TGF-ß2 Induces Ribosome Activity, Alters Ribosome Composition and Inhibits IRES-Mediated Translation in Chondrocytes.

Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.

An integrative platform for detection of RNA 2'-O-methylation reveals its broad distribution on mRNA.

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers.

Subverting the Canon: Novel Cancer-Promoting Functions and Mechanisms for snoRNAs.

Small nucleolar RNAs (snoRNAs) constitute a class of intron-derived non-coding RNAs ranging from 60 to 300 nucleotides. Canonically localized in the nucleolus, snoRNAs play a pivotal role in RNA modifications and pre-ribosomal RNA processing. Based on the types of modifications they involve, such as methylation and pseudouridylation, they are classified into two main families-box C/D and H/ACA snoRNAs. Recent investigations have revealed the unconventional synthesis and biogenesis strategies of snoRNAs, indicating their more profound roles in pathogenesis than previously envisioned. This review consolidates recent discoveries surrounding snoRNAs and provides insights into their mechanistic roles in cancer. It explores the intricate interactions of snoRNAs within signaling pathways and speculates on potential therapeutic solutions emerging from snoRNA research. In addition, it presents recent findings on the long non-coding small nucleolar RNA host gene (lncSNHG), a subset of long non-coding RNAs (lncRNAs), which are the transcripts of parental SNHGs that generate snoRNA. The nucleolus, the functional epicenter of snoRNAs, is also discussed. Through a deconstruction of the pathways driving snoRNA-induced oncogenesis, this review aims to serve as a roadmap to guide future research in the nuanced field of snoRNA-cancer interactions and inspire potential snoRNA-related cancer therapies.

2'-O-methylation (Nm) in RNA: progress, challenges, and future directions.

RNA 2'-O-methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5' cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs has been demonstrated to impact cellular processes, including translation and splicing. In addition, Nm modifications at the 5' cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self-mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field.

ZNF692 regulates nucleolar morphology by interacting with NPM1 and modifying its self-assembly properties.

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells.

Dissecting the oncogenic properties of essential RNA-modifying enzymes: a focus on NAT10.

Chemical modifications of ribonucleotides significantly alter the physicochemical properties and functions of RNA. Initially perceived as static and essential marks in ribosomal RNA (rRNA) and transfer RNA (tRNA), recent discoveries unveiled a dynamic landscape of RNA modifications in messenger RNA (mRNA) and other regulatory RNAs. These findings spurred extensive efforts to map the distribution and function of RNA modifications, aiming to elucidate their distribution and functional significance in normal cellular homeostasis and pathological states. Significant dysregulation of RNA modifications is extensively documented in cancers, accentuating the potential of RNA-modifying enzymes as therapeutic targets. However, the essential role of several RNA-modifying enzymes in normal physiological functions raises concerns about potential side effects. A notable example is N-acetyltransferase 10 (NAT10), which is responsible for acetylating cytidines in RNA. While emerging evidence positions NAT10 as an oncogenic factor and a potential target in various cancer types, its essential role in normal cellular processes complicates the development of targeted therapies. This review aims to comprehensively analyze the essential and oncogenic properties of NAT10. We discuss its crucial role in normal cell biology and aging alongside its contribution to cancer development and progression. We advocate for agnostic approaches to disentangling the intertwined essential and oncogenic functions of RNA-modifying enzymes. Such approaches are crucial for understanding the full spectrum of RNA-modifying enzymes and imperative for designing effective and safe therapeutic strategies.

Comparative Mitogenome Analyses of Fifteen Ramshorn Snails and Insights into the Phylogeny of Planorbidae (Gastropoda: Hygrophila).

Ramshorn snails from the family Planorbidae are important freshwater snails due to their low trophic level, and some of them act as intermediate hosts for zoonotic trematodes. There are about 250 species from 40 genera of Planorbidae, but only 14 species from 5 genera (Anisus, Biomphalaria, Bulinus, Gyraulus, and Planorbella) have sequenced complete mitochondrial genomes (mitogenomes). In this study, we sequenced and assembled a high-quality mitogenome of a ramshorn snail, Polypylis sp. TS-2018, which represented the first mitogenome of the genus. The mitogenome of Polypylis sp. TS-2018 is 13,749 bp in length, which is shorter than that of most gastropods. It contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA). We compared mitogenome characteristics, selection pressure, and gene rearrangement among all of the available mitogenomes of ramshorn snails. We found that the nonsynonymous and synonymous substitution rates (Ka/Ks) of most PCGs indicated purifying and negative selection, except for atp8 of Anisus, Biomphalaria, and Gyraulus, which indicated positive selection. We observed that transpositions and reverse transpositions occurred on 10 tRNAs and rrnS, which resulted in six gene arrangement types. We reconstructed the phylogenetic trees using the sequences of PCGs and rRNAs and strongly supported the monophyly of each genus, as well as three tribes in Planorbidae. Both the gene rearrangement and phylogenetic results suggested that Polypylis had a close relationship with Anisus and Gyraulus, while Bulinus was the sister group to all of the other genera. Our results provide useful data for further investigation of species identification, population genetics, and phylogenetics among ramshorn snails.

C2-methyladenosine in tRNA promotes protein translation by facilitating the decoding of tandem m2A-tRNA-dependent codons.

RNA modification C2-methyladenosine (m2A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m2A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m2A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m2A-modified chloroplast tRNAs and two m2A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m2A methyltransferases-RLMNL1, RLMNL2, and RLMNL3-which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m2A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m2A, uncovering a layer of translation regulation in plants.

Combinatorial and frequency properties of the ribosome ancestors.

BACKGROUND: The current ribosome has evolved from the primitive stages of life on Earth. Its function is to build proteins and on the basis of this role, we are looking for a universal common ancestor to the ribosome which could: i) present optimal combinatorial properties, and ii) have left vestiges in the current molecules composing the ribosome (rRNA or r-proteins) or helping in its construction and functioning. METHODS: Genomic public databases are used for finding the nucleotide sequences of rRNAs and mRNA of r-proteins and statistical calculations are performed on the occurrence in these genes of some pentamers belonging to the RNA proposed as optimal ribosome ancestor. RESULTS: After having exhibited a possible solution to the problem of an RNA capable of catalyzing peptide genesis, traces of this RNA are found in many rRNAs and mRNA of r-proteins, as well as in factors contributing to the construction of the current ribosome. CONCLUSIONS: The existence of an optimal primordial RNA whose function is to facilitate the creation of peptide bonds between amino acids may have contributed to accelerate the emergence of the first vital processes. Its traces should be found in many living species inside structures structurally and functionally close to the ribosome, which is already the case in the species studied in this article.

Phylogenetic relationships and adaptation in deep-sea carideans revealed by mitogenomes.

The deep-sea environment is characterized by extreme and inhospitable conditions, including oxygen depletion, low temperatures, high pressure, absence of light, and limited food availability. Mitochondria and mitogenomes play a crudial role in aerobic respiration to generate energy for eukaryotes. Here, using the Illumina Hiseq 4000 platform, we performed mitogenome sequencing for five deep-sea caridean species: Lebbeus shinkaiae, Lebbeus Formosus, Glyphocrangon regalis, Heterocarpus dorsalis, and Heterocarpus laevigatus, and five deep-sea caridean mitogenomes were assembled and identified. Each of the five mitogenomes contained 13 protein-coding genes, 2 rRNAs and 22 tRNAs. Specific elements, such as tandem repeats and AT-rich sequences, were observed in the control regions of Lebbeus formosus and Lebbeus shinkaiae, potentially take a role in regulating mitochondrial genome replication and transcription. The gene order of all obtained mitogenomes follows caridean ancestral type organization. Phylogenetic analysis shows a robustly supported phylogenetic tree for the infraorder Caridea. The monophyly of the families included in this study was strongly supported. This study supports the monophyly of Oplophoroidea, but rejects the monophyletic status of Nematocarcinoidea, Crangonoidea, and Alpheoidea. At the genus level, Plesionika is polyphyletic and Rimicaris is paraphyletic in our analysis. Furthermore, Paralebbeus may be considered invalid and synonymous with Lebbeus. Positive selection analysis reveals evidence for adaptive changes in the mitogenome of different deep-sea caridean lineages. Nine residues located in cox1, cox3, atp6, nad1, nad2, nad4, nad5, nad6 and cytb were determined to have undergone positive selection. Mitogenome of different deep-sea lineages experienced different positive selection, and the lineage represented by Alvinocarididae living in deep-sea hydrothermal vents experienced the strongest positive selection. This study provides valuable insights into the adaptive evolution of deep-sea shrimps at the mitochondrial, highlighting the mitogenomic strategy that contribute to their unique adaptations in the deep-sea environment.

MINRbase: a comprehensive database of nuclear- and mitochondrial-ribosomal-RNA-derived fragments (rRFs).

We describe the Mitochondrial and Nuclear rRNA fragment database (MINRbase), a knowledge repository aimed at facilitating the study of ribosomal RNA-derived fragments (rRFs). MINRbase provides interactive access to the profiles of 130 238 expressed rRFs arising from the four human nuclear rRNAs (18S, 5.8S, 28S, 5S), two mitochondrial rRNAs (12S, 16S) or four spacers of 45S pre-rRNA. We compiled these profiles by analyzing 11 632 datasets, including the GEUVADIS and The Cancer Genome Atlas (TCGA) repositories. MINRbase offers a user-friendly interface that lets researchers issue complex queries based on one or more criteria, such as parental rRNA identity, nucleotide sequence, rRF minimum abundance and metadata keywords (e.g. tissue type, disease). A 'summary' page for each rRF provides a granular breakdown of its expression by tissue type, disease, sex, ancestry and other variables; it also allows users to create publication-ready plots at the click of a button. MINRbase has already allowed us to generate support for three novel observations: the internal spacers of 45S are prolific producers of abundant rRFs; many abundant rRFs straddle the known boundaries of rRNAs; rRF production is regimented and depends on 'personal attributes' (sex, ancestry) and 'context' (tissue type, tissue state, disease). MINRbase is available at https://cm.jefferson.edu/MINRbase/.

Mitochondrial-derived peptides: Antidiabetic functions and evolutionary perspectives.

Mitochondrial-derived peptides (MDPs) are a novel class of bioactive microproteins encoded by short open-reading frames (sORF) in mitochondrial DNA (mtDNA). Currently, three types of MDPs have been identified: Humanin (HN), MOTS-c (Mitochondrial ORF within Twelve S rRNA type-c), and SHLP1-6 (small Humanin-like peptide, 1 to 6). The 12 S ribosomal RNA (MT-RNR1) gene harbors the sequence for MOTS-c, whereas HN and SHLP1-6 are encoded by the 16 S ribosomal RNA (MT-RNR2) gene. Special genetic codes are used in mtDNA as compared to nuclear DNA: (i) ATA and ATT are used as start codons in addition to the standard start codon ATG; (ii) AGA and AGG are used as stop codons instead of coding for arginine; (iii) the standard stop codon UGA is used to code for tryptophan. While HN, SHLP6, and MOTS-c are encoded by the H (heavy owing to high guanine + thymine base composition)-strand of the mtDNA, SHLP1-5 are encoded by the L (light owing to less guanine + thymine base composition)-strand. MDPs attenuate disease pathology including Type 1 diabetes (T1D), Type 2 diabetes (T2D), gestational diabetes, Alzheimer's disease (AD), cardiovascular diseases, prostate cancer, and macular degeneration. The current review will focus on the MDP regulation of T2D, T1D, and gestational diabetes along with an emphasis on the evolutionary pressures for conservation of the amino acid sequences of MDPs.

Molecular mechanism of human ISG20L2 for the ITS1 cleavage in the processing of 18S precursor ribosomal RNA.

The exonuclease ISG20L2 has been initially characterized for its role in the mammalian 5.8S rRNA 3' end maturation, specifically in the cleavage of ITS2 of 12S precursor ribosomal RNA (pre-rRNA). Here, we show that human ISG20L2 is also involved in 18S pre-rRNA maturation through removing the ITS1 region, and contributes to ribosomal biogenesis and cell proliferation. Furthermore, we determined the crystal structure of the ISG20L2 nuclease domain at 2.9 Å resolution. It exhibits the typical αßα fold of the DEDD 3'-5' exonuclease with a catalytic pocket located in the hollow near the center. The catalytic residues Asp183, Glu185, Asp267, His322 and Asp327 constitute the DEDDh motif in ISG20L2. The active pocket represents conformational flexibility in the absence of an RNA substrate. Using structural superposition and mutagenesis assay, we mapped RNA substrate binding residues in ISG20L2. Finally, cellular assays revealed that ISG20L2 is aberrantly up-regulated in colon adenocarcinoma and promotes colon cancer cell proliferation through regulating ribosome biogenesis. Together, these results reveal that ISG20L2 is a new enzymatic member for 18S pre-rRNA maturation, provide insights into the mechanism of ISG20L2 underlying pre-rRNA processing, and suggest that ISG20L2 is a potential therapeutic target for colon adenocarcinoma.

Roles of rRNA N-methyladenosine modification in the function of ribosomes.

The N-methyladenosine (m6A) modification of ribosomal RNA (rRNA) plays critical roles in regulating the function of ribosomes, the essential molecular machines that translate genetic information from mRNA into proteins. Specifically, m6A modification affects ribosome biogenesis, stability, and function by regulating the processing and maturation of rRNA, the assembly and composition of ribosomes, and the accuracy and efficiency of translation. Furthermore, m6A modification allows for dynamic regulation of translation in response to environmental and cellular signals. Therefore, a deeper understanding of the mechanisms and functions of m6A modification in rRNA will advance our knowledge of ribosome-mediated gene expression and facilitate the development of new therapeutic strategies for ribosome-related diseases.

SNORA56-mediated pseudouridylation of 28 S rRNA inhibits ferroptosis and promotes colorectal cancer proliferation by enhancing GCLC translation.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies and is characterized by reprogrammed metabolism. Ferroptosis, a programmed cell death dependent on iron, has emerged as a promising strategy for CRC treatment. Although small nucleolar RNAs are extensively involved in carcinogenesis, it is unclear if they regulate ferroptosis during CRC pathogenesis. METHODS: The dysregulated snoRNAs were identified using published sequencing data of CRC tissues. The expression of the candidate snoRNAs, host gene and target gene were assessed by real-time quantitative PCR (RT-qPCR), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and western blots. The biological function of critical molecules was investigated using in vitro and in vivo strategies including Cell Counting Kit-8 (CCK8), colony formation assay, flow cytometry, Fe2+/Fe3+, GSH/GSSG and the xenograft mice models. The ribosomal activities were determined by polysome profiling and O-propargyl-puromycin (OP-Puro) assay. The proteomics was conducted to clarify the downstream targets and the underlying mechanisms were validated by IHC, Pearson correlation analysis, protein stability and rescue assays. The clinical significance of the snoRNA was explored using the Cox proportional hazard model, receiver operating characteristic (ROC) and survival analysis. RESULTS: Here, we investigated the SNORA56, which was elevated in CRC tissues and plasma, and correlated with CRC prognosis. SNORA56 deficiency in CRC impaired proliferation and triggered ferroptosis, resulting in reduced tumorigenesis. Mechanistically, SNORA56 mediated the pseudouridylation of 28 S rRNA at the U1664 site and promoted the translation of the catalytic subunit of glutamate cysteine ligase (GCLC), an indispensable rate-limiting enzyme in the biosynthesis of glutathione, which can inhibit ferroptosis by suppressing lipid peroxidation. CONCLUSIONS: Therefore, the SNORA56/28S rRNA/GCLC axis stimulates CRC progression by inhibiting the accumulation of cellular peroxides, and it may provide biomarker and therapeutic applications in CRC.

Comparative mitochondrial genome analysis of three leafhopper species of the genus Abrus Dai & Zhang (Hemiptera: Cicadellidae: Deltocephalinae) from China with phylogenetic implication.

BACKGROUND: The phylogenetic position and classification of Athysanini are poorly defined, as it includes a large group of polyphyletic genera that have historically been assigned to it mainly because they still exhibit the most typical deltocephaline genitalic and external body characters but lack the distinctive characteristics that other tribes possess. The bamboo-feeding leafhopper genus Abrus belong to the tribe Athysanini of subfamily Deltocephalinae, which currently comprises 19 valid described species, and are limited to the Oriental and Palaearctic regions in China. Although the taxonomy of Abrus are well updated, the references on comparative mitogenomic analyses of Abrus species are only known for a single species. In this study, we sequenced and analyzed the complete mitochondrial genomes (mitogenomes) of Abrus daozhenensis Chen, Yang & Li, 2012 (16,391bp) and A. yunshanensis Chen, Yang & Li, 2012 (15,768bp) (Athysanini), and compared with published mitogenome sequence of A. expansivus Xing & Li, 2014 (15,904bp). RESULTS: These Abrus species shared highly conserved mitogenomes with similar gene order to that of the putative ancestral insect with 37 typical genes and a non-coding A + T-rich region. The nucleotide composition of these genomes is highly biased toward A + T nucleotides (76.2%, 76.3%, and 74.7%), AT-skews (0.091 to 0.095, and 0.095), negative GC-skews (- 0.138, - 0.161, and - 0.138), and codon usage. All 22 tRNA genes had typical cloverleaf secondary structures, except for trnS1 (AGN) which lacks the dihydrouridine arm, and distinctively trnG in the mitogenome of A. expansivus lacks the TψC arm. Phylogenetic analyses based on 13 PCGs, 2 rRNA genes, and 22 tRNA genes consistently recovered the monophyletic Opsiini, Penthimiini, Selenocephalini, Scaphoideini, and Athysanini (except Watanabella graminea, previously sequenced species as Chlorotettix nigromaculatus) based on limited available mitogenome sequence data of 37 species. CONCLUSION: At present, Abrus belongs to the tribe Athysanini based on both morphological and molecular datasets, which is strongly supported in present phylogenetic analyses in both BI and ML methods using the six concatenated datasets: amino acid sequences and nucleotides from different combinations of protein-coding genes (PCGs), ribosomal RNA (rRNAs), and transfer RNA (tRNAs). Phylogenetic trees reconstructed herein based on the BI and ML analyses consistently recovered monophylitic Athysanini, except Watanabella graminea (Athysanini) in Opsiini with high support values.

The Effects of Deregulated Ribosomal Biogenesis in Cancer.

Ribosomes are macromolecular ribonucleoprotein complexes assembled from RNA and proteins. Functional ribosomes arise from the nucleolus, require ribosomal RNA processing and the coordinated assembly of ribosomal proteins (RPs), and are frequently hyperactivated to support the requirement for protein synthesis during the self-biosynthetic and metabolic activities of cancer cells. Studies have provided relevant information on targeted anticancer molecules involved in ribosome biogenesis (RiBi), as increased RiBi is characteristic of many types of cancer. The association between unlimited cell proliferation and alterations in specific steps of RiBi has been highlighted as a possible critical driver of tumorigenesis and metastasis. Thus, alterations in numerous regulators and actors involved in RiBi, particularly in cancer, significantly affect the rate and quality of protein synthesis and, ultimately, the transcriptome to generate the associated proteome. Alterations in RiBi in cancer cells activate nucleolar stress response-related pathways that play important roles in cancer-targeted interventions and immunotherapies. In this review, we focus on the association between alterations in RiBi and cancer. Emphasis is placed on RiBi deregulation and its secondary consequences, including changes in protein synthesis, loss of RPs, adaptive transcription and translation, nucleolar stress regulation, metabolic changes, and the impaired ribosome biogenesis checkpoint.

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

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

Downregulation of ribosomal RNA (rRNA) genes in human head and neck squamous cell carcinoma (HNSCC) cells correlates with rDNA promoter hypermethylation.

Eukaryotes carry hundreds of ribosomal RNA (rRNA) genes as tandem arrays, which generate rRNA for protein synthesis. Humans carry âˆ¼ 400 rRNA gene copies and their expression is epigenetically regulated. Dysregulation of rRNA synthesis and ribosome biogenesis are characteristic features of cancers. Targeting aberrant rRNA expression for cancer therapy is being explored. Head and neck squamous cell carcinoma (HNSCC) is among the most prevalent cancers globally. Using quantitative PCR and bisulfite sequencing, we show that rRNA genes are downregulated and their promoters are hypermethylated in HNSCC cell lines. These findings may have relevance for prognosis and diagnosis of HNSCC.