Nucleolar Research Articles

Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion.

Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.

Tiny nuance leads to large difference: Construction of fluorescent probes to visualize early and late apoptotic stages

Apoptosis is an exquisitely controlled complex process which plays key roles in maintaining cell homeostasis. Abnormal regulation of apoptosis was directly related to severe diseases including cancer, neurodegenerative diseases, and cardiovascular diseases. Herein, we proposed a chain-engineering strategy for constructing probes to distinguish the different stages of apoptosis. Two fluorescent probes (CPI-2 and CPI-3) with very similar structure were developed to detect the late and early apoptotic stages, respectively. CPI-2 with short chain could not enter live cells due to its poor membrane permeability, and thus exclusively illuminated the nucleolus of late apoptotic cells with damaged plasma membrane. CPI-3 was permeable to biomembranes and stained mitochondria in living cells, which immigrated into the nucleolus during early apoptotic procedure due to the decrease of mitochondrial membrane potential. Furthermore, CPI-2 and CPI-3 were successfully used to monitor the different stages of apoptosis induced by different concentrations of H2O2, paclitaxel, and rotenone. Particularly, we studied the heavy metals poisoning and detoxification of cysteine by CPI-2 and CPI-3. This work provides a viable strategy for constructing probes to detect the different stages of apoptosis, which is expected to promote the development of probes to monitor apoptosis processes.

Toxic effect of Callistemon viminalis essential oil on the ovary of engorged Rhipicephalus microplus female ticks (Acari: Ixodidae).

Rhipicephalus microplus is the main ectoparasite of cattle and its parasitism can reduce weight gain of hosts, in addition to causing anemia, increasing the risk of myiasis, and resulting in the transmission of Babesia bovis and Anaplasma marginale, among other pathogens. The use of synthetic chemicals plays an important role in controlling these ticks. However, its frequent and indiscriminate use has contributed to the selection of resistant strains, resulting in greater interest in the search for natural-origin products. The shrub Callistemon viminalis (Myrtaceae), also known as weeping bottlebrush, is known for its antibacterial, acaricidal, repellent, and antifungal activities, but there are no reports in the literature about its effects on the internal morphology of ticks. This study aimed to extract and characterize the essential oil obtained from the leaves of C. viminalis. Additionally, its effects on the ovary morphology of engorged R. microplus were assessed through histological, histochemical, and morphometric techniques. Exposure to C. viminalis caused dose-dependent morphological changes, such as cellular alterations in the epithelial layer lining the ovary lumen and the pedicel, irregularity of the chorion and oocyte shape, changes in protein and carbohydrate content, decrease in oocyte size, reduction in the size of the nucleus as well as cytoplasmic and nucleolar vacuolation. Thus, C. viminalis essential oil exhibited a toxic effect on the reproductive system of R. microplus, which may result in the reproductive impairment of this tick species.

Platelet-Rich Plasma in Regenerative Medicine: Possible Applications in Management of Burns and Post-Burn Scars: A Review.

Contribution of platelets in tissue regeneration and their possible application in regenerative medicine, which is primarily mediated via secretion of granular components following platelet activation, has been well established in the recent decades. Therefore, platelet rich plasma (PRP), as a portion of plasma with higher concentrations of platelets than the baseline level, is now an attractive therapeutic option in various medical fields mainly for tissue repair and regeneration following injuries. Burn injuries are devastating trauma with high rate of morbidities affecting several aspects of the patient's life. They require a long-time medical care and high costs. However, even following the best treatment procedures, post-burn scars are inevitable consequence of burn healing process. Therefore, development of new treatment modalities for both burn healing and prevention of post-burn scar establishment seems to be necessary. Regarding the well-known role of PRP in wound healing, here we aimed to provide a comprehensive insight in the possible application of PRP as an adjuvant therapy for the management of burn injuries and subsequent scars. In terms of the following keywords (individually or in combination), original/review articles were searched in PubMed, Scopus, and Google Scholar databases from 2009 to 2021: platelet rich plasma, PRP therapy, platelet biology, platelet function, burn healing, burn scar, scar formation, burn management, wound healing, regenerative medicine. All type of articles or book chapters in English language and relevant data were included in this review. This review initially focused on PRP, its mechanisms of action, preparation methods, and available sources. Then, pathophysiology of burns and subsequent scars were discussed. Finally, their current conventional therapeutic modalities and implication of PRP in their healing process were highlighted.

Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells.

Styrene dyes are useful imaging probes and fluorescent sensors due to their strong fluorogenic responses to environmental changes or binding macromolecules. Previously, indole-containing styrene dyes have been reported to selectively bind RNA in the nucleolus and cytoplasm. However, the application of these indole-based dyes in cell imaging is limited by their moderate fluorescence enhancement and quantum yields, as well as relatively high background associated with these green-emitting dyes. In this work, we have investigated the positional and electronic effects of the electron donor by generating regioisomeric and isosteric analogues of the indole ring. Select probes exhibited large Stokes shifts, enhanced molar extinction coefficients, and bathochromic shifts in their absorption and fluorescence wavelengths. In particular, the indolizine analogues displayed high membrane permeability, strong fluorogenic responses upon binding RNA, compatibility with fluorescence lifetime imaging microscopy (FLIM), low cytotoxicity, and excellent photostability. These indolizine dyes not only give rise to rapid, sensitive, and intense staining of nucleoli in live cells but can also resolve subnucleolar structures enabling highly detailed studies of nucleolar morphology. Furthermore, our dyes can partition into RNA coacervates and resolve the formation of multiphase complex coacervate droplets. These indolizine-containing styrene probes offer the highest fluorescence enhancement among the RNA-selective dyes reported in the literature; thus, these new dyes are excellent alternatives to the commercially available RNA dye, SYTO RNASelect, for visualizing RNA in live cells and in vitro.

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs.

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM). To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs. First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.

Identifying potential targets for lung cancer intervention by analyzing the crosstalk of cancer-associated fibroblasts and immune and metabolism microenvironment.

Cancer-associated fibroblasts (CAFs) have been reported to play a crucial role in the tumor microenvironment and progression. The data used in this study were obtained from the Cancer Genome Atlas and Gene Expression Omnibus databases, and all analyses were performed using R software. We first quantified the CAFs infiltration through single sample gene set enrichment analysis in the TCGA and combined GEO cohort (GSE30219, GSE37745, and GSE50081). Our result showed that patients with high levels of CAF infiltration were associated with worse clinical features and poor prognosis. Immune microenvironment analysis indicated that high CAF infiltration might result in increased infiltration of immune cells, including aDC, B cells, CD8+ T cells, cytotoxic cells, DC, eosinophils, iDC, macrophages, mast cells, neutrophils, NK CD56dim cells, NK cells, pDC, and T cells. Correlation analysis showed a significant positive correlation between CAFs and M2 macrophages, while a negative correlation was found between CAFs and glycerophospholipid metabolism. Kaplan-Meier survival curves indicated that glycerophospholipid metabolism was a protective factor against lung cancer. Biological enrichment analysis showed that pathways such as allograft rejection, epithelial-mesenchymal transition, KRAS signaling, TNF-α signaling, myogenesis, IL6/JAK/STAT3 signaling, IL2/STAT5 signaling were upregulated in the patients with high CAF infiltration. Moreover, patients with high CAF infiltration had a lower proportion of immunotherapy responders. Genome analysis showed that low CAFs infiltration was associated with high genome instability. We identified FGF5 and CELF3 as key genes involved in the interaction between CAFs, M2 macrophages, and glycerophospholipid metabolism, and further analyzed FGF5. In vitro experiments showed that FGF5 promoted the proliferation, invasion and migration of lung cancer cells and was primarily localized in the nucleoli fibrillar center. Our study provides novel insights into the roles of CAFs in lung cancer progression and the underlying crosstalk of tumor metabolism and immune microenvironment.

Electromagnetic Field Effect on Bone Marrow Stem Cells Differentiation and Nucleoli AgNOR

Purpose: Numerous studies have described the effect of Electromagnetic Fields (EMFs) in the promotion of Bone Marrow Stem Cell (BMSC) differentiation. We aimed to investigate the influence of frequency (10 and 100 Hz) and different pulse shapes (sine, rectangular, and triangular) of EMF on rats' BMSCs.
 Materials and Methods: The BMSCs in 6 groups were exposed to EMF for 1 h/ 7 days. The BMSCs viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The cresyl violet labeled the Nissl bodies, and the silver nitrate staining was done to evaluate the BMSCs nucleoli AgNORs.
 Results: The MTT test verified that EMF and pulse shape did not affect cell viability. In Nissl bodies staining most of the large neurons were related to the rectangular 10 Hz EMF group. The majority of the differentiated BMSCs were astrocytes, microglia, and oligodendrocyte in the triangular 100 Hz EMF group. Although the silver nitrate staining confirmed the effect of 10 Hz EMF, pulse shape alteration did not affect AgNOR parameters. In conclusion, we presented a low-magnetic flux density EMF (400 µT) to assess the responses of BMSCs nuclei.
 Conclusion: The findings showed that BMSCs differentiation was frequency-dependent. Further investigations are recommended for recognizing the function of EMF on BMSCs.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

Duodenal gastric-type neoplasm with uncertain malignant potential in a lipoma.

The pathologic diagnosis of duodenal tumors is a developing field; however, its overview remains unclear. We describe a rare case of a duodenal gastric-type neoplasm in a 50-year-old woman. She visited her primary care doctor with complaints of upper abdominal pain, tarry stools, and shortness of breath on exertion. She was admitted owing to a stalked polyp with erosion and hemorrhage in the descending part of the duodenum. Endoscopic mucosal resection (EMR) was performed on the polyp. Histologically, the resected polyp was a lipomatous lesion in the submucosal layer, composed of mature adipose tissues. Scattered irregular lobules of Brunner's gland-like structures with well-preserved construction but mildly enlarged nuclei and occasional conspicuous nucleoli of the constituent cells were observed. The resection margin was negative. EMR findings of the duodenal polyp showed a gastric epithelial tumor within a lipoma, a rare histological type that has not been reported previously. This tumor may be classified as a "neoplasm with uncertain malignant potential" in a lipoma, an intermediate category between adenoma and invasive adenocarcinoma. There is no consensus on treatment, and careful follow-up is recommended. This is the first report of a duodenal gastric-type neoplasm with uncertain malignant potential in a lipoma.

Ferruginous bodies exert a strong proinflammatory effect

ABSTRACT One of the main problems related to ferruginous-asbestos bodies (ABs) exposure is their potential pathogenetic role in asbestos-related diseases. The aim of this study was to examine whether purified ABs, might stimulate inflammatory cells. ABs were isolated by exploiting their magnetic properties, therefore avoiding the strong chemical treatment usually employed for this purpose. This latter treatment, which is based upon the digestion of organic matter with concentrated hypochlorite, may markedly modify the AB structure and consequently also their “in vivo” manifestations. ABs were found to induce secretion of human neutrophil granular component myeloperoxidase, as well as stimulate rat mast cell degranulation. Data demonstrated that by triggering secretory processes in inflammatory cells, purified ABs may play a role in the pathogenesis of asbestos-related diseases by continuing and enhancing the pro-inflammatory activity of the asbestos fibers.

Nucleoli and the nucleoli-centromere association are dynamic during normal development and in cancer.

Centromeres are known to cluster around nucleoli in Drosophila and mammalian cells, but the significance of the nucleoli-centromere interaction remains underexplored. To determine whether the interaction is dynamic under different physiological and pathological conditions, we examined nucleolar structure and centromeres at various differentiation stages using cell culture models and the results showed dynamic changes in nucleolar characteristics and nucleoli-centromere interactions through differentiation and in cancer cells. Embryonic stem cells usually have a single large nucleolus, which is clustered with a high percentage of centromeres. As cells differentiate into intermediate states, the nucleolar number increases and the centromere association decreases. In terminally differentiated cells, including myotubes, neurons, and keratinocytes, the number of nucleoli and their association with centromeres are at the lowest. Cancer cells demonstrate the pattern of nucleoli number and nucleoli-centromere association that is akin to proliferative cell types, suggesting that nucleolar reorganization and changes in nucleoli-centromere interactions may play a role in facilitating malignant transformation. This idea is supported in a case of pediatric rhabdomyosarcoma, in which induced differentiation reduces the nucleolar number and centromere association. These findings suggest active roles of nucleolar structure in centromere function and genome organization critical for cellular function in both normal development and cancer.

Evaluation of a Developed MRI-Guided Focused Ultrasound System in 7 T Small Animal MRI and Proof-of-Concept in a Prostate Cancer Xenograft Model to Improve Radiation Therapy.

Focused ultrasound (FUS) can be used to physiologically change or destroy tissue in a non-invasive way. A few commercial systems have clinical approval for the thermal ablation of solid tumors for the treatment of neurological diseases and palliative pain management of bone metastases. However, the thermal effects of FUS are known to lead to various biological effects, such as inhibition of repair of DNA damage, reduction in tumor hypoxia, and induction of apoptosis. Here, we studied radiosensitization as a combination therapy of FUS and RT in a xenograft mouse model using newly developed MRI-compatible FUS equipment. Xenograft tumor-bearing mice were produced by subcutaneous injection of the human prostate cancer cell line PC-3. Animals were treated with FUS in 7 T MRI at 4.8 W/cm2 to reach ~45 °C and held for 30 min. The temperature was controlled via fiber optics and proton resonance frequency shift (PRF) MR thermometry in parallel. In the combination group, animals were treated with FUS followed by X-ray at a single dose of 10 Gy. The effects of FUS and RT were assessed via hematoxylin-eosin (H&E) staining. Tumor proliferation was detected by the immunohistochemistry of Ki67 and apoptosis was measured by a TUNEL assay. At 40 days follow-up, the impact of RT on cancer cells was significantly improved by FUS as demonstrated by a reduction in cell nucleoli from 189 to 237 compared to RT alone. Inhibition of tumor growth by 4.6 times was observed in vivo in the FUS + RT group (85.3%) in contrast to the tumor volume of 393% in the untreated control. Our results demonstrated the feasibility of combined MRI-guided FUS and RT for the treatment of prostate cancer in a xenograft mouse model and may provide a chance for less invasive cancer therapy through radiosensitization.

Oligomerization and sequence patterning can tune multiphasic condensate miscibility.

Endogenous biological condensates, comprised of a multitude of proteins and RNAs, are organized into multiphasic structures. This multiphasic organization is generally understood to be critical for facilitating the proper biological function of these condensates. However, the biophysical principles driving multiphase formation are poorly understood. Here, we utilize in vivo experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biological condensates. We demonstrate that increasing the oligomerization state of an IDR results in enhanced immiscibility and multiphase formation. Strikingly, we found that oligomerization can asymmetrically tune the miscibility of IDRs, with the effect being more potent when applied to stronger homotypic IDRs. Finally, we utilized the nucleolus as a model multiphasic condensate to demonstrate how perturbing the oligomerization of nucleolar components in the granular component can change their localization to the nucleolar periphery. Our findings lead us to propose oligomerization as a flexible biophysical mechanism which cells can exploit to tune the multiphasic organization of biological condensates and thereby regulate their biological function.

Elucidating the role of nucleophosmin in protein quality control mechanisms inside the nucleolus.

Irreversible protein misfolding and aggregation is associated with many human diseases, including neurodegenerative diseases such as Huntington's and Parkinson's disease. Complex quality control mechanisms and response pathways serve to maintain protein homeostasis in cells. One stress response pathway involves the nucleolus, which accumulates misfolded nuclear proteins under proteotoxic stress conditions. The nucleolar protein, nucleophosmin (NPM1), located in the granular component (GC) region of the nucleolus, interacts with misfolded proteins, reducing their mobility and thus mitigating irreversible aggregation. NPM1 undergoes liquid-liquid phase separation (LLPS) with nucleolar proteins and RNA via homotypic and heterotypic interactions involving its intrinsically disordered region, a process we propose mediates the nucleolar stress response pathway. We hypothesize that misfolded proteins interact with NPM1 through the oligomerization domain and/or the intrinsically disordered region of NPM1 and form phase-separated dynamic assemblies that prevent irreversible aggregation. Under heat stress, certain “model” proteins unfold and migrate to the GC phase of the nucleolus and interact with NPM1. To investigate how the nucleolar partitioning of the model proteins changes in response to their misfolding, we performed fluorescence confocal microscopy on the NPM1-mCherry fusion protein and different model proteins fused with mEGFP in transiently transfected HEK293T cells. We imaged under normal conditions, under heat stress, and during a recovery period to investigate the reversibility of the model proteins’ association with NPM1 in the nucleolus. Next, we will perform in vitro phase separation assays to understand how model protein misfolding affects LLPS with NPM1 in a reconstituted GC phase. The proposed studies will elucidate the structural mechanisms that promote the association of misfolded proteins with NPM1. Understanding the mechanisms underlying nuclear protein stress responses may provide new directions for the development of therapeutics against neurodegenerative diseases in the future.

Phase separation underlies interactions of the SARS-CoV-2 nucleocapsid protein with nucleoli.

RNA viruses hijack host cell functions to promote viral replication. During infection, viral components enter membrane-bound and membrane-less cellular organelles, including the phase-separated nucleolus, where they perturb nucleolar proteins to aid virus replication. Recently, viruses have been shown to utilize the process of liquid-liquid phase separation (LLPS) to package their genome and promote viral RNA synthesis. The viral nucleocapsid (N) protein is critically involved in several aspects of virus replication. Importantly, the nucleolar phosphoprotein, Nucleophosmin (NPM1), with roles in ribosome biogenesis, was reported to interact with the N protein of several different RNA viruses and facilitate viral replication. However, the impact of this interaction and SARS CoV-2 N protein (NCoV-2) nucleolar localization on ribosome biogenesis and viral life cycle remains poorly understood. Our in vitro studies show that NPM1 undergoes heterotypic phase separation with NCoV-2, causing the formation of liquid-like droplets or condensates with restricted NPM1 mobility. Sequence analysis of NCoV-2 revealed enrichment of positively charged (Arg and Lys) amino acids, an important “sequence feature” that governs nuclear and nucleolar localization. Importantly, our cellular studies reveal the colocalization of NCoV-2 with NPM1 in the nucleoli of Vero E6 cells. These results suggest that there is a direct interaction between NPM1 and NCoV-2. Strikingly, NCoV-2 displaces NPM1 from reconstituted, granular component-like condensates and from nucleoli. Based on our collective findings, we propose that displacement of regulatory proteins (e.g., NPM1) from nucleoli by NCoV-2 through interactions of its Arg residues with ribosomal RNA (rRNA), may promote protein synthesis and the viral life cycle. These mechanistic insights will provide new avenues for the development of novel antiviral therapies for inhibition of nucleolar localization of NCoV-2.

Low Temperature Scanning Electron Microscopy (LTSEM) Findings on the Ultrastructure of Trebouxia lynnae (Trebouxiophyceae, Lichenized Microalgae)

The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucleolar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.

Abstract A012: NAT10 promotes metastasis through enhancer remodeling of cancer cells

Abstract Distant organ metastases account for the majority of the cancer-related death. However, the detailed mechanism underlying the metastatic potential of cancer cells is poorly understood. We have previously shown than NUP210-dependent nuclear pore-associated chromatin-bound protein complex is involved in cellular mechanotransduction and metastasis in breast cancer. Although NAT10, an RNA cytidine acetyl transferase has been shown to interact with this protein complex, specific function of NAT10 in metastasis is not well understood. Using allograft and genetically engineered highly metastatic PyMT mouse model, we found that the loss of NAT10 significantly decreases lung metastasis in mice. NAT10 is mainly localized to the nucleolus and nucleoplasmic compartment of the cancer cells. Current understanding suggests a role of NAT10 in acetylation of mRNAs and ribosomal RNAs. In contrast to its role as an RNA acetylase, we uncovered a new mechanism of NAT10 in cancer cells. Using co-immunoprecipitation followed by mass spectrometry and chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we found that NAT10 is interacting with super-enhancer associated protein complex. RNA-seq analysis revealed that the loss of NAT10 leads to dramatic transcriptional suppression of super-enhancer driven genes including MYC. Mechanistically, using mass spectrometry-based quantification of protein acetylation, we showed that the loss of NAT10 leads to decreased acetylation of enhancer-associated Histone H4 and H3 modification. NAT10 loss also leads to increased heterochromatin-forming histone methylation within the cell nuclei. Our study revealed a distinct role of NAT10 as an enhancer remodeler in metastatic cancer cells. Therefore, NAT10 could be a potential therapeutic target in preventing metastatic disease. Citation Format: Ruhul Amin, Ngoc-Han Ha, Jordan L. Meier, Kent W. Hunter. NAT10 promotes metastasis through enhancer remodeling of cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A012.

PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10 (NOL10), binds to polyglutamine tract sequences and is expressed in the nucleolus. Using dynamic imaging of high-speed atomic force microscopy, we show that PQBP5/NOL10 is an intrinsically disordered protein. Super-resolution microscopy and correlative light and electron microscopy method show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus, constituting the granule meshwork in the granular component area, which is distinct from other nucleolar substructures, such as the fibrillar center and dense fibrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins. Droplet and thermal shift assays show that the biophysical features of PQBP5/NOL10 remain stable under stress conditions, explaining the spatial role of this protein. PQBP5/NOL10 can be functionally depleted by sequestration with polyglutamine disease proteins in vitro and in vivo, leading to the pathological deformity or disappearance of the nucleolus. Taken together, these findings indicate that PQBP5/NOL10 is an essential protein needed to maintain the structure of the nucleolus.

Blood transcriptome of Rasa Aragonesa rams with different sexual behavior phenotype reveals CRYL1 and SORCS2 as genes associated with this trait.

Reproductive fitness of rams is seasonal, showing the highest libido during short days coinciding with the ovarian cyclicity resumption in the ewe. However, the remarkable variation in sexual behavior between rams impair farm efficiency and profitability. Intending to identify in vivo sexual behavior biomarkers that may aid farmers to select active rams, transcriptome profiling of blood was carried out by analyzing samples from 6 sexually active (A) and 6 nonactive (NA) Rasa Aragonesa rams using RNA-Seq technique. A total of 14,078 genes were expressed in blood but only four genes were differentially expressed (FDR < 0.10) in the A vs. NA rams comparison. The genes, acrosin inhibitor 1 (ENSOARG00020023278) and SORCS2, were upregulated (log2FC > 1) in active rams, whereas the CRYL1 and immunoglobulin lambda-1 light chain isoform X47 (ENSOARG00020025518) genes were downregulated (log2FC < -1) in this same group. Gene set Enrichment Analysis (GSEA) identified 428 signaling pathways, predominantly related to biological processes. The lysosome pathway (GO:0005764) was the most enriched, and may affect fertility and sexual behavior, given the crucial role played by lysosomes in steroidogenesis, being the SORCS2 gene related to this signaling pathway. Furthermore, the enriched positive regulation of ERK1 and ERK2 cascade (GO:0070374) pathway is associated with reproductive phenotypes such as fertility via modulation of hypothalamic regulation and GnRH-mediated production of pituitary gonadotropins. Furthermore, external side of plasma membrane (GO:0009897), fibrillar center (GO:0001650), focal adhesion (GO:0005925), and lamellipodium (GO:0030027) pathways were also enriched, suggesting that some molecules of these pathways might also be involved in rams' sexual behavior. These results provide new clues for understanding the molecular regulation of sexual behavior in rams. Further investigations will be needed to confirm the functions of SORCS2 and CRYL1 in relation to sexual behavior.