucleolus evolve and that whether the nucleolus remains playing a single role in ribosomebiogenesis along the evolution.Increasing data support new nucleolus functions,including signal recognition particleassembly,small RNA modification,telomerase maturation,cell-cycle and aging control,and cell stress sensor.Multiplefunctions of the nucleolus possibly result from the plurifunctionality of nucleolar proteins,such as nucleolin and Nopp 140.Proteomic analyses of human and Arabidopsis nucleolus lead a remarkable progress in understanding the evolution andnew functions of nucleoli.In this review,we present a brief history of nucleolus research and new concepts and unre-solved questions.Also,we introduce hepatitis D virus for studying the communication between the nucleolus and othersubnuclear compartments,and Caenorhabditis elegans for the role of nucleolus in the development and the epistaticcontrol of nucleologenesis.     

Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC‐MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus‐infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus‐infected cells.     

Optical control of protein delivery and partitioning in the nucleolus

The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline–arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.     

Changes in nucleolar morphology and proteins during infection with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear structure involved in ribosome subunit biogenesis, cell cycle control and mediating responses to cell stress, among other functions. While many different viruses target proteins to the nucleolus and recruit nucleolar proteins to facilitate virus replication, the effect of infection on the nucleolus in terms of morphology and protein content is unknown. Previously we have shown that the coronavirus nucleocapsid protein will localize to the nucleolus. In this study, using the avian infectious bronchitis coronavirus, we have shown that virus infection results in a number of changes to the nucleolus both in terms of gross morphology and protein content. Using confocal microscopy coupled with fluorescent labelled nucleolar marker proteins we observed changes in the morphology of the nucleolus including an enlarged fibrillar centre. We found that the tumour suppressor protein, p53, which localizes normally to the nucleus and nucleolus, was redistributed predominately to the cytoplasm.     

核仁和核仁蛋白

核仁是位于细胞核内的非膜结构。电子显微镜下的核仁从形态上可以分为三层结构包括纤维中心区(FC)、高密度纤维区(DFC)和颗粒区(GC)。核仁内的蛋白有核糖体蛋白和非核糖体蛋白两种。利用蛋白质组学方法已经鉴定了350多种核仁蛋白,其中包括80多种核糖体蛋白。核仁是核糖体合成的场所,核仁中的非核糖体蛋白对核糖体的生物合成起关键调控作用。核仁不仅是细胞内通讯和核糖体：RNA加工的中心,而且在细胞周期、细胞增殖和衰老中起重要调控作用;核仁也是tRNA、mRNA和其它类型小分子RNA加工的场所。因此核仁是一个多功能的细胞生命活动中心。     

RNA viruses: hijacking the dynamic nucleolus

The nucleolus is a dynamic subnuclear structure with roles in ribosome subunit biogenesis, mediation of cell-stress responses and regulation of cell growth. The proteome and structure of the nucleolus are constantly changing in response to metabolic conditions. RNA viruses interact with the nucleolus to usurp host-cell functions and recruit nucleolar proteins to facilitate virus replication. Investigating the interactions between RNA viruses and the nucleolus will facilitate the design of novel anti-viral therapies, such as recombinant vaccines and therapeutic molecular interventions, and also contribute to a more detailed understanding of the cell biology of the nucleolus.     

Structure and function of the nucleolus.

The activity of the ribosomal RNA genes generates a distinct subnuclear structure, the nucleolus, which is the site of ribosome biogenesis. The signals that target proteins and snoRNAs (small nucleolar RNAs) to the nucleolus, the nuclear import of ribosomal proteins, the export of the completed ribosomal subunits and the molecular organization of the nucleolus have been the subject of intense research during the past year. Evidence is accumulating that nucleoli functionally interact with coiled bodies and are also involved in the maturation of non-ribosomal RNA species.     

The multifunctional nucleolus

The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.     

Tryptophans 286 and 288 in the C-terminal region of protein B23.1 are important for its nucleolar localization

Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein B23, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon.     

Tryptophans 286 and 288 in the C-terminal Region of Protein B23.1 are Important for Its Nucleolar Localization

Nucleolar protein B23 can shuttle between the nucleolus and cytoplasm. However, the mechanism involved in the protein moving and staying in the nucleolus is not fully understood. To identify the nucleolar localization signal sequence of protein B23, we examined the subnuclear location of B23.1 mutant proteins fused with green fluorescent protein in HeLa cells. The results suggested that the two C-terminal tryptophan residues (Trp-286 and Trp-288) of protein B23.1 were important in this phenomenon.     

The Nucleolus and Viral Infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus p...     

Nucleolar targeting: the hub of the matter

The nucleolus is a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Such functions are frequently mediated by the sequestration or release of nucleolar proteins. Our understanding of protein targeting to the nucleolus is much less complete than our knowledge of membrane‐spanning translocation systems—such as those involved in nuclear targeting—and the experimental evidence reveals that few parallels exist with these better‐characterized systems. Here, we discuss the current understanding of nucleolar targeting, explore the types of sequence that control the localization of a protein to the nucleolus, and speculate that certain subsets of nucleolar proteins might act as hub proteins that are able to bind to multiple protein targets. In parallel to other subnuclear structures, such as PML bodies, the proteins that are involved in the formation and maintenance of the nucleolus are inexorably linked to nucleolar trafficking.     

Interaction of the coronavirus nucleoprotein with nucleolar antigens and the host cell

Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.     

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site.     

The nucleolus and viral infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.     

The HIV Tat protein affects processing of ribosomal RNA precursor

Background  

Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.