Role In Ribosome Biogenesis Research Articles

Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders

PurposeThis study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear. MethodsWe identified 105 affected individuals, including 39 previously reported cases, and systematically analyzed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis. ResultsBiallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability, infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe global developmental delay/intellectual disability, absent speech, and autistic features, whereas seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, and parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, particularly in pre-rRNA processing. ConclusionThis study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of “ribosomopathies.”

SnoRNAs in making “snoman” ribosomes

In a recent study in Cell, Cheng and Wang etal.1 show that the small nucleolar RNA (snoRNA) SNORA13 has a non-canonical role in ribosome biogenesis and senescence by acting directly on RPL23 and regulating its assembly into the 60S ribosomal subunit.

Complete list of canonical post-transcriptional modifications in the Bacillus subtilis ribosome and their link to RbgA driven large subunit assembly.

Ribosomal RNA modifications in prokaryotes have been sporadically studied, but there is a lack of a comprehensive picture of modification sites across bacterial phylogeny. Bacillus subtilis is a preeminent model organism for gram-positive bacteria, with a well-annotated and editable genome, convenient for fundamental studies and industrial use. Yet remarkably, there has been no complete characterization of its rRNA modification inventory. By expanding modern MS tools for the discovery of RNA modifications, we found a total of 25 modification sites in 16S and 23S rRNA of B. subtilis, including the chemical identity of the modified nucleosides and their precise sequence location. Furthermore, by perturbing large subunit biogenesis using depletion of an essential factor RbgA and measuring the completion of 23S modifications in the accumulated intermediate, we provide a first look at the order of modification steps during the late stages of assembly in B. subtilis. While our work expands the knowledge of bacterial rRNA modification patterns, adding B. subtilis to the list of fully annotated species after Escherichia coli and Thermus thermophilus, in a broader context, it provides the experimental framework for discovery and functional profiling of rRNA modifications to ultimately elucidate their role in ribosome biogenesis and translation.

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

ABSTRACT Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation.

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.

Copy number and sequence variation in rDNA of Daphnia pulex from natural populations: insights from whole-genome sequencing.

Ribosomal DNA (rDNA) has a vital role in ribosome biogenesis as it contains the genes that encode ribosomal RNA (rRNA) separated by intergenic spacers (IGSs). The rRNA genes occur in hundreds to tens of thousands of copies per haploid genome in eukaryotes and are generally highly conserved with low variation within species. Due to the repetitive nature and large size of rDNA arrays, detecting intraindividual variation can be difficult. In this study, we use whole-genome sequences of 169 Daphnia pulex individuals from 10 natural populations to measure the copy number and sequence variation in rDNA. This revealed that variation in rDNA copy number between individuals spans an order of magnitude. We further observed a substantial level of sequence variation within individual genomes. As expected, single-nucleotide polymorphisms occurred in regions of lower functional constraint such as the IGS and expansion segments of the rRNA genes. The presence of strong linkage disequilibrium among variants facilitated identification of haplotypes within each population. Although there was evidence of recombination among haplotypes from different populations, it is insufficient to eliminate linkage disequilibrium within populations. Estimating copy number and haplotype diversity within individuals revealed that the level of intraindividual sequence variation is not strongly correlated with copy number. The observed patterns of variation highlight a complex evolutionary history of rDNA in D. pulex. Future research should explore the functional implications of rDNA copy number and sequence variation on organismal phenotypes.

Molecular dynamics simulation studies and dynamic network analysis of Bacillus subtilis YsxC in GDP and GTP-Mg 2+ bound states

Ribosomes are essential cellular organelles made up of rRNA and rproteins found in both prokaryotic and eukaryotic cells, and play an important role in protein synthesis. The functional ribosomes are synthesized through the process called ribosome biogenesis, which proceeds with the help of certain factors such as chaperones, ribosomal proteins, and GTPases. GTPases bind to the premature ribosomal subunit and function as a checkpoint, ensuring the proper assembly of other proteins. To aid this process, GTPase undergoes conformational changes, alternating between an active GTP-bound state and an inactive GDP bound state. YsxC is a GTPase that functions this way, to help in the maturation of 50S subunit. Although YsxC plays an important role in ribosome biogenesis, a detailed knowledge of how the GDP and GTPMg2+ states of YsxC assist in the switching process is yet to be realized. Therefore, a study on the GDP and GTP-Mg2+ bound states is done for YsxC GTPase of Bacillus subtilis by all-atom molecular dynamics simulation for a period of 500 ns. The simulations aided the analysis of the RMSD from which it was noticed that both the GDP and GTP-Mg2+ states of YsxC attained equilibration after 200ns. The analysis of the Rg for the systems showed that the GTP-Mg2+ system showed higher values, in comparison to the GDP system, indicating conformational changes in the two systems. Thus, a residue-wise calculation for the difference in the SASA (solvent accessible surface area) of the GTP-Mg2+ and GDP system is done, and it is noticed that the GTP-Mg2+ system, especially in the regions recognized as Switch I and Switch II had a higher difference in SASA value. In which Switch I’s Lys55 (positive residue) and Switch II’s Arg89 (positive residue) showed a distinct difference, indicating that Switch I has more accessibility to solvent. Giving rise to a supposition that the residue Lys55 attracts the negative rRNA and aids in the binding of YsxC with the premature 50S subunit in the GTP-Mg2+ bound state by associating with the rRNA. To fathom the role of the GTP-Mg2+ bound state in increasing the SASA value of Switch I region, a community network is constructed to extract the shortest path. More number of paths are observed between the selected residue in the GTP-Mg2+ bound state inferring that the increased SASA in the Lys55 is due to the stronger connection with the nucleotide. Overall this study suggest that enhanced surface exposure of YsxC’s Sw-I in the GTP-Mg2+ bound state facilitate the GTP-Mg2+ bound system to involve in ribosome biogenesis by interacting with ribosomal constituents (rRNA and rproteins).

Clinical significance of PNO1 as a novel biomarker and therapeutic target of hepatocellular carcinoma.

The RNA-binding protein PNO1 plays an essential role in ribosome biogenesis. Recent studies have shown that it is involved in tumorigenesis; however, its role in hepatocellular carcinoma (HCC) is not well understood. The purpose of this study was to examine whether PNO1 can be used as a biomarker of HCC and also examine the therapeutic potential of PNO1 knockout for the treatment of HCC. PNO1 expression was upregulated in HCC and associated with poor prognosis. PNO1 expression was positively associated with tumour stage, lymph node metastasis and poor survival. PNO1 expression was significantly higher in HCC compared to that in fibrolamellar carcinoma or normal tissues. Furthermore, HCC tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53. PNO1 knockout suppressed cell viability, colony formation and EMT of HCC cells. Since activation of Notch signalling pathway promotes HCC, we measured the effects of PNO1 knockout on the components of Notch pathway and its targets. PNO1 knockout suppressed Notch signalling by modulating the expression of Notch ligands and their receptors, and downstream targets. PNO1 knockout also inhibited genes involved in surface adhesion, cell cycle, inflammation and chemotaxis. PNO1 knockout also inhibited colony and spheroid formation, cell migration and invasion, and markers of stem cells, pluripotency and EMT in CSCs. Overall, our data suggest that PNO1 can be used as a diagnostic and prognostic biomarker of HCC, and knockout of PNO1 by CRISPR/Cas9 can be beneficial for the management of HCC by targeting CSCs.

Abstract 381: DUSP12 in glioblastoma: Insights into nucleolar stress and DNA damage response

Abstract Glioblastoma (GBM), an extremely aggressive brain tumor, presents molecular complexities that require enormous challenges for its better understanding. The relatively unexplored dual-specificity phosphatase 12 (DUSP12) is an attractive target due to its zinc finger-like domain suggesting potential interactions with nucleic acids and ribonucleoproteins. This highly conserved enzyme plays a pivotal role in ribosome biogenesis across eukaryotes. Importantly, cell cycle arrest induced by DNA damage often coincides with alterations in ribosome biogenesis, a phenomenon termed nucleolar stress, linked to changes in the localization and activity of nucleophosmin (NPM) and p53 proteins. Bioinformatic analysis of public databases revealed elevated DUSP12 expression in glioblastoma samples compared to normal tissue, displaying strong positive correlations with p53 and NPM. While DUSP12 expression did not impact tumor progression in wild-type p53 patients, its lower expression was associated with increased tumor progression in p53-mutant cases, emphasizing the significance of p53 status in understanding DUSP12's roles in GBM. Gene ontology analysis of DUSP12-correlated genes in glioblastoma patients showed robust enrichment in biological processes like ribosome biogenesis, chromosome organization, cell cycle, and DNA repair, with a substantial number of nucleoplasmic and nucleolar proteins being recently identified by our group through mass spectrometry as DUSP12 partners in other tumor models. A substantial difference in DUSP12 protein levels between the GBM cell lines U87MG (wild-type p53) and U138MG (mutant p53) was observed, with the latter presenting higher levels of this phosphatase. Some biological responses of these cell lines were assessed after exposure to the topoisomerase inhibitors doxorubicin (DX) and camptothecin (CPT) through viability assays in adherent and spheroid cultures, γH2AX foci formation, and ATM/Chk2 activation, showing marginal differences between the cell lines. Notably, DX treatment displayed strong nucleolar stress features, such as alterations in the number and size of nucleoli observed by NPM subcellular distribution changes in both cell lines, and induction of p21 expression only in the p53-profficient U87MG. These alterations were accompanied by nuclear accumulation of DUSP12, underlining its potential regulatory role in this process. The knockdown of DUSP12 with specific siRNA led to a substantial decrease of the treatment responsiveness in U138MG, consistent with the lower levels of this phosphatase associated with a more aggressive phenotype in mutant p53 patients. In summary, DUSP12 emerges as a pivotal player in glioblastoma, influencing nucleolar stress dynamics and tumor aggressiveness, particularly in the context of p53 mutations. Citation Format: Viktor Kalbermatter Boell, Fábio Luís Forti. DUSP12 in glioblastoma: Insights into nucleolar stress and DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 381.

Abstract A037: Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS)

Abstract Heterozygous germline variants in DDX41 are identified in ~5% of patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). Recognition of the hereditary nature of such cancers and a better understanding of the factors that affect progression to malignancy has deep implications for health surveillance of patients and relatives that share a pathogenic DNA variant. DDX41 has a role in ribosome biogenesis and in opposing R-loop accumulation at promoters, but the mechanisms through which DDX41 loss drives cancer development are poorly understood. In a collaboration between Cambridge, UK and Mayo Clinic, USA we identified 85 patients with germline DDX41 variants [73/85 with germline pathogenic variants (GPV) and 12 with variants of uncertain significance (VUS)]. The majority were identified through targeted myeloid NGS upon presentation with AML (30/85) or MDS (44/85). Average age at diagnosis was 65 (35-93) with male predominance (57/85). The most common GPV was the NM_016222.4(DDX41):c.415_418dup; p.D140Gfs*2 (15/85). The majority (53.4% 39/73), also acquired a somatic variants in the other DDX41 allele (referred to as GPV+Som). The most common somatic variant (82% 32/39) was the R525H, a variant predicted to reduce the ATPase function of the helicase domain. We hypothesise that the markedly reduced DDX41 activity in cells with biallelic variants, is a key cause of progression to overt malignancy. In our cohort, we observed that somatic variants occurred at a similar frequency in association with start loss, truncating, splice, in-frame deletion, and missense GPV. However, whereas MDS was the predominant diagnosis within GPV (67.6% 23/34, AML 26.4% 9/34), AML was the most frequent diagnosis within GPV+Som (AML 51%.2 20/39, MDS 38.4% 15/39). Overall, there was a significantly higher likelihood of AML in the presence of a somatic variant (p<0.05), suggesting that there might be a dosage effect on the phenotype. This pattern was observed across all the GPV, with the exception of missense, suggesting that many might be benign polymorphysms. In addition, although somatic variants are typically subclonal (overall mean variant allele fraction VAF=9.87%, 3-27.8%), we observed a higher somatic VAF in AML over MDS cases (12.4% vs 7.7% respectively p<0.05). A lower bone marrow cellularity was also characteristic of GPV+Som vs GPV alone (mean observed/expected cellularity for age 0.9 vs 1.32 respectively p<0.05). In two cases, serial bone marrow morphology and NGS data were available, and we observed marked reduction in bone marrow cellularity upon acquisition of the R525H clone. The mechanisms of AML evolution appear different in DDX41 HMMS compared to sporadic cases. Interestingly, over half of our patients (21/34 GPV and 23/39 GPV+Som) did not exhibit additional somatic drivers. Our work suggests that acquisition of a somatic variant is a critical event in disease progression. Furthermore, the near universally subclonal nature of somatic variants suggest that they may affect disease phenotype through non-cell autonomous mechanisms. Citation Format: Ludovica Marando, Talha Badar, Yael Kusne, Audrey Morris, Terra Lasho, Abhishek Mangaonkar, Christy Finke, Carles Crawley, James M. Foran, Aref Al-Kali, Andrew King, Hassan Alkhateeb, Naseema Gangat, Chi Wong, Rong He, David Viswanatha, Faisal Basheer, Mark Litzow, Alejandro Ferrer, George Vassiliou, Mrinal Patnaik. Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A037.

New Functional Motifs for the Targeted Localization of Proteins to the Nucleolus in Drosophila and Human Cells.

The nucleolus is a significant nuclear organelle that is primarily known for its role in ribosome biogenesis. However, emerging evidence suggests that the nucleolus may have additional functions. Particularly, it is involved in the organization of the three-dimensional structure of the genome. The nucleolus acts as a platform for the clustering of repressed chromatin, although this process is not yet fully understood, especially in the context of Drosophila. One way to study the regions of the genome that cluster near the nucleolus in Drosophila demands the identification of a reliable nucleolus-localizing signal (NoLS) motif(s) that can highly specifically recruit the protein of interest to the nucleolus. Here, we tested a series of various NoLS motifs from proteins of different species, as well as some of their combinations, for the ability to drive the nucleolar localization of the chimeric H2B-GFP protein. Several short motifs were found to effectively localize the H2B-GFP protein to the nucleolus in over 40% of transfected Drosophila S2 cells. Furthermore, it was demonstrated that NoLS motifs derived from Drosophila proteins exhibited greater efficiency compared to that of those from other species.

Cellular and Molecular Analysis of Hematopoietic Stem and Progenitor Cells in Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an inherited multi-organ disorder with bone marrow failure (BMF), which may include neutropenia, anemia, and thrombocytopenia, as well as a high risk of myelodysplasia and leukemia. Hematological complications are common causes of morbidity and mortality in SDS. Around 90% of SDS patients have biallelic mutations in the Shwachman-Bodian-Diamond Syndrome ( SBDS) gene, which plays an important role in ribosome biogenesis and hematopoietic cell survival. In a collaborative study led by Dr. John Dick (Science 2016) novel hematopoietic progenitor subsets were identified (noted as F1, F2 and F3), of which the abundance, clonal capacity, and function in disease states remain unknown. We hypothesized that cellular and molecular analysis of hematopoietic stem and progenitor cells (HSPC) including these novel progenitors would provide valuable insights into the pathogenesis of BMF in SDS. Using multiparametric flow cytometry we quantified the following HSPC populations: hematopoietic stem cells (HSC), multipotent progenitors (MPP F1-F3), common myeloid progenitors (CMP F1-F3), megakaryocyte-erythroid progenitors (MEP F1-F3) and granulocyte-monocyte progenitor (GMP) in 13 SDS patients with SBDS mutations and 10 normal control donors. We found a significant reduction in CD34+/CD38- and CD34+/CD38+ cells in SDS patients. It is particularly noteworthy that the sub-populations of CMPs (F1 and F2), and MEPs (F2 and F3) were prominently decreased. These cells contribute to the production of myeloid, erythroid, and megakaryocytic cells with different proportions. Using single-cell colony-forming assays, we assessed the cloning efficiency of each HSPC, revealing that HSCs, MPPs and GMPs from SDS patients exhibited a significantly lower cloning efficiency compared to those from control subjects. Notably, the typical erythroid-committing progenitor cells (CMP F2, CMP F3, MEP F2, and MEP F3) in SDS collectively produced approximately 46% fewer erythroid cells per colony compared to control progenitors, while myeloid-committing progenitor cells (CMP F1, MEP F1) from these patients produced approximately 86% fewer myeloid cells, including neutrophils, per colony compared to the corresponding control cells. Further, HSCs and MPPs-F1 cells from SDS patients had a reduced cloning efficiency of approximately 22% and 27%, respectively, compared to control HSCs and MPP-F1 progenitors. These data suggest that multiple HSPC populations from SDS patients are characterized by reduced cell numbers, functional impairments, and dysregulation compared to normal subjects. Next, we performed transcriptome analysis of each HSPC by RNA sequencing. We revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS. Using KEGG pathway analysis, we found that downregulation of the ribosome pathway reached statistical significance in HSC, CMP F1 and MEP F3. Downregulation of the hematopoietic cell lineage pathway reached statistical significance in MEP F1-2 and MPP F1-2. Additionally, the TGF-beta signaling pathway was suppressed in CMP F1-3, MPP F1, and GMP, while the NF-kappa B signaling pathway, crucial for regulating the survival, activation and differentiation of innate immune cells and inflammatory T cells, was also found to be decreased in GMP. Interrogation of specific dysregulate genes, we found that the hematopoietic transcription factor, ZFP36 (ZFP36 ring finger protein) which involved in cellular response to cytokines and growth factors, was significantly downregulated in five subpopulations of SDS HSPCs, particularly noticeable in the four progenitors (CMP F1-3, MEP F2, and GMP) which produced substantially fewer colonies in SDS patients compared to controls. Additionally, we observed downregulation of TGFB2 and upregulation of NFKBIA and NFKBIB may inhibit the NF-kappa B signaling pathway in SDS patients. These alterations have the potential to influence the hematopoietic cell lineage pathway, leading to BMF. Functional assays are ongoing and aim to assess the impact of these dysregulated genes on formation and behavior of HSPCs. In summary, our data suggest that multiple SDS HSPC populations are characterized by reduced cell numbers and functional impairments. As well, we revealed dysregulated pathways and genes with the potential to disrupt hematopoiesis and mediate BMF in SDS.

The Signaling of Neuregulin-Epidermal Growth Factor Receptors and Its Impact on the Nervous System

The activation of members of the Epidermal Growth Factor Receptor (EGFR) family (including ErbB) triggers pathways that have significant effects on cellular processes and have profound consequences both in physiological and pathological conditions. Within the nervous system, the neuregulin (NRG)/ErbB3 signaling plays a crucial role in promoting the formation and maturation of excitatory synapses. Noteworthy is ErbB3, which is actively involved in the process of cerebellar lamination and myelination. All members of the ErbB-family, in particular ErbB3, have been observed within the nuclei of various cell types, including both full-length receptors and alternative variants. One of these variants was detected in Schwann cells and in glioblastoma primary cells where it showed a neuregulin-dependent expression. It binds to promoters’ chromatin associated with genes, like ezrin, involved in the formation of Ranvier’s node. Its nucleolar localization suggests that it may play a role in ribosome biogenesis and in cell proliferation. The regulation of ErbB3 expression is a complex and dynamic process that can be influenced by different factors, including miRNAs. This mechanism appears to play a significant role in glioblastoma and is often associated with a poor prognosis. Altogether, the targeting of ErbB3 has emerged as an active area of research in glioblastoma treatment. These findings highlight the underappreciated role of ErbB3 as a significant receptor that can potentially play a pivotal role in diverse pathologies, implying the existence of a shared and intricate mechanism that warrants further investigation.

Nucleolin is required for multiple centrosome-associated functions in early vertebrate mitosis.

Nucleolin is a multifunctional RNA-binding protein that resides predominantly not only in the nucleolus, but also in multiple other subcellular pools in the cytoplasm in mammalian cells, and is best known for its roles in ribosome biogenesis, RNA stability, and translation. During early mitosis, nucleolin is required for equatorial mitotic chromosome alignment prior to metaphase. Using high resolution fluorescence imaging, we reveal that nucleolin is required for multiple centrosome-associated functions at the G2-prophase boundary. Nucleolin depletion led to dissociation of the centrosomes from the G2 nuclear envelope, a delay in the onset of nuclear envelope breakdown, reduced inter-centrosome separation, and longer metaphase spindles. Our results reveal novel roles for nucleolin in early mammalian mitosis, establishing multiple important functions for nucleolin during mammalian cell division.

Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity

Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used. Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

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.

Inhibition of ribosome assembly factor PNO1 by CRISPR/Cas9 technique suppresses lung adenocarcinoma and Notch pathway: Clinical application.

Growth is crucially controlled by the functional ribosomes available in cells. To meet the enhanced energy demand, cancer cells re-wire and increase their ribosome biogenesis. The RNA-binding protein PNO1, a ribosome assembly factor, plays an essential role in ribosome biogenesis. The purpose of this study was to examine whether PNO1 can be used as a biomarker for lung adenocarcinoma and also examine the molecular mechanisms by which PNO1 knockdown by CRISPR/Cas9 inhibited growth and epithelial-mesenchymal transition (EMT). The expression of PNO1 was significantly higher in lung adenocarcinoma compared to normal lung tissues. PNO1 expression in lung adenocarcinoma patients increased with stage, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53, suggesting the influence of Tp53 status on PNO1 expression. PNO1 knockdown inhibited cell viability, colony formation, and EMT, and induced apoptosis. Since dysregulated signalling through the Notch receptors promotes lung adenocarcinoma, we measured the effects of PNO1 inhibition on the Notch pathway. PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors, their ligands, and downstream targets. PNO1 knockdown also suppressed CCND1, p21, PTGS-2, IL-1α, IL-8, and CXCL-8 genes. Overall, our data suggest that PNO1 can be used as a diagnostic biomarker, and also can be an attractive therapeutic target for the treatment of lung adenocarcinoma.

Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA

The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low.

NPM1 Mutation Quantitation Evaluated By a Commercially Available Assay and the Cepheid Prototype‡ Xpert® NPM1 Mutation Assay

Aims The objective of this study was to compare the performance of the novel Cepheid Prototype Xpert® NPM1 Mutation Assay to the Qiagen ipsogen® NPM1 mut A, B&D MutaQuant Kits using samples contrived using cell line-spiked peripheral blood. Our goal was to evaluate the comparative performance of a faster and easier to use assay than commercially available products for use in the monitoring of minimal residual disease (MRD) in NPM1-positive AML patients. Background A common mutation in AML (20-30% of cases) is a four base pair insertion in the NPM1 gene.¹ NPM1 plays a role in ribosome biogenesis, maintaining genomic stability, and modulating growth suppression by binding and stabilizing p53.² NPM1 mutant A consists of about 80% of NPM1 mutations, while mutants B or D are present in a sizeable minority of patients.¹² Monitoring of NPM1 mutations can provide prognostic value and can assist with choice of treatment.³ Methods Three NPM1-positive cell lines, OCI-AML3 MutA, OCI-AML3 MutB, and OCI-AML3 MutD, which express NPM1 MutA, NPM1 Mut B, and NPM1 Mut D, respectively, were grown and isolated. After processing the cells into cell lysate, the cell-lysates were spiked into NPM1-negative EDTA whole blood lysate and serially diluted to target 13 or 14 NPM1/ABL levels from approximately 2500% (log reduction (LR) 1.4) to approximately 0.01% (LR -4.0). A subset of each level underwent hands-off sample preparation, total RNA extraction, and RT-qPCR all within the Cepheid GeneXpert automated cartridge system, and another subset had total RNA extracted using the Zymo Direct-zol™ RNA Miniprep Plus Kit. RNA was qualified and quantified using the Thermo Scientific NanoDrop™ 2000 Spectrophotometer before being tested per the comparator assay vendor's recommendations. The Cepheid Prototype assay utilized 4.5mL of lysate, whereas the Qiagen kits used 100 ng of RNA per test. Specimens spiked with cell lines expressing different NPM1 mutations were tested using the respective ipsogen kit designed for that mutation. The Cepheid Prototype assay was evaluated against the comparator assay for each subtype using Deming regressions. Bland-Altman plots were utilized to evaluate bias between assay quantitation outputs. Results Deming regressions and associated Bland-Altman plots showed a high degree of correlation and agreement between comparator assays and the Cepheid Prototype assay (Figure 1). Conclusions The novel Cepheid Prototype assay showed a high level of agreement with Qiagen kits in this study using whole blood EDTA lysate samples spiked with three NPM1-positive cell-lysates. Agreement tended to decline at low input levels, and a small but acceptable degree of nonlinearity was present in MutA and MutD. Total time to result (TTR) was minimized in the Cepheid Prototype assay to under 170 minutes, while the comparator assay had a TTR of approximately six hours plus RNA extraction. While RNA extraction is performed within Cepheid Prototype assay cartridge and is included in this 170-minute TTR, the Qiagen kit requires additional expensive and labor-intensive extraction steps that can last one-hour to multiple days, which are not included in the six-hour TTR estimate. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal