Integrated analysis of RNA-seq datasets reveals novel targets and regulators of COVID-19 severity.

During the COVID-19 pandemic, RNA-seq datasets were produced to investigate the virus-host relationship. However, much of these data remains underexplored. To improve the search for molecular targets and biomarkers, we performed an integrated analysis of multiple RNA-seq datasets, expanding the cohort and including patients from different countries, encompassing severe and mild COVID-19 patients. Our analysis revealed that severe COVID-19 patients exhibit overexpression of genes coding for proteins of extracellular exosomes, endomembrane system, and neutrophil granules (e.g., S100A9, LY96, and RAB1B), which may play an essential role in the cellular response to infection. Concurrently, these patients exhibit down-regulation of genes encoding components of the T cell receptor complex and nucleolus, including TP53, IL2RB, and NCL Finally, SPI1 may emerge as a central transcriptional factor associated with the up-regulated genes, whereas TP53, MYC, and MAX were associated with the down-regulated genes during COVID-19. This study identified targets and transcriptional factors, lighting on the molecular pathophysiology of syndrome coronavirus 2 infection.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Trypanosoma cruzi Fibrillarins: Two paralogous proteins with non-identical signals for nuclear transport.

Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.

The nucleolus of Giardia and its ribosomal biogenesis.

Giardia duodenalis is a protozoan intestinal parasite that causes a significant number of infections worldwide each year, particularly in low-income and developing countries. Despite the availability of treatments for this parasitic infection, treatment failures are alarmingly common. As a result, new therapeutic strategies are urgently needed to effectively combat this disease. On the other hand, within the eukaryotic nucleus, the nucleolus stands out as the most prominent structure. It plays a crucial role in coordinating ribosome biogenesis and is involved in vital processes such as maintaining genome stability, regulating cell cycle progression, controlling cell senescence, and responding to stress. Given its significance, the nucleolus presents itself as a valuable target for selectively inducing cell death in undesirable cells, making it a potential avenue for anti-Giardia treatments. Despite its potential importance, the Giardia nucleolus remains poorly studied and often overlooked. In light of this, the objective of this study is to provide a detailed molecular description of the structure and function of the Giardia nucleolus, with a primary focus on its involvement in ribosomal biogenesis. Likewise, it discusses the targeting of the Giardia nucleolus as a therapeutic strategy, its feasibility, and the challenges involved.

Caracterização do papel de Nop53 na montagem da subunidade ribossomal maior em Saccharomyces cerevisiae / Characterization of the role of Nop53 in the assembly of the large ribosomal subunit in Saccharomyces cerevisiae

Os ribossomos são complexos ribonucleoproteicos conservados formados por duas subunidades assimétricas (40S e 60S em eucariotos) responsáveis pela tradução da informação genética e catálise da síntese proteica. A montagem destes complexos em eucariotos é mais bem descrita em S. cerevisiae, constituindo um processo celular energeticamente dispendioso e com múltiplas etapas. Ela tem origem no nucléolo com a transcrição do pré-rRNA 35S e requer o recrutamento hierárquico e transiente de cerca de 200 fatores de montagem para garantir a formação correta dos centros funcionais aptos à tradução. Neste processo, que se estende no núcleo e citoplasma, 79 proteínas ribossomais associam-se gradativamente à medida que o prérRNA é dobrado, modificado e processado. O processamento do pré-rRNA 35S consiste na remoção progressiva de espaçadores internos (ITS1 e ITS2) e externos (5ETS e 3ETS), que separam e flanqueiam os rRNAs maduros componentes de ambas subunidades ribossomais. A clivagem do ITS1 separa as vias de maturação do pré-60S e do pré-40S. O ITS2, que, em associação a fatores de montagem, forma uma estrutura denominada ITS2-foot, é o último espaçador do pré-60S a ser removido. A composição do ITS2-foot permanece inalterada no nucléolo até a transição entre o estado E nucleolar e a formação da partícula Nog2 nuclear. Nesta etapa, a liberação do fator Erb1 permite o recrutamento do fator de montagem conservado e essencial Nop53. Na base do ITS2-foot, Nop53 recruta o exossomo via RNA helicase Mtr4 para a clivagem 3-5 exonucleolítica de parte do ITS2 levando à desmontagem do ITS2-foot. O fato de Nop53 atuar como ponte entre dois grandes complexos e apresentar uma estrutura flexível e estendida nos levou a aprofundar a caracterização de seu papel durante a maturação do pré60S. Neste trabalho, usando análise proteômica quantitativa label-free baseada em espectrometria de massas, caracterizou-se o interactoma de Nop53, e avaliou-se o impacto da depleção de Nop53 no interactoma da subunidade catalítica do exossomo Rrp6 e na composição de pré-ribossomos representativos de quase todas as etapas de maturação do pré-60S. Em paralelo, foram caracterizados mutantes truncados de Nop53 e avaliada por pull-down a interação de Nop53 com componentes do exossomo. Os resultados obtidos mostraram que Nop53 é capaz de interagir com o cofator do exossomo Mpp6, sugerindo pontos adicionais de interação durante o recrutamento do exossomo ao pré-60S. A análise do interactoma de Rrp6 mostrou uma associação precoce do exossomo aos intermediários pré-ribossomais nucleolares mais iniciais, anteriores aos previamente descritos. Mudanças na composição dos intermediários pré-60S revelaram que a depleção de Nop53 afeta a transição entre o estado E e a partícula Nog2, afetando eventos tardios de maturação como o recrutamento de Yvh1. Comparando-se o efeito da depleção de Nop53 com o de mutantes nop53 desprovidos da região de recrutamento do exossomo, obtivemos evidências bioquímicas do papel estrutural de Nop53 na base do ITS2- foot. Em conjunto, estas observações, à luz de estruturas de intermediários pré-ribossomais recentemente descritas, nos permitiram concluir que o recrutamento de Nop53 ao pré-60S contribui para a estabilização de eventos de remodelamento do rRNA que antecedem a formação da partícula Nog2

Ribosomes are conserved ribonucleoprotein complexes formed by two asymmetric subunits (the 40S and the 60S in eukaryotes) responsible for translating the genetic information and catalyzing protein synthesis. The assembly of these complexes in eukaryotes is best described in S. cerevisiae. It is an energetically demanding, multi-step cellular process, that starts in the nucleolus with the transcription of the 35S pre-rRNA. It requires the hierarchical and transient recruitment of about 200 assembly factors to ensure the correct formation of the functional centers suitable for translation. In this process, which extends into the nucleus and cytoplasm, 79 ribosomal proteins gradually associate as the pre-rRNA is folded, modified, and processed. The 35S pre-rRNA processing happens with the progressive removal of internal (ITS1 and ITS2) and external (5'ETS and 3'ETS) transcribed spacers, which separate and flank the mature rRNA components of both ribosomal subunits. The cleavage at the ITS1 separates the pre-60S and pre40S maturation pathways. The ITS2, which in association with assembly factors constitutes a structure called ITS2-foot, is the last pre-60S spacer to be removed. The composition of the ITS2- foot remains unchanged in the nucleolus until the transition between the nucleolar state E and the nuclear Nog2 particle. At this stage, the release of Erb1 allows the recruitment of the conserved and essential assembly factor Nop53. At the base of the ITS2-foot, Nop53 recruits the exosome via the RNA helicase Mtr4 for the ITS2 3'-5' exonucleolytic cleavage leading to the ITS2-foot disassembly. The fact that Nop53 acts as a bridge between these two large complexes and exhibits a flexible and extended structure led us to further characterize its role in the pre-60S maturation. In this work, using mass spectrometry-based label-free quantitative proteomics, we characterized the interactome of Nop53, as well as the impact of the depletion of Nop53 on the interactome of the exosome catalytic subunit Rrp6 and on the composition of pre-ribosomes representative of almost all pre-60S maturation stages. In parallel, we characterized nop53 truncated mutants and evaluated the interaction of Nop53 with exosome components by pulldown assays. The results showed that Nop53 can interact with the exosome cofactor Mpp6, suggesting the contribution of additional points of interaction during the exosome recruitment to the pre-60S. The analysis of the Rrp6 interactome revealed an early association of the exosome with pre-ribosomal intermediates at very early nucleolar stages, before those previously described. Changes in the composition of pre-60S intermediates revealed that Nop53 depletion affects the transition between the state E and the Nog2 particle, affecting late pre-60S maturation events, such as the Yvh1 recruitment. Comparing the effect of Nop53 depletion with that of nop53 mutants lacking the exosome interacting region, we obtained biochemical evidence of the structural role of Nop53 at the base of the ITS2-foot. Altogether, and in light of recently described structures of pre-ribosomal intermediates, these observations allowed us to conclude that the recruitment of Nop53 to the pre-60S contributes to the stabilization of rRNA remodeling events that precede the formation of the Nog2 particle

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.

The ribosome assembly factor Nop53 has a structural role in the formation of nuclear pre-60S intermediates, affecting late maturation events.

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome.

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Dramatic nucleolar dispersion in the salivary gland of Schwenkfeldina sp. (Diptera: Sciaridae).

Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar bodies contain ribosomal DNA and RNA, are transcriptionally active and may appear free in the nucleoplasm or associated with specific chromosome regions in salivary gland nuclei. This report deals with an extreme case of nucleolar fragmentation/dispersion detected in the salivary gland of Schwenkfeldina sp. Such a phenomenon in this species was found to be restricted to cell types undergoing polyteny and seems to be differentially controlled according to the cell type. Furthermore, transcriptional activity was detected in virtually all the micronucleolar bodies generated in the salivary gland. The relative proportion of the rDNA in polytene and diploid tissues showed that rDNA under-replication did not occur in polytene nuclei suggesting that the nucleolar and concomitant rDNA dispersion in Schwenkfeldina sp. may reflect a previously hypothesised process in order to counterbalance the rDNA loss due to the under-replication. The chromosomal distribution of epigenetic markers for the heterochromatin agreed with early cytological observations in this species suggesting that heterochromatin is spread throughout the chromosome length of Schwenkfeldina sp. A comparison made with results from another sciarid species argues for a role played by the heterochromatin in the establishment of the rDNA topology in polytene nuclei of Sciaridae.

Expression of the tumor-expressed protein MageB2 enhances rRNA transcription.

An essential requirement for cells to sustain a high proliferating rate is to be paired with enhanced protein synthesis through the production of ribosomes. For this reason, part of the growth-factor signaling pathways, are devoted to activate ribosome biogenesis. Enhanced production of ribosomes is a hallmark in cancer cells, which is boosted by different mechanisms. Here we report that the nucleolar tumor-protein MageB2, whose expression is associated with cell proliferation, also participates in ribosome biogenesis. Studies carried out in both siRNA-mediated MageB2 silenced cells and CRISPR/CAS9-mediated MageB2 knockout (KO) cells showed that its expression is linked to rRNA transcription increase independently of the cell proliferation status. Mechanistically, MageB2 interacts with phospho-UBF, a protein which causes the recruitment of RNA Pol I pre-initiation complex required for rRNA transcription. In addition, cells expressing MageB2 displays enhanced phospho-UBF occupancy at the rDNA gene promoter. Proteomic studies performed in MageB2 KO cells revealed impairment in ribosomal protein (RPs) content. Functionally, enhancement in rRNA production in MageB2 expressing cells, was directly associated with an increased dynamic in protein synthesis. Altogether our results unveil a novel function for a tumor-expressed protein from the MAGE-I family. Findings reported here suggest that nucleolar MageB2 might play a role in enhancing ribosome biogenesis as part of its repertoire to support cancer cell proliferation.

Nucleolar disruption, activation of P53 and premature senescence in POLR3A-mutated Wiedemann-Rautenstrauch syndrome fibroblasts.

Recently, mutations in the RNA polymerase III subunit A (POLR3A) have been described as the cause of the neonatal progeria or Wiedemann-Rautenstrauch syndrome (WRS). POLR3A has important roles in transcription regulation of small RNAs, including tRNA, 5S rRNA, and 7SK rRNA. We aim to describe the cellular and molecular features of WRS fibroblasts. Cultures of primary fibroblasts from one WRS patient [monoallelic POLR3A variant c.3772_3773delCT (p.Leu1258Glyfs*12)] and one control patient were cultured in vitro. The mutation caused a decrease in the expression of wildtype POLR3A mRNA and POLR3A protein and a sharp increase in mutant protein expression. In addition, there was an increase in the nuclear localization of the mutant protein. These changes were associated with an increase in the number and area of nucleoli and to a high increase in the expression of pP53 and pH2AX. All these changes were associated with premature senescence. The present observations add to our understanding of the differences between Hutchinson-Gilford progeria syndrome and WRS and opens new alternatives to study cell senesce and human aging.

Nucleolar localization of the yeast RNA exosome subunit Rrp44 hints at early pre-rRNA processing as its main function.

The RNA exosome is a multisubunit protein complex involved in RNA surveillance of all classes of RNA, and is essential for pre-rRNA processing. The exosome is conserved throughout evolution, present in archaea and eukaryotes from yeast to humans, where it localizes to the nucleus and cytoplasm. The catalytically active subunit Rrp44/Dis3 of the exosome in budding yeast (Saccharomyces cerevisiae) is considered a protein present in these two subcellular compartments, and here we report that it not only localizes mainly to the nucleus, but is concentrated in the nucleolus, where the early pre-rRNA processing reactions take place. Moreover, we show by confocal microscopy analysis that the core exosome subunits Rrp41 and Rrp43 also localize largely to the nucleus and strongly accumulate in the nucleolus. These results shown here shed additional light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex, which seems to be primarily in early pre-rRNA processing and surveillance.

Reproductive Aspects of Chagas Disease Vectors: Evidence of Transcriptional Activity during the Nucleolar Persistence Phenomenon in the Spermatogenesis of Triatomines.

The subfamily Triatominae currently consists of 154 species, most of them being of great importance for public health because they are considered potential vectors of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease. In addition to their epidemiological importance, these insects are considered important biological models for cell studies because they have peculiar characteristics in their cells, for example, persistence of the nucleolus during spermatogenesis. This phenomenon is characterized by the presence of the nucleolus or nucleolar corpuscles during all phases of meiosis. To date, all knowledge is restricted to the study of the presence/absence of the nucleolus during the triatomine meiosis, so the present work aimed to analyze if this persistent nucleolar material has transcriptional activity. Analysis of the meiotic metaphases of Rhodnius montenegrensis and Panstrongylus megistus by using fluorochrome acridine orange made it possible to characterize the presence of RNA in the nucleolar material. Thus, it was demonstrated, for the first time, that the persistent nucleolar material during triatomine meiosis is transcriptionally active, supporting the hypothesis of the relationship between nucleolar persistence during meiosis of these insects and the formation of the chromoid body, an organelle responsible for the support of all transcriptional activities during spermiogenesis.

Nucleolar Structure and Function in Trypanosomatid Protozoa.

The nucleolus is the conspicuous nuclear body where ribosomal RNA genes are transcribed by RNA polymerase I, pre-ribosomal RNA is processed, and ribosomal subunits are assembled. Other important functions have been attributed to the nucleolus over the years. Here we review the current knowledge about the structure and function of the nucleolus in the trypanosomatid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania ssp., which represent one of the earliest branching lineages among the eukaryotes. These protozoan parasites present a single nucleolus that is preserved throughout the closed nuclear division, and that seems to lack fibrillar centers. Trypanosomatids possess a relatively low number of rRNA genes, which encode rRNA molecules that contain large expansion segments, including several that are trypanosomatid-specific. Notably, the large subunit rRNA (28S-type) is fragmented into two large and four small rRNA species. Hence, compared to other organisms, the rRNA primary transcript requires additional processing steps in trypanosomatids. Accordingly, this group of parasites contains the highest number ever reported of snoRNAs that participate in rRNA processing. The number of modified rRNA nucleotides in trypanosomatids is also higher than in other organisms. Regarding the structure and biogenesis of the ribosomes, recent cryo-electron microscopy analyses have revealed several trypanosomatid-specific features that are discussed here. Additional functions of the nucleolus in trypanosomatids are also reviewed.

The intracellular domain of ß-dystroglycan mediates the nucleolar stress response by suppressing UBF transcriptional activity.

ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.

Nucleolar alterations are reliable parameters to determine the cytogenotoxicity of environmental pollutants.

Pollution generated by deposition of industrial activity waste in the environment without due care can lead to serious environmental consequences. Bioassays in higher plants are means of understanding the cytogenotoxic effects of these substances. In the present work, Allium cepa L. was used as a model species to assess nucleolar changes induced by environmental pollutants. The substances used were Methyl Methane Sulfonate (MMS), cadmium (Cd), Spent Potliner (SPL) and the herbicide Atrazine. Water was used as a negative control. The silver-stained nucleolar organizer region (AgNOR) assay was used making it possible to evaluate how nucleolar parameters (number of nucleoli per nucleus and nucleoli area) behave when facing stress caused by such pollutants. The results obtained showed a variation in the observed parameters: an increase in the number of nucleoli in the treated cells and tendency to a reduction in nucleolar area, indicating that the tested pollutants may have impaired nucleolar activity. In addition, it was possible to establish a relationship between the behavior of the nucleolus with other changes as plantlet growth, cell proliferation, and DNA damage.

Nucleolar Expression and Chromosomal Associations in Robertsonian Spermatocytes of Mus musculus domesticus.

We studied and compared the nucleolar expression or nucleoli from specific bivalents in spermatocytes of the standard Mus musculus domesticus 2n=40, of Robertsonian (Rb) homozygotes 2n = 24 and heterozygotes 2n = 32. We analyzed 200 nuclear microspreads of each specific nucleolar chromosome and spermatocyte karyotype, using FISH to identify specific nucleolar bivalents, immunofluorescence for both fibrillarin of the nucleolus and the synaptonemal complex of the bivalents, and DAPI for heterochromatin. There was nucleolar expression in all the chromosomal conditions studied. By specific nucleolar bivalent, the quantitative relative nucleolar expression was higher in the bivalent 12 than in its derivatives, lower in the bivalent 15 than in its derivatives and higher in the bivalent 16 than its Rb derivatives. In the interactions between non-homologous chromosomal domains, the nucleolar bivalents were preferentially associated through pericentromeric heterochromatin with other bivalents of similar morphology and sometimes with other nucleolar bivalents. We suggest that the nucleolar expression in Rb nucleolar chromosomes is modified as a consequence of different localization of ribosomal genes (NOR) in the Rb chromosomes, its proximity to heterochromatin and its associations with chromosomes of the same morphology.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity.

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

Nucleolar Division in the Promastigote Stage of Leishmania major Parasite: A Nop56 Point of View.

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56) during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56 in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical, to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out by an analogous mechanism.

Synaptic configuration of quadrivalents and their association with the XY bivalent in spermatocytes of Robertsonian heterozygotes of Mus domesticus.

BACKGROUND: The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains and consequently is prone to modification by chromosomal rearrangements. We have shown that nuclear architecture is modified in spermatocytes of Robertsonian (Rb) homozygotes of Mus domesticus. In this study we analyse the synaptic configuration of the quadrivalents formed in the meiotic prophase of spermatocytes of mice double heterozygotes for the dependent Rb chromosomes: Rbs 11.16 and 16.17. RESULTS: Electron microscope spreads of 60 pachytene spermatocytes from four animals of Mus domesticus 2n = 38 were studied and their respective quadrivalents analysed in detail. Normal synaptonemal complex was found between arms 16 of the Rb metacentric chromosomes, telocentrics 11 and 17 and homologous arms of the Rb metacentric chromosomes. About 43% of the quadrivalents formed a synaptonemal complex between the heterologous short arms of chromosomes 11 and 17. This synaptonemal complex is bound to the nuclear envelope through a fourth synapsed telomere, thus dragging the entire quadrivalent to the nuclear envelope. About 57% of quadrivalents showed unsynapsed single axes in the short arms of the telocentric chromosomes. About 90% of these unsynapsed quadrivalents also showed a telomere-to-telomere association between one of the single axes of the telocentric chromosome 11 or 17 and the X chromosome single axis, which was otherwise normally paired with the Y chromosome. Nucleolar material was associated with two bivalents and with the quadrivalent. CONCLUSIONS: The spermatocytes of heterozygotes for dependent Rb chromosomes formed a quadrivalent where four chromosomes are synapsed together and bound to the nuclear envelope through four telomeres. The nuclear configuration is determined by the fourth shortest telomere, which drags the centromere regions and heterochromatin of all the chromosomes towards the nuclear envelope, favouring the reiterated encounter and eventual rearrangement between the heterologous chromosomes. The unsynapsed regions of quadrivalents are frequently bound to the single axis of the X chromosome, possibly perturbing chromatin condensation and gene expression.