Nucleolar targeting in an early-branching eukaryote suggests a general physicochemical mechanism for ribosome protein sorting

The eukaryotic cell targets proteins to the organelles in which they function, both membrane-bound (like the nucleus) and non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals, and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in an early-branching eukaryote, Trypanosoma brucei, to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and protein physical properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins in comparison to mitochondrial ribosome proteins followed the same pattern - pointing to adaptation of physicochemical properties to assist segregation.

Nucleolar Proteins and Non-Coding RNAs: Roles in Renal Cancer

Renal cell cancer is the most frequent kidney malignancy. Most RCC cases are classified as clear cell renal cell carcinoma (ccRCC), characterized by high aggressiveness and poor prognosis for patients. ccRCC aggressiveness is defined by classification systems based on changes in morphology of nucleoli, the membraneless substructures of nuclei. The latter act as the sites of ribosome biogenesis as well as the hubs that trap and immobilize proteins, preventing their action in other cellular compartments. Thereby, nucleoli control cellular functioning and homeostasis. Nucleoli are also the sites of activity of multiple noncoding RNAs, including snoRNAs, IGS RNA, and miRNAs. Recent years have brought several remarkable discoveries regarding the role of nucleolar non-coding RNAs, in particular snoRNAs, in ccRCC. The expression of snoRNAs is largely dysregulated in ccRCC tumors. snoRNAs, such as SNHG1, SNHG4 and SNHG12, act as miRNA sponges, leading to aberrant expression of oncogenes and tumor suppressors, and directly contributing to ccRCC development and progression. snoRNAs can also act without affecting miRNA functioning, by altering the expression of key oncogenic proteins such as HIF1A. snoRNAs are also potentially useful biomarkers of ccRCC progression. Here, we comprehensively discuss the role of nucleolar proteins and non-coding RNAs in ccRCC.

Phase Separation of Intrinsically Disordered Nucleolar Proteins Relate to Localization and Function

The process of phase separation allows for the establishment and formation of subcompartmentalized structures, thus enabling cells to perform simultaneous processes with precise organization and low energy requirements. Chemical modifications of proteins, RNA, and lipids alter the molecular environment facilitating enzymatic reactions at higher concentrations in particular regions of the cell. In this review, we discuss the nucleolus as an example of the establishment, dynamics, and maintenance of a membraneless organelle with a high level of organization.

Proteome Analysis of Condensed Barley Mitotic Chromosomes

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

The intrinsically disorderly story of Ki-67

Ki-67 is one of the most famous marker proteins used by histologists to identify proliferating cells. Indeed, over 30 000 articles referring to Ki-67 are listed on PubMed. Here, we review some of the current literature regarding the protein. Despite its clinical importance, our knowledge of the molecular biology and biochemistry of Ki-67 is far from complete, and its exact molecular function(s) remain enigmatic. Furthermore, reports describing Ki-67 function are often contradictory, and it has only recently become clear that this proliferation marker is itself dispensable for cell proliferation. We discuss the unusual organization of the protein and its mRNA and how they relate to various models for its function. In particular, we focus on ways in which the intrinsically disordered structure of Ki-67 might aid in the assembly of the still-mysterious mitotic chromosome periphery compartment by controlling liquid–liquid phase separation of nucleolar proteins and RNAs.

Localization of METTL16 at the Nuclear Periphery and the Nucleolus Is Cell Cycle-Specific and METTL16 Interacts with Several Nucleolar Proteins

METTL16 methyltransferase is responsible for the methylation of N6-adenosine (m6A) in several RNAs. In mouse cells, we showed that the nuclear distribution of METTL16 is cell cycle-specific. In the G1/S phases, METTL16 accumulates to the nucleolus, while in the G2 phase, the level of METTL16 increases in the nucleoplasm. In metaphase and anaphase, there is a very low pool of the METTL16 protein, but in telophase, residual METTL16 appears to be associated with the newly formed nuclear lamina. In A-type lamin-depleted cells, we observed a reduction of METTL16 when compared with the wild-type counterpart. However, METTL16 does not interact with A-type and B-type lamins, but interacts with Lamin B Receptor (LBR) and Lap2α. Additionally, Lap2α depletion caused METTL16 downregulation in the nuclear pool. Furthermore, METTL16 interacted with DDB2, a key protein of the nucleotide excision repair (NER), and also with nucleolar proteins, including TCOF, NOLC1, and UBF1/2, but not fibrillarin. From this view, the METTL16 protein may also regulate the transcription of ribosomal genes because we observed that the high level of m6A in 18S rRNA appeared in cells with upregulated METTL16.

Manipulation of Cellular Processes via Nucleolus Hijaking in the Course of Viral Infection in Mammals

Due to their exceptional simplicity of organization, viruses rely on the resources, molecular mechanisms, macromolecular complexes, regulatory pathways, and functional compartments of the host cell for an effective infection process. The nucleolus plays an important role in the process of interaction between the virus and the infected cell. The interactions of viral proteins and nucleic acids with the nucleolus during the infection process are universal phenomena and have been described for almost all taxonomic groups. During infection, proteins of the nucleolus in association with viral components can be directly used for the processes of replication and transcription of viral nucleic acids and the assembly and transport of viral particles. In the course of a viral infection, the usurpation of the nucleolus functions occurs and the usurpation is accompanied by profound changes in ribosome biogenesis. Recent studies have demonstrated that the nucleolus is a multifunctional and dynamic compartment. In addition to the biogenesis of ribosomes, it is involved in regulating the cell cycle and apoptosis, responding to cellular stress, repairing DNA, and transcribing RNA polymerase II-dependent genes. A viral infection can be accompanied by targeted transport of viral proteins to the nucleolus, massive release of resident proteins of the nucleolus into the nucleoplasm and cytoplasm, the movement of non-nucleolar proteins into the nucleolar compartment, and the temporary localization of viral nucleic acids in the nucleolus. The interaction of viral and nucleolar proteins interferes with canonical and non-canonical functions of the nucleolus and results in a change in the physiology of the host cell: cell cycle arrest, intensification or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and the modification of signaling cascades involved in the stress response. The nucleolus is, therefore, an important target during viral infection. In this review, we discuss the functional impact of viral proteins and nucleic acid interaction with the nucleolus during infection.

­­ Histone H2B.V demarcates strategic regions in the Trypanosoma cruzi genome, associates with a bromodomain factor and affects parasite differentiation and host cell invasion

Histone variants play a crucial role in chromatin structure organization and gene expression. Trypanosomatids have an unusual H2B variant (H2B.V) that is known to dimerize with the variant H2A.Z generating unstable nucleosomes. Previously, we found that H2B.V protein is enriched in nonreplicative life forms of Trypanosoma cruzi , suggesting that this variant may contribute to the differences in chromatin structure and global transcription rates observed among parasite life forms. Here, we performed the first genome-wide profiling of histone localization in T. cruzi using replicative and nonreplicative life forms, and we found that H2B.V was preferentially located at the edges of divergent switch regions, which encompass putative transcriptional start regions; at some tDNA loci; and between the conserved and disrupted genome compartments, mainly at trans-sialidase, mucin and MASP genes. Remarkably, the chromatin of nonreplicative forms was depleted of H2B.V-enriched peaks in comparison to replicative forms. Interactome assays indicated that H2B.V associated specifically with H2A.Z, bromodomain factor 2, nucleolar proteins and a histone chaperone, among others. Parasites expressing reduced H2B.V levels were associated with higher rates of parasite differentiation and mammalian cell infectivity. Taken together, H2B.V demarcates critical genomic regions and associates with regulatory chromatin proteins, suggesting a scenario wherein local chromatin structures associated with parasite differentiation and invasion are regulated during the parasite life cycle.

The GAR/RGG motif defines a family of nuclear alarmins

The nucleus is the target of autoantibodies in many diseases, which suggests intrinsic nuclear adjuvants that confer its high autoimmunogenicity. Nucleolin (NCL) is one abundant nucleolar autoantigen in systemic lupus erythematosus (SLE) patients and, in lupus-prone mice, it elicits autoantibodies early. With purified NCL, we observed that it was a potent alarmin that activated monocytes, macrophages and dendritic cells and it was a ligand for TLR2 and TLR4. NCL released by necrotic cells also exhibited alarmin activity. The NCL alarmin activity resides in its glycine/arginine-rich (GAR/RGG) motif and can be displayed by synthetic GAR/RGG peptides. Two more GAR/RGG-containing nucleolar proteins, fibrillarin (FBRL) and GAR1, were also confirmed to be novel alarmins. Therefore, the GAR/RGG alarmin motif predicts a family of nucleolar alarmins. The apparent prevalence of nucleolar alarmins suggests their positive contribution to tissue homeostasis by inducing self-limiting tissue inflammation with autoimmunity only occurring when surveillance is broken down.

The uniqueBrucellaeffectors NyxA and NyxB target SENP3 to modulate the subcellular localisation of nucleolar proteins

The cell nucleus is a primary target for intracellular bacterial pathogens to counteract immune responses and hijack host signalling pathways to cause disease. The mechanisms controlling nuclear protein localisation in the context of stress responses induced upon bacterial infection are still poorly understood. Here we show that theBrucella abortuseffectors NyxA and NyxB interfere with the host sentrin specific protease 3 (SENP3), which is essential for intracellular replication. Translocated Nyx effectors directly interact with SENP3viaa defined acidic patch identified from the crystal structure of NyxB, preventing its nucleolar localisation at the late stages of the infection. By sequestering SENP3, the Nyx effectors induce the cytoplasmic accumulation of the nucleolar AAA-ATPase NVL, the large subunit ribosomal protein L5 (RPL5) and the ribophagy receptor NUFIP1 in Nyx-enriched structures in the vicinity of replicating bacteria. This shuttling of ribosomal biogenesis-associated nucleolar proteins is negatively regulated by SENP3 and dependent on the autophagy-initiation protein Beclin1, indicative of a ribophagy-derived process induced duringBrucellainfection. Our results highlight a new nucleomodulatory function by two uniqueBrucellaeffectors, and reveal that SENP3 is a critical regulator of the subcellular localisation of multiple nucleolar proteins duringBrucellainfection, promoting intracellular replication.