RNA Function Research Articles

M6A Methylated Long Noncoding RNA LOC339803 Regulates Intestinal Inflammatory Response.

Cytokine mediated sustained inflammation increases the risk to develop different complex chronic inflammatory diseases, but the implicated mechanisms remain unclear. Increasing evidence shows that long noncoding RNAs (lncRNAs) play key roles in the pathogenesis of inflammatory disorders, while inflammation associated variants are described to affect their function or essential RNA modifications as N6-methyladenosine (m6A) methylation, increasing predisposition to inflammatory diseases. Here, the functional implication of the intestinal inflammation associated lncRNA LOC339803 in the production of cytokines by intestinal epithelial cells is described. Allele-specific m6A methylation is found to affect YTHDC1 mediated protein binding affinity. LOC339803-YTHDC1 interaction dictates chromatin localization of LOC339803 ultimately inducing the expression of NFκB mediated proinflammatory cytokines and contributing to the development of intestinal inflammation. These findings are confirmed using human intestinal biopsy samples from different intestinal inflammatory conditions and controls. Additionally, it is demonstrated that LOC339803 targeting can be a useful strategy for the amelioration of intestinal inflammation in vitro and ex vivo. Overall, the results support the importance of the methylated LOC339803 lncRNA as a mediator of intestinal inflammation, explaining genetic susceptibility and presenting this lncRNA as a potential novel therapeutic target for the treatment of inflammatory intestinal disorders.

KARR-seq reveals cellular higher-order RNA structures and RNA-RNA interactions.

RNA fate and function are affected by their structures and interactomes. However, how RNA and RNA-binding proteins (RBPs) assemble into higher-order structures and how RNA molecules may interact with each other to facilitate functions remain largely unknown. Here we present KARR-seq, which uses N3-kethoxal labeling and multifunctional chemical crosslinkers to covalently trap and determine RNA-RNA interactions and higher-order RNA structures inside cells, independent of local protein binding to RNA. KARR-seq depicts higher-order RNA structure and detects widespread intermolecular RNA-RNA interactions with high sensitivity and accuracy. Using KARR-seq, we show that translation represses mRNA compaction under native and stress conditions. We determined the higher-order RNA structures of respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV) and identified RNA-RNA interactions between the viruses and the host RNAs that potentially regulate viral replication.

Intracellular RNA Labeling Technologies for the Analysis of RNA Biology†

Comprehensive SummaryAs a cornerstone of the central dogma of molecular biology, RNA plays vital roles in living organisms. Over the past few decades, many RNA labeling technologies have been developed to elucidate the biological function of RNA. These technologies have significantly advanced our understanding of RNA secondary structure, localization, and turnover. Additionally, taking advantage of these innovative RNA labeling approaches, plenty of tool kits have been devised for the regulation of RNA‐related biological process, such as gene expression and gene editing. In this review, we primarily focus on an array of intracellular RNA labeling methods, encompassing chemical probes‐based labeling, metabolic labeling, and proximity‐dependent labeling. We also provide a brief overview of their applications in the research of RNA biology. Finally, the perspectives of RNA labeling are also discussed.Key Scientists4SU was first used in1978 as a safer way to directly examine RNA synthesis. However, the enrichment of 4SU‐labeled RNA seriously relied on mercurated cellulose columns, which are toxic. In 2005, Boothroyd et al. introduced a novel covalent‐modification method of 4SU based on thiol exchange reaction. Then, Salic et al. applied 5EU, which is clickable, for RNA metabolic labeling in animals. Later, Spitale et al. designed 5EC and 2’AzU, which enabled cell specific RNA metabolic labeling under the help of specific enzyme. In 2013, directly chemical RNA labeling in living cell for RNA structure mapping was realized by Chang and Kool et al. using SHAPE reagent, which was further modified with an azide handle in 2015. Weissman, Bevilacqua and Assmann et al. introduced DMS probe that targets adenine and cytosine of RNA for RNA structure mapping in 2014. Then, Chang et al. exploited psoralen for RNA duplex mapping in 2016. Later, He, Zhou and Zhang et al. described another clickable probe N3‐kethoxal targeting guanine for RNA secondary structure mapping. Besides, Chang and Ting et al. described a proximity‐dependent RNA labeling method based on APEX in 2019, revealing subcellular RNA localization. Meanwhile, Zou and Wang et al. achieved proximity‐dependent RNA labeling using miniSOG oxidase, which provided an alternative method to investigate the spatial transcriptome. Spitale et al. utilizing photosensitizer dibromofluorescein (DBF) to realize proximity‐dependent RNA labeling and sequencing.

Abstract B069: Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing

Abstract Human satellite II (HSATII) composed of tandem repeats in pericentromeric regions in the human genome is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of the repetitive elements in cancer tissues can facilitate incidental double-stranded RNA formation. However, it remains controversial whether double-stranded RNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the formation of double-stranded HSATII (dsHSATII) RNA and explored its molecular functions. The overexpression of dsHSATII RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, STRBP, which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. Depletion of STRBP induced the mesenchymal transition and enhanced the invasiveness. Conversely, the epithelial–mesenchymal transition (EMT) of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with the EMT events, which was interfered by the overexpression of dsHSATII RNA. Finally, we confirmed that isoform switching of CLSTN1, one of the target genes of STRBP-associated alternative splicing, induced EMT-like morphological changes. These findings reveal a novel cell-autonomous function of dsHSATII RNA in enhancing malignant potential, providing a better understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors. Citation Format: Takahiro Kishikawa, Takuma Iwata, Motoyuki Otsuka, Mitsuhiro Fujishiro. Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B069.

FTO attenuates LPS-induced acute kidney injury by inhibiting autophagy via regulating SNHG14/miR-373-3p/ATG7 axis

N6‐methyladenosine (m6A) is a master driver of RNA function and implicates in the pathogenesis of renal injury. LncRNA SNHG14 is highly expressed in sepsis patients with acute kidney injury (AKI) and aggravates kidney cell dysfunction. This study aimed to explore whether demethylase FTO affect m6A methylation of SNHG14 in AKI injury and its underlying mechanism. The expression level of FTO was obviously downregulated in sepsis-associated AKI patients compared with normal controls. Mechanistically, FTO overexpression impeded SNHG14 expression by decreasing the stability of SNHG14 in an m6A-dependent manner in LPS-induced HK-2 cells. Additionally, FTO overexpression inhibited cell autophagy and apoptosis while promoting cell viability of LPS-induced HK-2 cells. Moreover, overexpression of FTO inhibited SNHG14 expression and autophagy in LPS-induced AKI mice. Functionally, SNHG14 acts as a competing endogenous RNA (ceRNA) via directly sponging miR-373-3p in LPS induced HK-2 cells. Additionally, miR-373-3p directly targets ATG7. Inhibition of SNHG14 suppresses NF-κB signaling pathway and production of inflammatory cytokines (TNF-α, IL-6, and IL-1β) via miR-373-3p/ATG7 in LPS-induced HK-2 cells. Furthermore, the SNHG14/miR-373-3p/ATG7 interaction network contributes to the regulatory effect of FTO on LPS-induced HK-2 cell viability, apoptosis and autophagy. These results suggested demethylase FTO suppressed the m6A modification of lncRNA SNHG14 and inhibits autophagy in LPS-induced AKI via regulating miR-373-3p/ATG7, which provided an important novel perspective for understanding sepsis-associated AKI and is conducive for developing new therapeutic targets and strategies.

Influence of ion and hydration atmospheres on RNA structure and dynamics: insights from advanced theoretical and computational methods.

RNA, a highly charged biopolymer composed of negatively charged phosphate groups, defies electrostatic repulsion to adopt well-defined, compact structures. Hence, the presence of positively charged metal ions is crucial not only for RNA's charge neutralization, but they also coherently decorate the ion atmosphere of RNA to stabilize its compact fold. This feature article elucidates various modes of close RNA-ion interactions, with a special emphasis on Mg2+ as an outer-sphere and inner-sphere ion. Through examples, we highlight how inner-sphere chelated Mg2+ stabilizes RNA pseudoknots, while outer-sphere ions can also exert long-range electrostatic interactions, inducing groove narrowing, coaxial helical stacking, and RNA ring formation. In addition to investigating the RNA's ion environment, we note that the RNA's hydration environment is relatively underexplored. Our study delves into its profound interplay with the structural dynamics of RNA, employing state-of-the-art atomistic simulation techniques. Through examples, we illustrate how specific ions and water molecules are associated with RNA functions, leveraging atomistic simulations to identify preferential ion binding and hydration sites. However, understanding their impact(s) on the RNA structure remains challenging due to the involvement of large length and long time scales associated with RNA's dynamic nature. Nevertheless, our contributions and recent advances in coarse-grained simulation techniques offer insights into large-scale structural changes dynamically linked to the RNA ion atmosphere. In this connection, we also review how different cutting-edge computational simulation methods provide a microscopic lens into the influence of ions and hydration on RNA structure and dynamics, elucidating distinct ion atmospheric components and specific hydration layers and their individual and collective impacts.

Exonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA.

Pseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio. This method, called RNA exonuclease-assisted identification of pseudouridine sites (RIPS), combines specific CMC-labeling of Ψ sites with an exonuclease-assisted digestion strategy for the detection of Ψ sites. Utilizing exonuclease XRN1 to digest RNA strands not labeled by CMC, RIPS significantly reduces the background signal from unlabeled strands and enhances the positive signal of Ψ sites labeled by CMC, which terminates exonuclease digestion. As a result, we can enrich Ψ sites and identify them at single-base resolution. Considering the unique functions of single-stranded RNA (ssRNA), we employed RIPS to distinguish Ψ sites in single-stranded and double-stranded regions of RNA. Our results indicated that CMC could specifically label Ψ sites in ssRNA under natural conditions, enabling RIPS to selectively identify Ψ sites in ssRNA, which may facilitate the study on the functions of Ψ sites.

Tumor Promoting Effect of Long Non-Coding RNA RP11-556E13.1 and its Clinical Significance in Hepatocellular Carcinoma.

The aim of this study was to reveal the function of the long non-coding RNA (lncRNA) RP11-556E13.1 (RP11) and its clinical significance in hepatocellular carcinoma (HCC). LncRNA and mRNA expression profiling was performed using lncRNA and mRNA microarrays in HCC and adjacent tissues. Human tissue samples were analyzed by semiquantitative real-time polymerase chain reaction (sqRT-PCR) to evaluate the expression of RP11. Smart silencer RNA (siRNA) was used to knockdown the expression of RP11 in HCC cells. The function of RP11 was determined by some cell function experiments in HCC cells. Western blotting (WB) was performed to detect proteins that were presumably associated with these function changes. An Affymetrix Human HTA2.0 microarray was used to detect the underlying mechanism of RP11 in HCC. lncRNA RP11 was the most significantly upregulated lncRNA in HCC tissues compared with the adjacent tissues (p < 0.05, fold change = 20.24). The expression of RP11 was significantly higher in HCC tissues compared to adjacent tissues in 112 tissue pairs (p < 0.05). The higher the expression of RP11 in HCC tissues, the bigger the tumor size, the poorer the histological differentiation, and the lower the overall survival rate of the patients (all p < 0.05). After the knockdown of RP11, HCC cells displayed inhibited proliferation, increased apoptosis rate, and G1/S arrest. Moreover, the expression of cleaved PARP1 and cleaved caspase-3 was increased. GO enrichment and KEGG pathway enrichment analysis showed some important pathways that might be related to the knockdown of RP11 in HCC cells. lncRNA RP11 is an HCC-promoting gene and a potential prognostic predictor of HCC.

Preservation of functionality, immunophenotype, and recovery of HIV RNA from PBMCs cryopreserved for more than 20 years.

Many research laboratories have long-term repositories of cryopreserved peripheral blood mononuclear cells (PBMC), which are costly to maintain but are of uncertain utility for immunological studies after decades in storage. This study investigated preservation of cell surface phenotypes and in-vitro functional capacity of PBMC from viraemic HIV+ patients and healthy seronegative control subjects, after more than 20 years of cryopreservation. PBMC were assessed by 18-colour flow cytometry for major lymphocyte subsets within T, B, NK, and dendritic cells and monocytes. Markers of T-cell differentiation and activation were compared with original immunophenotyping performed in 1995/1996 on fresh blood at the time of collection. Functionality of PBMC was assessed by culture with influenza antigen or polyclonal T-cell activation, to measure upregulation of activation-induced CD25 and CD134 (OX40) on CD4 T cells and cytokine production at day 2, and proliferative CD25+ CD4 blasts at day 7. RNA was extracted from cultures containing proliferating CD4+ blast cells, and intracellular HIV RNA was measured using short amplicons for both the Double R and pol region pi code assays, whereas long 4-kbp amplicons were sequenced. All major lymphocyte and T-cell subpopulations were conserved after long-term cryostorage, except for decreased proportions of activated CD38+HLA-DR+ CD4 and CD8 T cells in PBMC from HIV+ patients. Otherwise, differences in T-cell subpopulations between recent and long-term cryopreserved PBMC primarily reflected donor age-associated or HIV infection-associated effects on phenotypes. Proportions of naïve, memory, and effector subsets of T cells from thawed PBMC correlated with results from the original flow cytometric analysis of respective fresh blood samples. Antigen-specific and polyclonal T-cell responses were readily detected in cryopreserved PBMC from HIV+ patients and healthy control donors. Intracellular HIV RNA quantitation by pi code assay correlated with original plasma viral RNA load results. Full-length intracellular and supernatant-derived amplicons were generated from 5/12 donors, and sequences were ≥80% wild-type, consistent with replication competence. This unique study provides strong rationale and validity for using well-maintained biorepositories to support immunovirological research even decades after collection.

Synthesis of Nucleotides Bearing the 2'-O-Trifluoromethyl Group and Their Application in RNA Analogs Preparation.

The integration of fluorine atoms into biologically active organic compounds has proved to be a vital technique in small molecule drugs. This technique can substantially enhance crucial properties, including metabolic stability, lipophilicity, and bioavailability, often with a mere addition of a single fluorine atom or a trifluoromethyl group. Over the past few decades, this concept has also been applied in nucleic acid chemistry. A commonly employed 2'-OH substitution is the introduction of a 2'-deoxy-2'-fluoro (2'-F) group. The strong electronegativity of fluorine prompts the modified siRNA to readily adopt a C3'-endo conformation, resulting in significant advantages in terms of binding affinity. To enrich the toolbox of chemical modification of oligonucleotides, the replacement of the 2'-OH with the 2'-O-trifluoromethyl group has been developed in RNA analog synthesis. Oligodeoxynucleotides containing the 2'-O-trifluoromethyl group can greatly increase the thermal stability of DNA/RNA duplexes depending on the position and amount of the modification. Moreover, 2'-O-trifluoromethylated oligodeoxynucleotide also exhibited a slightly higher resistance to snake venom phosphodiesterase than the unmodified oligodeoxynucleotide. The 2'-O-trifluoromethylated oligonucleotides can emerge as a label to study RNA structure and function as well, or to develop DNA/RNA-based diagnostics. Hence, it is necessary to report an effective method for the synthesis, deprotection, purification, and characterization of oligonucleotides bearing a 2'-O-trifluoromethyl group. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Preparation of 6-N-benzoyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl adenosine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 2: Preparation of 4-N-acetyl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl cytidine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 3: Preparation of 2-N-isobutyryl-5'-O-dimethoxytrityl-2'-O-trifluoromethyl guanine 3'-(2-cyanoethyl N,N-diisopropyl)phosphoramidite Basic Protocol 4: Preparation of 5'-O-dimethoxytrityl-2'-O-2-trifluoromethyl uridine 3'-(2-cyanoethyl N,N-diisopropyl) phosphoramidite Basic Protocol 5: Solid-phase synthesis of 2'-O-trifluoromethylated RNA analogs Basic Protocol 6: Deprotection and purification of 2'-O-trifluoromethyl-RNAs.

Identification of bona fide RNA G-quadruplex binding proteins.

RNAs often accomplish their diverse functions through direct interactions with RNA-binding proteins (RBPs) in a sequence- and/or structure-dependent manner. RNA G-quadruplexes (rG4s) are unique secondary structures formed by guanine-rich RNA sequences which impact RNA function independently and in combination with RBPs. Efforts from several labs have identified dozens of rG4 specific RBPs (rG4BPs), although the research is still in the growing phase. Here we present methods for the systematic identification of rG4BPs using a pull-down approach that takes advantage of the chemical modification of guanine bases. This allows abolishing the rG4 structures while still maintaining the base composition intact, and hence helps in recognizing true rG4BPS (in contrast to G-rich motif binders). In combination with other biochemical assays, such an approach can be efficiently used for the identification and characterization of bona fide rG4BPs.

Global characterization of RNA modifying enzymes with RNA-mediated activity-based protein profiling (RNABPP).

Post-transcriptional RNA modifications can regulate RNA function and play an important role in gene expression. Studying RNA modifying enzymes and their associated modifications remains a considerable challenge. Here we describe the RNA-mediated activity-based protein profiling (RNABPP) methodology, a chemoproteomic strategy for profiling the activity of RNA modifying enzymes in their native context. RNABPP relies upon metabolic RNA labeling with modified ribonucleoside-based probes, combined with protein-RNA enrichment and quantitative proteomics. The RNABPP approach is a general strategy based on chemical reactivity and enzyme mechanism, making it suitable for probing multiple classes of RNA modifying enzymes across diverse biological systems.

A user guide to RT-based mapping of RNA modifications.

Chemical modifications to RNA nucleotides are both a naturally occurring layer of biological regulation and an increasingly prevalent approach to synthetically alter RNA function in therapeutic applications. Detection of their presence, prevalence, and stoichiometry across different RNAs is critical to understanding their underlying functions. However, this remains challenging due to the technical barriers involved in differentiating chemically similar modification species, and in detecting rare or low stoichiometry modifications. Reverse transcription-based techniques rely on the introduction of a predictable mutation, truncation, or deletion signature when a reverse transcriptase encounters a modified nucleotide of interest. Previous studies have shown promise in detecting modifications to single nucleotide resolution, but the low efficiency and processivity of many commercially available reverse transcriptases has resulted in discordant conclusions in some cases. Here, we present guidelines and best practices for applying the highly processive MarathonRT enzyme to reverse transcription-based modification sequencing. These guidelines include recommendations for controls and example protocols to help users plan robust experiments for mapping modification(s) of choice, as well as discussion of the limitations for the methods described.

Accurate identification of 8-oxoguanine in RNA with single-nucleotide resolution using ligase-dependent qPCR.

8-oxoguanine (o8G), a prevalent oxidative modification in RNA induced by reactive oxygen species (ROS), plays a pivotal role in regulating RNA functions. Accurate detection and quantification of o8G modifications is critical to understanding their biological significance and potential as disease biomarkers, but effective detection methods remain limited. Here, we have developed a highly specific T3 DNA ligase-dependent qPCR assay that exploits the enzyme's ability to discriminate o8G from guanine (G) with single-nucleotide resolution. This method can detect o8G in RNA at levels as low as 500 fM, with an up to 18-fold higher selectivity for discriminating o8G from G. By simulating oxidative stress conditions in SH-SY5Y and HS683 cell lines treated with rotenone, we successfully identified site-specific o8G modifications in key miRNAs associated with neuroprotective responses, including miR-124, let-7a and miR-29a. The developed assay holds significant promise for the practical identification of o8G, facilitating its potential for detailed studies of o8G dynamics in various biological contexts and diseases.

Simultaneous In Situ Detection of m6A-Modified and Unmodified RNAs Using DART-FISH.

N6-methyladenosine (m6A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m6A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m6A-modified and unmodified target transcripts. DART-FISH combines m6A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m6A stoichiometry and methylated mRNA localization in single cells.

RNA m5C methylation: a potential modulator of innate immune pathways in hepatocellular carcinoma.

RNA 5-methylcytosine (m5C) methylation plays a crucial role in hepatocellular carcinoma (HCC). As reported, aberrant m5C methylation is closely associated with the progression, therapeutic efficacy, and prognosis of HCC. The innate immune system functions as the primary defense mechanism in the body against pathogenic infections and tumors since it can activate innate immune pathways through pattern recognition receptors to exert anti-infection and anti-tumor effects. Recently, m5C methylation has been demonstrated to affect the activation of innate immune pathways including TLR, cGAS-STING, and RIG-I pathways by modulating RNA function, unveiling new mechanisms underlying the regulation of innate immune responses by tumor cells. However, research on m5C methylation and its interplay with innate immune pathways is still in its infancy. Therefore, this review details the biological significance of RNA m5C methylation in HCC and discusses its potential regulatory relationship with TLR, cGAS-STING, and RIG-I pathways, thereby providing fresh insights into the role of RNA methylation in the innate immune mechanisms and treatment of HCC.

Structural Analysis of the Hepatitis B Virus RNA Encapsidation Signal ε by Selective 2'-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE).

RNA structure is crucial for RNA function, including in viral cis-elements such as the hepatitis B virus (HBV) RNA encapsidation signal ε. Interacting with the viral polymerase ε mediates packaging of the pregenomic (pg) RNA into capsids, initiation of reverse transcription, and it affects the mRNA functions of pgRNA. As free RNA, the 61-nucleotide (nt) ε sequence adopts a bipartite stem-loop structure with a central bulge and an apical loop. Due to stable Watson-Crick base pairing, this was already predicted by early RNA folding programs and confirmed by classical enzymatic and chemical structure probing. A newer, high-resolution probing technique exploits the selective acylation of solvent-accessible 2'-hydroxyls in the RNA backbone by electrophilic compounds such as 2-methylnicotinic acid imidazolide (NAI), followed by mapping of the modified sites by primer extension. This SHAPE principle has meanwhile been extended to numerous applications. Here we provide a basic protocol for NAI-based SHAPE of isolated HBV ε RNA which already provided insights into the impact of mutations, and preliminarily, of polymerase binding on the RNA structural dynamics. While the focus is on NAI modification, we also briefly cover target RNA preparation by in vitro transcription, primer extension using a radiolabeled primer, and analysis of the resulting cDNAs by denaturing polyacrylamide gelelectrophoresis (PAGE). Given the high tolerance of SHAPE chemistry to different conditions, including applicability in live cells, we expect this technique to greatly facilitate deciphering the conformational dynamics underlying the various functions of the ε element, especially in concert with the recently solved three-dimensional structure of the free RNA.

Chitotriose Enhanced Antitumor Activity of Doxorubicin through Egr1 Upregulation in MDA-MB-231 Cells.

Dietary supplementation is proposed as a strategy to reduce the side effects of conventional chemotherapy for triple-negative breast cancer (TNBC). Chitosan oligosaccharides (COS), a functional carbohydrate, have been identified to potentially inhibit cancer cell proliferation. However, a detailed investigation is required to fully understand its exact influence, particularly in terms of COS composition. The antitumor activities of COS oligomers and its monomer of glucosamine, when combined with doxorubicin separately, were evaluated in MDA-MB-231 cells. Chitotriose was identified to have the most significant synergistic effect. Preincubation with chitotriose was observed to promote the entry of doxorubicin into the cell nuclei and induce morphological changes in the cells. Mechanism analysis at the transcriptional level revealed that the early growth response 1 (Egr1) gene was a key regulator in enhancing the suppressive effect. This gene was found to modulate the activity of its downstream gene, growth arrest, and DNA damage-inducible alpha (Gadd45a). The role of Egr1 was confirmed through a small interfering RNA test and function assay. These findings provide insight into the effect and underlying mechanism of chitotriose supplementation for TNBC therapy.

NOC1 is a direct MYC target, and its protein interactome dissects its activity in controlling nucleolar function.

The nucleolus is a subnuclear compartment critical in ribosome biogenesis and cellular stress responses. These mechanisms are governed by a complex interplay of proteins, including NOC1, a member of the NOC family of nucleolar proteins responsible for controlling rRNA processing and ribosomal maturation. This study reveals a novel relationship between NOC1 and MYC transcription factor, known for its crucial role in controlling ribosomal biogenesis, cell growth, and proliferation. Here, we demonstrate that NOC1 functions as a direct target of MYC, as it is transcriptionally induced through a functional MYC-binding E-box sequence in the NOC1 promoter region. Furthermore, protein interactome analysis reveals that NOC1-complex includes the nucleolar proteins NOC2 and NOC3 and other nucleolar components such as Nucleostemin1 Ns1 transporters of ribosomal subunits and components involved in rRNA processing and maturation. In response to MYC, NOC1 expression and localization within the nucleolus significantly increase, suggesting a direct functional link between MYC activity and NOC1 function. Notably, NOC1 over-expression leads to the formation of large nuclear granules and enlarged nucleoli, which co-localize with nucleolar fibrillarin and Ns1. Additionally, we demonstrate that NOC1 expression is necessary for Ns1 nucleolar localization, suggesting a role for NOC1 in maintaining nucleolar structure. Finally, the co-expression of NOC1 and MYC enhances nucleolus size and maintains their co-localization, outlining another aspect of the cooperation between NOC1 and MYC in nucleolar dynamics. This study also reveals an enrichment with NOC1 with few proteins involved in RNA processing, modification, and splicing. Moreover, proteins such as Ythdc1, Flacc, and splenito are known to mediate N6-methyladenosine (m6A) methylation of mRNAs in nuclear export, revealing NOC1's potential involvement in coordinating RNA splicing and nuclear mRNA export. In summary, we uncovered novel roles for NOC1 in nucleolar homeostasis and established its direct connection with MYC in the network governing nucleolar structure and function. These findings also highlight NOC1's interaction with proteins relevant to specific RNA functions, suggesting a broader role in addition to its control of nucleolar homeostasis and providing new insight that can be further investigated.

LncRNA PSMA3-AS1 activates the progression of triple-negative breast cancer cells by blocking miR-186-5p-mediated PSME3 inhibition.

Triple-negative breast cancer (TNBC) is an aggressive malignant tumor with a high death rate in the whole world. This cancer mainly occurs in young women group possessed with poor prognoses. Long noncoding RNAs (lncRNAs) are known for regulating human diseases and cancers. Even though growing researches have illuminated that lncRNAs have a close relation with TNBC progression, the function of lncRNAPSMA3 antisense RNA 1 (PSMA3-AS1) in TNBC has not been discussed and exposed yet. In the present research, the expression pattern and functional role of PSMA3-AS1 were analyzed and unveiled with the help of RT-qPCR and functional assays. The findings demonstrated that PSMA3-AS1 was notably upregulated in TNBC cells. Silencing of PSMA3-AS1 had suppressing effects on TNBC cell growth and migration. Mechanistically, PSMA3-AS1 induced upregulation of proteasome activator subunit 3 (PSME3) by functioning as a miR-186-5p sponge. Furthermore, rescue assays certified that overexpression of PSME3 or inhibition of miR-186-5p could abrogate the inhibiting role of silenced PSMA3-AS1 on TNBC cell functions. To summarize, PSMA3-AS1 abolishes miR-186-5p-mediated suppression on PSME3 to accelerate TNBC progression.