Showing papers on "Nucleolus published in 2022"

RNA is required for the integrity of multiple nuclear and cytoplasmic membrane‐less RNP granules

Abstract: Numerous membrane‐less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super‐enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane‐less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Abstract: Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease.

Viscoelastic RNA entanglement and advective flow underlie nucleolar form and function

Abstract: The nucleolus facilitates transcription, processing, and assembly of ribosomal RNA (rRNA), the most abundant RNA in cells. Nucleolar function is facilitated by its multiphase liquid properties, but nucleolar fluidity and its connection to ribosome biogenesis remain unclear. Here, we used quantitative imaging, mathematical modeling, and pulse-chase nucleotide labelling to map nucleolar rRNA dynamics. Inconsistent with a purely diffusive process, rRNA steadily expands away from the transcriptional sites, moving in a slow (~1Å/s), radially-directed fashion. This motion reflects the viscoelastic properties of a highly concentrated gel of entangled rRNA, whose constant polymerization drives steady outward flow. We propose a new viscoelastic rRNA release model, where nucleolar rRNA cleavage and processing reduce entanglement, fluidizing the nucleolar periphery to facilitate release of mature pre-ribosomal particles.

Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

Abstract: Eukaryotic chromosomes are folded into hierarchical domains, forming functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks contributing to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for the identification of nucleolar associated domains (NADs). Here we have established DamID- and HiC-based methodologies to generate accurate genome-wide maps of NADs in embryonic stem cells (ESCs) and neural progenitor cells (NPCs), revealing layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs show higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation into NPCs, chromosomes around the nucleolus acquire a more compact, rigid architecture with neural genes moving away from nucleoli and becoming unlocked for later activation. Further, histone modifications and the interaction strength within A and B compartments of NADs and LADs in ESCs set the choice to associate with NL or nucleoli upon dissociation from their respective compartments during differentiation. The methodologies here developed will make possible to include the nucleolar contribution in nuclear space and genome function in diverse biological systems.

Cytoplasmic forces functionally reorganize nuclear condensates in oocytes

Abstract: Abstract Cells remodel their cytoplasm with force-generating cytoskeletal motors. Their activity generates random forces that stir the cytoplasm, agitating and displacing membrane-bound organelles like the nucleus in somatic and germ cells. These forces are transmitted inside the nucleus, yet their consequences on liquid-like biomolecular condensates residing in the nucleus remain unexplored. Here, we probe experimentally and computationally diverse nuclear condensates, that include nuclear speckles, Cajal bodies, and nucleoli, during cytoplasmic remodeling of female germ cells named oocytes. We discover that growing mammalian oocytes deploy cytoplasmic forces to timely impose multiscale reorganization of nuclear condensates for the success of meiotic divisions. These cytoplasmic forces accelerate nuclear condensate collision-coalescence and molecular kinetics within condensates. Disrupting the forces decelerates nuclear condensate reorganization on both scales, which correlates with compromised condensate-associated mRNA processing and hindered oocyte divisions that drive female fertility. We establish that cytoplasmic forces can reorganize nuclear condensates in an evolutionary conserved fashion in insects. Our work implies that cells evolved a mechanism, based on cytoplasmic force tuning, to functionally regulate a broad range of nuclear condensates across scales. This finding opens new perspectives when studying condensate-associated pathologies like cancer, neurodegeneration and viral infections.

Nucleolin: a cell portal for viruses, bacteria, and toxins

Abstract: The main localization of nucleolin is the nucleolus, but this protein is present in multiple subcellular sites, and it is unconventionally secreted. On the cell surface, nucleolin acts as a receptor for various viruses, some bacteria, and some toxins. Aim of this review is to discuss the characteristics that make nucleolin able to act as receptor or co-receptor of so many and different pathogens. The important features that emerge are its multivalence, and its role as a bridge between the cell surface and the nucleus. Multiple domains, short linear motifs and post-translational modifications confer and modulate nucleolin ability to interact with nucleic acids, with proteins, but also with carbohydrates and lipids. This modular multivalence allows nucleolin to participate in different types of biomolecular condensates and to move to various subcellular locations, where it can act as a kind of molecular glue. It moves from the nucleus to the cell surface and can accompany particles in the reverse direction, from the cell surface into the nucleus, which is the destination of several pathogens to manipulate the cell in their favour.

Nucleolar-based Dux repression is essential for embryonic two-cell stage exit

Abstract: In this study, Xie et al. investigated the mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation in mammalian embryos, and report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. Their findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development.

Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly

Abstract: High content image analysis, single molecule tracking, modeling, and DBA patient analysis revealed that ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly. The nucleolus is the site of ribosome assembly and formed through liquid–liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid–liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond–Blackfan anemia patient harboring a heterozygous, large deletion in RPL5. Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

RNA folding and functions of RNA helicases in ribosome biogenesis

Abstract: ABSTRACT Eukaryotic ribosome biogenesis involves the synthesis of ribosomal RNA (rRNA) and its stepwise folding into the unique structure present in mature ribosomes. rRNA folding starts already co-transcriptionally in the nucleolus and continues when pre-ribosomal particles further maturate in the nucleolus and upon their transit to the nucleoplasm and cytoplasm. While the approximate order of folding of rRNA subdomains is known, especially from cryo-EM structures of pre-ribosomal particles, the actual mechanisms of rRNA folding are less well understood. Both small nucleolar RNAs (snoRNAs) and proteins have been implicated in rRNA folding. snoRNAs hybridize to precursor rRNAs (pre-rRNAs) and thereby prevent premature folding of the respective rRNA elements. Ribosomal proteins (r-proteins) and ribosome assembly factors might have a similar function by binding to rRNA elements and preventing their premature folding. Besides that, a small group of ribosome assembly factors are thought to play a more active role in rRNA folding. In particular, multiple RNA helicases participate in individual ribosome assembly steps, where they are believed to coordinate RNA folding/unfolding events or the release of proteins from the rRNA. In this review, we summarize the current knowledge on mechanisms of RNA folding and on the specific function of the individual RNA helicases involved. As the yeast Saccharomyces cerevisiae is the organism in which ribosome biogenesis and the role of RNA helicases in this process is best studied, we focused our review on insights from this model organism, but also make comparisons to other organisms where applicable.

An oomycete effector targets a plant RNA helicase involved in root development and defense

Abstract: Oomycete plant pathogens secrete effector proteins to promote disease. The damaging soilborne legume pathogen Aphanomyces euteiches harbors a specific repertoire of Small Secreted Protein effectors (AeSSPs), but their biological functions remain unknown. Here we characterize AeSSP1256. The function of AeSSP1256 is investigated by physiological and molecular characterization of Medicago truncatula roots expressing the effector. A potential protein target of AeSSP1256 is identified by yeast-two hybrid, co-immunoprecipitation, and fluorescent resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM) assays, as well as promoter studies and mutant characterization. AeSSP1256 impairs M. truncatula root development and promotes pathogen infection. The effector is localized to the nucleoli rim, triggers nucleoli enlargement and downregulates expression of M. truncatula ribosome-related genes. AeSSP1256 interacts with a functional nucleocytoplasmic plant RNA helicase (MtRH10). AeSSP1256 relocates MtRH10 to the perinucleolar space and hinders its binding to plant RNA. MtRH10 is associated with ribosome-related genes, root development and defense. This work reveals that an oomycete effector targets a plant RNA helicase, possibly to trigger nucleolar stress and thereby promote pathogen infection.

Alternative splicing of an exitron determines the subnuclear localization of the Arabidopsis DNA-glycosylase MBD4L under heat stress.

Abstract: DNA glycosylases are critical enzymes that recognize small base lesions in DNA and trigger their repair to preserve genome integrity. The Arabidopsis MBD4-like (MBD4L) DNA glycosylase improves tolerance to genotoxic stress. This enzyme is encoded by a single gene carrying an exitron at its 5´ region. Although alternative splicing (AS) of exitrons (protein-coding cryptic introns within exons) is suspected to increase protein diversity, phenotypes associated to exitron removal or retention are only known for a few genes. Here, we show that alternative splicing of the MBD4L exitron determines the generation of two enzyme isoforms with different subnuclear localization. Both isoforms conserve the catalytic domain but are directed either to the nucleoplasm or the nucleolus. Interestingly, heat-stress regulates the AS of the MBD4L exitron and increases the relative abundance of the nucleolar variant. This process depends on the splicing factors NTR1 and RS31. Notoriously, we generated transgenic plants expressing a mutated MBD4L-GFP gene version that abolished exitron splicing and found that nucleolar protein targeting was impaired in these plants. Our findings suggest that AS of the MBD4L exitron can function as a mechanism to drive the enzyme to the nucleolus during heat stress. Several DNA repair enzymes reach the nucleolus under particular conditions although AS of exitrons has not been so far associated with this process. To our knowledge, this is the first example of an exitron mediating enzyme localization in eukaryotes.

A Carbonized Fluorescent Nucleolus Probe Discloses RNA Reduction in the Process of Mitophagy

Abstract: Open AccessCCS ChemistryRESEARCH ARTICLE5 Aug 2022A Carbonized Fluorescent Nucleolus Probe Discloses RNA Reduction in the Process of Mitophagy Hua Liu, Xin Geng, Xin Wang, Lin Wei, Zhaohui Li, Shen Lin and Lehui Xiao Hua Liu College of Chemistry, Zhengzhou University, Zhengzhou 450001 State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author , Xin Geng College of Chemistry, Zhengzhou University, Zhengzhou 450001 Google Scholar More articles by this author , Xin Wang State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author , Lin Wei College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081 Google Scholar More articles by this author , Zhaohui Li College of Chemistry, Zhengzhou University, Zhengzhou 450001 Google Scholar More articles by this author , Shen Lin State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author and Lehui Xiao *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Medicinal Chemical Biology, Tianjin Key Laboratory of Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin 300071 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101371 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Mitophagy is a complicated process of cell metabolism that exhibits dynamic spatiotemporal coordination between multiple organelles. Despite its relevance to nucleoli, visualizing the distribution of nucleolar RNA in the process of mitophagy remains a great challenge because of the difficulty in specifically labeling RNA. Herein, we demonstrate a robust carbonized fluorescent probe with folic acid (FA) and m-phenylenediamine (m-PD) as the precursors, which displays superior RNA-anchoring ability and fast cellular permeability to tag nucleolar RNA in living cells. More importantly, this probe possesses spontaneous blinking behavior in physiological conditions, which is suitable for nanoscopic imaging of subtle changes in the distribution of nucleolar RNA. Overall, this work presents a new way for in situ observation of nanoscopic behavior of nucleolar RNA in living cells, providing a powerful approach for further exploration of cellular metabolism in mitophagy. Download figure Download PowerPoint Introduction Mitochondria are membrane-bound organelles that play important roles in supplying energy for cellular metabolic processes.1,2 The reactive oxygen species (ROS) accompanying the energy generating process can induce oxidative damage of mitochondria.3 To maintain a healthy population of mitochondria, the mitophagy process, a specialized form of autophagy, is required to selectively eliminate damaged mitochondria. Typically, damaged mitochondria are engulfed by autophagosomes and digested in lysosomes, which is an important cellular mechanism to sustain cellular metabolism. To further understand this process, various fluorescent probes targeting lysosome-related organelles have been designed to visualize the spatiotemporal coordination among various organelles.4–9 Nucleoli, consisting of ribosomal DNA (rDNA), ribosomal RNA (rRNA), and proteins, are important and highly dynamic subnuclear structures inside the cell that manipulate the synthesis of lysosome-related hydrolases and respond to the cellular environment.10 Despite the significant roles of nucleoli in the mitophagy process, little attention is paid to the dynamic change of nucleoli. Inhibition of the mammalian target of rapamycin (mTOR) signaling can promote mitophagy and diminish nucleolar size and function.11 Upon nutrient starvation, mTOR signaling is inhibited and nucleolar stress triggers several response pathways to maintain cell homeostasis. For instance, the nucleolus is depopulated of proteins in response to cellular stress, driving its reorganization.12 Therefore, having a good understanding of the crosstalk between the reorganization of the nucleolus and mitophagy is significant. However, it is still challenging to reveal the dynamic variation of nucleoli during the mitophagy process because of the targeting difficulty and compact structure of nucleoli. rRNA is synthesized and processed in the nucleoli. The compartmentalization, segregation, aggregation, and cleavage of nucleolar RNA are critical indicators for cell transcription, division, and apoptosis. One of the strategies to elucidate the dynamic behavior of nucleoli in the mitophagy process is to visualize nucleolar RNA by RNA-targeted fluorescent probes. However, monitoring their spatiotemporal distribution is difficult because of the imperfection of current fluorescent probes in differentiating between DNA and RNA, crossing cellular barriers, and being biocompatible.13 Recently, carbonized fluorescent probes, such as carbon dots (CDs), have emerged as an important class of fluorescent probes and been widely applied in live cell imaging due to their excellent biocompatibility, facile preparation, and high cell permeability.14–20 Additionally, CDs have abundant precursors available making them advantageous in designing fluorescent probes with specific targeting ability.21 To achieve RNA-specific targeting capability, an ingenious solution is to synthesize CDs with RNA affinity materials. It has been reported that folic acid (FA) can bind with RNA by interacting with its major groove and terminal loop region.22,23 Moreover, m-phenylenediamine (m-PD) has been widely employed as a precursor in the preparation of CDs.24–26 Therefore, in this work, FA and m-PD were used as precursors to prepare RNA-CDs with RNA-anchoring ability and fluorescent emission properties. Because of the tiny size and highly compressed structure of nucleoli, some subtle but important changes may be missed when using conventional fluorescence microscopy due to the diffraction resolution barrier. Advances in super-resolution microscopy exceed the diffraction resolution barrier and can be employed to observe unresolvable details, providing novel insights into microcosmos and a chance to visualize the subtle changes in the higher-order organization of RNA.27,28 Single-molecule localization microscopy (SMLM) based on the accurate localization of the point spreading function (PSF) from an individual fluorophore has received much attention because it can achieve nanoscopic images without further modification of the microscope.29–32 To get a nanoscopic image with SMLM, the primary task is to satisfy the requirement of the blinking property. Some approaches regulate the blinking behavior of fluorescent probes by, for instance, using imaging buffer devoid of oxygen but containing additives such as thiols, ascorbic acid, and oxygen scavenger.33 However, the addition of additives may change the physiological environment of the cell, disturbing cellular metabolism. Thus, developing fluorescent probes with spontaneous blinking behaviors is important for nanoscopic imaging. In this work, FA and m-PD were used as the precursors to prepare RNA-CDs. As expected, the prepared RNA-CDs exhibit excellent RNA-anchoring ability and display yellow-green fluorescence. Because of the high cell permeability, RNA-CDs accumulate in nucleoli, enabling the imaging of nucleoli in live cells. Interestingly, the prepared RNA-CDs exhibit remarkable blinking property without any additives. They were employed to visualize the dynamic changes of nucleolar morphology and the distribution of RNA in the mitophagy process induced by nutrient starvation (Schemes 1a,b). We found that mitophagy has a significant effect on the nucleolar morphology and nucleolar RNA distribution. The RNA network in a single nucleolus was gradually destroyed in mitophagy. This work provides deep insight into the relation between the dynamic changes of nucleolar RNA and mitophagy, which is also helpful to comprehend other nucleolus-related processes in pathological situations. Scheme 1 | (a) Synthesis of RNA-CDs and their nucleolar RNA imaging capability. (b) In situ tracking of nucleolar RNA with nanoscopic imaging in the mitophagy process. Download figure Download PowerPoint Experimental Methods Fabrication of RNA-CDs RNA-CDs are prepared by one-step hydrothermal synthesis with FA and m-PD as the precursors. Specifically, FA aqueous solution (2.0 mg/mL) and m-PD aqueous solution (5.2 mg/mL) were mixed in equal volumes. After sonication for 10 min, the mixture was transferred to the reaction kettle and reacted at 200 °C for 12 h. After being cooled in the air, the mixture was centrifuged at 10,000 rpm for 10 min and the supernatant was collected. Subsequently, the supernatant was further purified by silica gel column chromatography with a mixture of ethyl acetate and methanol (1:1, v/v) as the eluent. Finally, the prepared RNA-CDs were dissolved in deionized water to a final concentration of 5.0 mg/mL. The quantum yield of the prepared RNA-CDs in water was calculated to be 21.8% by using rhodamine 6G as the reference. Cytotoxicity assay The cytotoxicity assay of RNA-CDs was performed based on a standard methylthiazolyldiphenyl-tetrazolium bromide (MTT) test. In brief, HepG2 cells were first seeded in 96-well plates with a density of 3 × 104 cells per well and cultured for 16 h. Afterwards, the cells were incubated with RNA-CDs (final concentration of 0, 0.1, 0.5, 1, 5, and 10 μg/mL) for 24 h at 37 °C. Then 20 μL of MTT solution (5 mg/mL) was added to each well and incubated with cells for another 4 h. Finally, supernatants were discarded and sediments were dissolved in 150 μL of dimethyl sulfoxide (DMSO). The optical density at 490 nm was measured by a microplate reader. The relative cell viability was determined by the equation: cell viability = (A – Ablank)/(Acontrol – Ablank), where A is the absorbance of the experimental group (cells and RNA-CDs), Ablank is the absorbance of the blank group (without cells and RNA-CDs), and Acontrol is the absorbance of the control group (cells only). Single-particle fluorescence imaging Single-particle fluorescence imaging was carried out by a Ti-U inverted epi-fluorescence microscope (Nikon, Tokyo, Japan) equipped with a single-mode fiber-coupled semiconductor laser. RNA-CDs were excited with a 473 nm laser (Changchun New Industries Optoelectronics Technology Co., Ltd., Changchun, China). The fluorescent signals were collected by a 100× (numerical aperture (NA) 1.49) total internal reflection fluorescence (TIRF) objective. For the nanoscopic imaging of nucleolar RNA, HepG2 cells were seeded in confocal dishes for 24 h and washed with phosphate buffered saline (PBS) twice. Dulbecco’s modified eagle medium containing 10% fetal bovine serum and RNA-CDs (1 ng/mL) was added to the confocal dishes and incubated for 15 min at 37 °C, and then serum-free medium was added to the cells to induce mitophagy. The fluorescent images were recorded at different time points, such as 0, 3, 6, and 9 h. The exposure time was 50 ms and the electron multiplying gain of the electron multiplying charge coupled device (EMCCD) (Andor iXon Ultra 897) was set to 5–60. Image J was used to process the results, and appropriate references were introduced to eliminate the system drift. Results and Discussion Fabrication and characterization of RNA-CDs Developing RNA-targeted fluorescent probes is the cornerstone to observe the spatial distribution of nucleolar RNA. It is reported that RNA-targeted fluorescent probes commonly consist of aromatic rings, nitrogen heterocyclic rings, and amine groups.34,35 Phenylenediamines are commonly used precursors for preparing CDs to provide heterocyclic compounds and polymers. According to previous reports, CDs prepared by m-PD have the potential to target nucleolar RNA.36,37 However, an additional precursor is required to improve the RNA targeting capability. FA is a water-soluble vitamin with pteridine, p-aminobenzoic acid, and glutamate moieties, which is involved in the synthesis of nucleic acids. It was reported that FA can bind with RNA by interacting with its major groove and terminal loop region.22,23 Therefore, FA and m-PD were used as the precursors to prepare RNA-CDs by a one-step hydrothermal method to incorporate both merits of FA and m-PD. To translocate through the nuclear pore, probes of small size are a prerequisite for nucleolar RNA imaging. Transmission electron microscopy (TEM) images show that the prepared RNA-CDs have a homogeneous morphology distribution and a small size dimension with an average diameter of 2.2 ± 0.2 nm (Figures 1a and 1b). The optical properties of prepared RNA-CDs were characterized, and RNA-CDs exhibited a maximum absorption at 445 nm and fluorescence emission at 535 nm (Figures 1c and 1e). Excitation-independent fluorescence emission spectra confirmed the homogeneous size distribution of RNA-CDs. To explore the structure of RNA-CDs, Fourier transform infrared (FTIR) spectra of m-PD, FA, and RNA-CDs were obtained (Figure 1f). Some characteristic signals were observed, such as stretching vibrations from O–H (3442 cm−1), N–H (3337 cm−1), C–H (2922 cm−1), C=O (1682 cm−1), C=C (1632 cm−1), C=N (1601 cm−1), and C–N (1400 cm−1).25 X-ray photoelectron spectroscopy (XPS) analysis was performed to further explore the functional groups on the surface of RNA-CDs. In the XPS spectrum (Figure 1g), three peaks that appeared at 284.8, 399.2, and 531.3 eV were attributed to C 1s, N 1s, and O 1s, respectively. In the N 1s spectrum, the binding energies of 399.2 and 400.4 eV confirmed the presence of pyridinic and pyrrolic N groups, respectively.38 The peaks at 284.8, 286.4, and 288.4 eV from C–C/C=C, C–O/C–N, and C=O groups in the C 1s spectrum and the peaks at 531.3 and 532.7 eV from C=O and C–O groups in the O 1s spectrum implied the presence of carbonyl groups and nitrogen-containing compounds on the surface of RNA-CDs.39 These results are in good agreement with the FTIR spectral measurements. Figure 1 | (a) TEM image of RNA-CDs. (b) Size distribution of RNA-CDs. (c) UV–vis absorption spectra of the RNA-CDs. The inset shows the photographs of RNA-CDs under daylight (left) and 365 nm UV light illumination (right). (d) Zeta potential of RNA-CDs. (e) Fluorescence excitation (λem = 535 nm) and emission spectra (λex = 390, 410, 430, 450, 470, and 480 nm) of the RNA-CDs. (f) FTIR spectra of FA, m-PD, and RNA-CDs. (g) XPS spectra of RNA-CDs. Download figure Download PowerPoint Blinking behaviors of single RNA-CDs One of the solutions to achieve nanoscopic resolution with fluorescence microscopy is SMLM ( Supporting Information Figure S1). Random blinking of individual probes is the foundation of nanoscopic imaging. Although the blinking property of fluorophores can be modulated by adding chemical reagents, probes with spontaneous blinking property are still the best choice for nanoscopic imaging. To ascertain whether RNA-CDs can be employed for nanoscopic imaging based on SMLM, the single particle fluorescent behaviors of RNA-CDs without any additives were investigated. Typical fluorescence intensity trajectories of single RNA-CDs are shown in Supporting Information Figure S2a. Spontaneous blinking behavior was evident. After several switches between fluorescent (“on”) and dark (“off”) states, RNA-CDs maintained high fluorescence emission under 25 s continuous illumination. However, under the same conditions, the fluorescence of single rhodamine B was quenched after blinking a few times ( Supporting Information Figure S2b). To quantitatively understand the blinking performance of single RNA-CDs, the duty cycle (proportion of time in “on” states), the frequency of “on” time (τon), and the frequency of photon-number of each switching event from single RNA-CDs were statistically analyzed from hundreds of trajectories. Fluorescent probes with long-lived “on” states correlate to simultaneous illumination of adjacent fluorophores. If their PSFs overlap, fluorophores cannot be positioned accurately based on the Gaussian fitting. High duty cycle increases the probability of localizing many probes within a diffraction-limited area simultaneously. A long “on” state also triggers similar inaccuracy. Though the “on” time of probes should be as small as possible in theory, the “on” time far less than exposure time will impair the collection of sufficient photons, which is not conducive for nanoscopic imaging. RNA-CDs with low duty cycle (0.06) and “on” times within the range of 0.1–0.3 s are suitable for nanoscopic imaging ( Supporting Information Figures S2c and S2e). As described in previous studies, the photon-number of single fluorescent probes greatly affects precise localization.29–31 Photon distribution of single RNA-CDs in the “on” state is shown in Supporting Information Figure S3. The photon-number from single RNA-CDs is ∼3000 per switching event, which is sufficient for nanoscopic imaging. Despite the remaining debate of the nanocrystals fluorescence blinking, it is consistent to contribute the fluorescence blinking behavior to charge carrier trapping,40 which can impede charge transport and recombination in nanocrystals. Analysis of the duration in the “on” and “off” states is a powerful method to probe charge trapping dynamics. From Supporting Information Figure S4, the probability density of τoff follows an inverse power law distribution, P(τ)off ∝ τ − α off , with a power law coefficient, αoff = 1.3. However, exponential truncation is observed in the probability density of τon, which is fit to a truncated power law, P ( τ ) on ∝ τ − α on × e ( − τ / τ c ) , with αon = 1.4 and cross-over time (τc) is 3.0 s. τc indicates the start of exponential truncation. The distribution of τoff and τon is rationalized. The truncated power law behavior of τon is interpreted as an increase in charge trapping rate. The blinking behaviors of single RNA-CDs can contribute to energetic diffusion of the corresponding charge trapping states or trap states undergoing a process of activation-deactivation. Evaluation of the RNA targeting ability of RNA-CDs RNA is a negatively charged polymer composed of nitrogenous bases, five-carbon sugars, and phosphate groups. Therefore, the common strategy for RNA binding is to construct probes with positively charged aromatic structures based on hydrogen bond interactions and electrostatic interactions. Herein, zeta potential was measured to estimate the surface functional groups of RNA-CDs. Different than common RNA-targeted fluorescent probes, RNA-CDs exhibit a negative zeta potential (−18.5 mV), which might originate from the –COOH group of RNA-CDs (Figure 1d). Compared with those probes with high positive charges, the prepared RNA-CDs can avoid undesirable interactions with negatively charged intracellular organelles and proteins. The effect of various biomolecules on the fluorescent spectra of RNA-CDs, including histidine (His), glycine (Gly), glucose (Glc), galactose (Gal), adenosine triphosphate (ATP), bovine serum albumin (BSA), double-stranded λ-DNA (dsDNA), single-stranded DNA (ssDNA), and RNA, were explored to assess the RNA-selectivity of RNA-CDs (Figure 2e). Generally, the isoelectric points of His, Gly, and BSA are 7.59, 5.97, and 4.9, respectively. Under the test conditions, the pH value is above the isoelectric points of Gly and BSA; therefore, Gly and BSA release protons and carry negative charges. Interestingly, the fluorescence intensity of RNA-CDs increases instantly by more than 10-fold after binding to RNA. Under the same experimental conditions, the fluorescence intensity of RNA-CDs mixed with other biomolecules exhibited negligible fluctuation even in the presence of dsDNA. In the presence of ssDNA, the fluorescence intensity of RNA-CDs increased 2.7 times, which is also far lower than that in the presence of RNA. Previous studies have demonstrated that FA binds with DNA and RNA at different locations.23 The distinct fluorescence enhancement of RNA-CDs may be due to the different binding modes with DNA and RNA. From Figures 2b and 2c, it is found that the fluorescence intensity of RNA-CDs increases with the addition of RNA. Upon treatment with ribonuclease (RNase), the fluorescence intensity of RNA-CDs drops to the initial value (Figure 2d). Figure 2 | (a) Photophysical transitions in solution states. Abbreviations: A, absorption, F, fluorescence, NRD, non-radiative decay, IC, internal conversion, ISC, intersystem crossing. (b) Fluorescence (FL) emission spectra of RNA-CDs (5 μg/mL) in the presence of various concentrations of RNA. (c) The relationship between the relative fluorescence intensity (I/I0, I0, and I are the fluorescence intensities at 535 nm in the absence and presence of RNA, respectively) and concentrations of RNA. (d) Fluorescence emission spectra of RNA-CDs (5 μg/mL) in different situations. (e) Selectivity studies of RNA-CDs (5 μg/mL) in the presence of potential interferences, including 100 μM His, 100 μM Gly, 100 μM Glc, 100 μM Gal, 100 μM ATP, 1 mg/mL BSA, 5 μM dsDNA, 5 μM ssDNA, and 5 μM RNA. (f) Normalized fluorescence emission spectra of RNA-CDs in solvents with different polarity. Download figure Download PowerPoint As shown in Figure 2a, fluorescent probes are normally activated from the ground state (S0) to the excited state (S1 and S2) upon photoexcitation. The high-energy excited states are unstable and revert to the ground state mainly through three processes, including radiative emission, vibrational relaxation, and intersystem crossing.41–43 To explore the fluorescence enhancement mechanism of RNA-CDs after binding with RNA, the fluorescence spectra of RNA-CDs in solutions with different viscosities were investigated by using a mixture of glycerol and water. While the solution viscosities gradually increased, the fluorescence intensities of RNA-CDs in the mixture also rose accordingly. Around 3-fold enhancement was noted in the 90% glycerol solution ( Supporting Information Figures S5a and S5b). Meanwhile, the fluorescence excitation spectra of RNA-CDs exhibited an obvious red-shift with increasing solution viscosity, consistent with that of RNA-CDs in RNA solution ( Supporting Information Figures S6a and S6b). Thus, restricted intramolecular motion of RNA-CDs after binding with RNA should be the key source of fluorescence enhancement. The fluorescence emission of RNA-CDs in solvent with different polarity was further explored. Generally, fluorescent probes exhibit solvatochromism, quenching in polar protic media. However, the fluorescence intensities of RNA-CDs dropped with a decrease in solvent polarity (Figure 2f). The anti-solvatochromic fluorescence emission can be explained by hydrogen-bond enhanced emission. It is reported that a hydrogen-bond network can minimize the vibrational and rotational energy loss, leading to the fluorescence enhancement.44 Specific targeting of nucleolar RNA Encouraged by the excellent RNA-selectivity of RNA-CDs in solution, HepG2 cells were co-stained with both RNA-CDs and Hoechst 33342 (a DNA-specific dye) to assess the imaging capability of RNA-CDs in living cells. Figure 3a shows that the RNA-CDs specifically accumulate in the nucleoli, demonstrating the potential RNA-anchoring ability of RNA-CDs. The corresponding intensity profile of the overlay (as shown in Figures 3b and 3c) demonstrates that RNA-CDs locate in the nucleoli and do not overlap with the DNA region stained by Hoechst 33342. Similar results are also observed with other cell lines, including HeLa cells (carcinoma cell line) and NCTC1469 cells (normal cell line), indicating the universal nucleolar targeting capability of RNA-CDs in different cell lines ( Supporting Information Figure S7). Confocal images of HepG2 cells stained with RNA-CDs at different sections through the z-axis scanning further demonstrate the nucleolar targeting ability ( Supporting Information Figure S8). To verify whether the RNA-CDs accumulated in the nucleolar region is indeed derived from RNA, fixed cells untreated and treated with RNase solution stained by RNA-CDs were imaged, respectively. First, HepG2 cells were fixed by 4% paraformaldehyde for 20 min to maintain their structures and shapes, and permeabilized by PBS containing 0.5% Triton X-100 for 2 min. After the fixed HepG2 cells were incubated with 0.5 μg/mL RNA-CDs for 15 min, RNA-CDs exhibited specific accumulation in the nucleolar regions and did not overlay with the DNA regions, consistent with the results from live cells ( Supporting Information Figure S9). Then, 25 μg/mL RNase solution was employed to specifically hydrolyze RNA by incubating with the fixed HepG2 cells at 37 °C for 2 h. As shown in Figures 3d and 3e, a dramatic decrease in the fluorescence intensity of RNA-CDs occurred in the nucleoli after RNase treatment, indicating that the accumulation of RNA-CDs in the nucleolar region is based on the RNA-anchoring ability of RNA-CDs. Figure 3 | (a) Confocal images of HepG2 cells stained with RNA-CDs (0.5 μg/mL) and Hoechst 33342 (0.5 μg/mL). (b) Corresponding 3D intensity profiles of the fluorescence images from the green box in (a). (c) Signal intensity profiles along the red lines in (a). (d) Confocal images of HepG2 cells stained with Hoechst 33342 (0.5 μg/mL) and RNA-CDs (0.5 μg/mL) after untreated or treated with RNase. (e) Signal intensity profiles along the red lines in (d). Download figure Download PowerPoint Monitoring RNA dynamics can provide detailed insights into the complex cellular mechanisms under different cellular states. Therefore, the cytotoxicity of RNA-CDs was studied to evaluate the suitability for in situ imaging of RNA in living cells (MTT test, Supporting Information Figure S10c). After HepG2 cells were seeded and cultured for 16 h, they were treated with RNA-CDs (0, 0.1, 0.5, 1, 5, and 10 μg/mL) for 24 h, respectively. More than 90% of these cells survived at the concentration of 0.5 μg/mL for the imaging experiments, indicating an excellent biocompatibility and capability to image RNA in living cells. Cellular internalization and intracellular transport of RNA-CDs The internalization and intracellular transport mechanism of RNA-CDs are crucial to elucidate their possible hazards and potential for studying biological behaviors. After HepG2 cells were seeded in the confocal dish for 24 h, 0.5 μg/mL RNA-CDs were added into the dish to investigate the uptake efficiency. Time-lapse images over a period of 15 min are shown in Supporting Information Figures S10a and S10b. RNA-CDs enter the cell within 2 min and the fluorescence intensities increase gradually with the increase of incubation time. A plateau is gradually achieved when the incubation time reaches 10 min. The high-speed cellular internalization efficiency of RNA-CDs may be attributed to their ultrasmall size. It is worth noting that, within 30 s, RNA-CDs already exist in the nucleoli as shown in Supporting Information Figure S11. To understand the intracellular transport mechanism of RNA-CDs, HepG2 cells were co-cultured with 0.5 μg/mL RNA-CDs at physiological temperature (37 °C) and low temperature (4 °C) for 15 min, respectively. The results showed that cellular internalization of RNA-CDs was partly blocked at low temperature, which might be caused by the decrease in activities of various enzymes for ATP production at low temperature, demonstrating that energy-dependent endocytosis is involved in the cellular internalization process. The clathrin- and caveolae-mediated endocytosis processes are the most extensively studied endocytosis pathways for non-phagocytic eukaryotic cells. Therefore, effects of dynasore (an inhibitor of clathrin-mediated e

Seneca Valley Virus 3Cpro Mediates Cleavage and Redistribution of Nucleolin To Facilitate Viral Replication

Abstract: The nucleolus is a subnuclear cellular compartment, and nucleolin (NCL) resides predominantly in the nucleolus. NCL participates in viral replication, translation, internalization, and also serves as a receptor for virus entry. ABSTRACT Seneca Valley virus (SVV) is a recently discovered pathogen that poses a significant threat to the global pig industry. It has been shown that many viruses are reliant on nucleocytoplasmic trafficking of nucleolin (NCL) for their own replication. Here, we demonstrate that NCL, a critical protein component of the nucleolus, is cleaved and translocated out of the nucleoli following SVV infection. Furthermore, our data suggest that SVV 3C protease (3Cpro) is responsible for this cleavage and subsequent delocalization from the nucleoli, and that inactivation of this protease activity abolished this cleavage and translocation. SVV 3Cpro cleaved NCL at residue Q545, and the cleavage fragment (aa 1 to 545) facilitated viral replication, which was similar to the activities described for full-length NCL. Small interfering RNA-mediated knockdown indicated that NCL is required for efficient viral replication and viral protein expression. In contrast, lentivirus-mediated overexpression of NCL significantly enhanced viral replication. Taken together, these results indicate that SVV 3Cpro targets NCL for its cleavage and redistribution, which contributes to efficient viral replication, thereby emphasizing the potential target of antiviral strategies for the control of SVV infection. IMPORTANCE The nucleolus is a subnuclear cellular compartment, and nucleolin (NCL) resides predominantly in the nucleolus. NCL participates in viral replication, translation, internalization, and also serves as a receptor for virus entry. The interaction between NCL and SVV is still unknown. Here, we demonstrate that SVV 3Cpro targets NCL for its cleavage and nucleocytoplasmic transportation, which contributes to efficient viral replication. Our results reveal novel function of SVV 3Cpro and provide further insight into the mechanisms by which SVV utilizes nucleoli for efficient replication.

Nucleolus and Nucleolar Stress: From Cell Fate Decision to Disease Development

Abstract: Besides the canonical function in ribosome biogenesis, there have been significant recent advances towards the fascinating roles of the nucleolus in stress response, cell destiny decision and disease progression. Nucleolar stress, an emerging concept describing aberrant nucleolar structure and function as a result of impaired rRNA synthesis and ribosome biogenesis under stress conditions, has been linked to a variety of signaling transductions, including but not limited to Mdm2-p53, NF-κB and HIF-1α pathways. Studies have uncovered that nucleolus is a stress sensor and signaling hub when cells encounter various stress conditions, such as nutrient deprivation, DNA damage and oxidative and thermal stress. Consequently, nucleolar stress plays a pivotal role in the determination of cell fate, such as apoptosis, senescence, autophagy and differentiation, in response to stress-induced damage. Nucleolar homeostasis has been involved in the pathogenesis of various chronic diseases, particularly tumorigenesis, neurodegenerative diseases and metabolic disorders. Mechanistic insights have revealed the indispensable role of nucleolus-initiated signaling in the progression of these diseases. Accordingly, the intervention of nucleolar stress may pave the path for developing novel therapies against these diseases. In this review, we systemically summarize recent findings linking the nucleolus to stress responses, signaling transduction and cell-fate decision, set the spotlight on the mechanisms by which nucleolar stress drives disease progression, and highlight the merit of the intervening nucleolus in disease treatment.

The nucleoplasmic phase of pre-40S formation prior to nuclear export

Abstract: Abstract Biogenesis of the small ribosomal subunit in eukaryotes starts in the nucleolus with the formation of a 90S precursor and ends in the cytoplasm. Here, we elucidate the enigmatic structural transitions of assembly intermediates from human and yeast cells during the nucleoplasmic maturation phase. After dissociation of all 90S factors, the 40S body adopts a close-to-mature conformation, whereas the 3' major domain, later forming the 40S head, remains entirely immature. A first coordination is facilitated by the assembly factors TSR1 and BUD23–TRMT112, followed by re-positioning of RRP12 that is already recruited early to the 90S for further head rearrangements. Eventually, the uS2 cluster, CK1 (Hrr25 in yeast) and the export factor SLX9 associate with the pre-40S to provide export competence. These exemplary findings reveal the evolutionary conserved mechanism of how yeast and humans assemble the 40S ribosomal subunit, but reveal also a few minor differences.

Targeting selenoprotein H in the nucleolus suppresses tumors and metastases by Isovalerylspiramycin I

Abstract: Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells.We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway.ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells.We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

FGF12 is a novel component of the nucleolar NOLC1/TCOF1 ribosome biogenesis complex

Abstract: Abstract Among the FGF proteins, the least characterized superfamily is the group of fibroblast growth factor homologous factors (FHFs). To date, the main role of FHFs has been primarily seen in the modulation of voltage-gated ion channels, but a full picture of the function of FHFs inside the cell is far from complete. In the present study, we focused on identifying novel FGF12 binding partners to indicate its intracellular functions. Among the identified proteins, a significant number were nuclear proteins, especially RNA-binding proteins involved in translational processes, such as ribosomal processing and modification. We have demonstrated that FGF12 is localized to the nucleolus, where it interacts with NOLC1 and TCOF1, proteins involved in the assembly of functional ribosomes. Interactions with both NOLC1 and TCOF1 are unique to FGF12, as other FHF proteins only bind to TCOF1. The formation of nucleolar FGF12 complexes with NOLC1 and TCOF1 is phosphorylation-dependent and requires the C-terminal region of FGF12. Surprisingly, NOLC1 and TCOF1 are unable to interact with each other in the absence of FGF12. Taken together, our data link FHF proteins to nucleoli for the first time and suggest a novel and unexpected role for FGF12 in ribosome biogenesis.

Targeting selenoprotein H in the nucleolus suppresses tumors and metastases by Isovalerylspiramycin I

Abstract: Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells.We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway.ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells.We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

Intranuclear Nanoribbons for Selective Killing of Osteosarcoma Cells.

Abstract: Here we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L- or D-phosphopentapeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides firstly transformed into micelles and then convert into nanoribbons. The peptides/assemblies firstly aggregate on cell membrane, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly, and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus-targeting, but also may lead a new way to selectively kill osteosarcoma cells and overcome drug resistance.

Nucleolar localization of the ErbB3 receptor as a new target in glioblastoma

Abstract: The nucleolus is a subnuclear, non-membrane bound domain that is the hub of ribosome biogenesis and a critical regulator of cell homeostasis. Rapid growth and division of cells in tumors are correlated with intensive nucleolar metabolism as a response to oncogenic factors overexpression. Several members of the Epidermal Growth Factor Receptor (EGFR) family, have been identified in the nucleus and nucleolus of many cancer cells, but their function in these compartments remains unexplored.We focused our research on the nucleolar function that a specific member of EGFR family, the ErbB3 receptor, plays in glioblastoma, a tumor without effective therapies. Here, Neuregulin 1 mediated proliferative stimuli, promotes ErbB3 relocalization from the nucleolus to the cytoplasm and increases pre-rRNA synthesis. Instead ErbB3 silencing or nucleolar stress reduce cell proliferation and affect cell cycle progression.These data point to the existence of an ErbB3-mediated non canonical pathway that glioblastoma cells use to control ribosomes synthesis and cell proliferation. These results highlight the potential role for the nucleolar ErbB3 receptor, as a new target in glioblastoma.

APE1 assembles biomolecular condensates to promote the ATR–Chk1 DNA damage response in nucleolus

Abstract: Multifunctional protein APE1/APEX1/HAP1/Ref-1 (designated as APE1) plays important roles in nuclease-mediated DNA repair and redox regulation in transcription. However, it is unclear how APE1 regulates the DNA damage response (DDR) pathways and influences genome integrity directly or indirectly. Here we show that siRNA-mediated APE1-knockdown or APE1 inhibitor treatment attenuates the ATR-Chk1 DDR under stress conditions in multiple immortalized cell lines. Congruently, APE1 overexpression (APE1-OE) activates the ATR DDR under unperturbed conditions, which is independent of APE1 nuclease and redox functions. Structural and functional analysis reveals a direct requirement of the extreme N-terminal 33 amino acids (NT33) within APE1 in the assembly of distinct biomolecular condensates in vitro and DNA/RNA-independent activation of the ATR DDR. Overexpressed APE1 co-localizes with nucleolar NPM1 and assembles biomolecular condensates in nucleoli in cancer but not non-malignant cells, which recruits ATR and its direct activator molecules TopBP1 and ETAA1. APE1 W119R mutant is deficient in nucleolar condensation and liquid-liquid phase separation and is incapable of activating nucleolar ATR DDR. Lastly, APE1-OE-induced nucleolar ATR DDR activation leads to compromised ribosomal RNA transcription and reduced cell viability. Taken together, we propose distinct mechanisms by which APE1 regulates ATR DDR pathways and functions in genome integrity maintenance.

Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana

Abstract: ABSTRACT Transcription and processing of 45S rRNAs in the nucleolus are keystones of ribosome biogenesis. While these processes are severely impacted by stress conditions in multiple species, primarily upon heat exposure, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. We show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, the accuracy of transcription initiation and cleavage at the primary P site in the 5’ETS (5’ External Transcribed Spacer) are not affected but the levels of primary 45S and 35S transcripts are, respectively, increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress. Remarkably, nucleolar structure, pre-rRNAs from major ITS1 processing pathway and ribosome profiles are restored after returning to optimal conditions, shedding light on the extreme plasticity of nucleolar functions in plant cells. Further genetic and molecular analysis to identify molecular clues implicated in these nucleolar responses indicate that cleavage rate at P site and nucleolin protein expression can act as a checkpoint control towards a productive pre-rRNA processing pathway.