As a member of the RNA-binding motif protein (RBM) family, RBM5 is a characterized tumor suppressor in lung and prostate cancers, with critical roles in alternative splicing of apoptosis- and cell cycle-related genes. However, its direct capacity to regulate gene transcription remains unreported. Here, we identified that RBM5 was significantly downregulated in breast cancer cells and clinical specimens, especially in the basal-like subtype. RBM5 overexpression attenuated breast cancer cell malignancy, while RBM5 knockdown exerted opposite effects. Among genes with promoter G-quadruplex (G4) motifs, RBM5 was positively correlated with multiple tumor suppressors, including BAP1, but uncorrelated with the oncogene MYC. RBM5 bound to G4 motifs in both MYC and BAP1 promoters, but differentially modulated G4 structure stability: it destabilized MYC-G4 while stabilizing BAP1-G4. Mechanistically, either the RRM1 or the RRM2 domain was sufficient for MYC-G4 binding, whereas both domains were required for BAP1-G4 interaction. RBM5 manipulation regulated endogenous BAP1 but not MYC expression. BAP1 overexpression reversed the protumorigenic effects of RBM5 knockdown in cellulo and tumor growth in a xenograft mouse model. Collectively, we reveal that RBM5 acts as a breast cancer tumor suppressor via directly binding the BAP1 promoter G4 to transcriptionally activate BAP1 expression.

ABSTRACT Among non‐classical nucleic acid secondary structures, G‐quadruplexes (G4s) play diverse roles in cellular functions and disease pathogenesis. However, the molecular mechanisms underlying the assembly of endogenous G4s into punctate condensates in cells remain unclear. Biomolecular condensates can arise from weak multivalent intermolecular interactions involving proteins and/or nucleic acids; this phenomenon is frequently linked to liquid–liquid phase separation. Recent research has provided compelling evidence for G4s driving biomolecular condensation. In this review, we first summarize the latest breakthroughs in the structural classification of G4s. In addition to frequently reported intramolecular G4s, intermolecular G4s have also been observed in cellular environments. Next, we discuss the regulatory role of G4s in condensation. Although G4s can independently form condensates, they primarily serve as structural platforms that facilitate condensate formation and regulate their phase transitions. Ultimately, this review reveals the multifaceted physiological and pathological functions of G4‐driven condensates, including chromatin organization, assembly of stress granules and paraspeckles, abnormal transcriptional activation, telomere maintenance, neurodegenerative disease‐associated protein aggregation, and viral inclusion body formation.

The immune system plays a vital role to prevent cancers from occurrence and development. The eventual onset and progression of cancers necessitate the compromise of immune surveillance, and the dysregulation of many key regulators contribute to this process. Understanding the mechanisms and identifying key players regulating immune evasion is crucial for effective anticancer therapies that revive the immune system. As a multifunctional transcription factor, Yin Yang 1 (YY1) is increasingly expressed in various cancers, and can both stimulate oncogenic development and promote immune evasion of cancer cells. In this review, we categorize the YY1-regulated cancer immune evasion into a previously defined framework, including camouflage, coercion, and cytoprotection with a focus on gene expression network in different cancers. We first outline the studies of how YY1 enables cancer cells to avoid immune system recognition. Second, we review the research related to how YY1 attenuates the tumor suppressive functions of immune effector cells. Third, we elaborate YY1-mediated mechanisms to protect cancer cells from cytotoxicity of immune cells. Finally, we discuss the complexity of the YY1-involved regulatory network of cancer immune evasion.

Competitive endogenous RNAs (ceRNAs) absorb microRNAs and subsequently promote corresponding mRNA and long noncoding RNA (lncRNA) expression, which may alter cancer cell malignancy. Thus, dissecting ceRNA networks may reveal novel targets in cancer therapies. In this study, we analyzed differentially expressed genes (DEGs) of mRNAs and lncRNAs, and differentially expressed microRNAs (DE-miRNAs) and circular RNAs (DE-circRNAs) extracted from high-throughput sequencing datasets of hepatocellular carcinoma patients. Based on these data, we identified 26 gene modules using weighted gene co-expression network analysis (WGCNA), of which 5 were associated with tumor differentiation. In these modules, 269 genes were identified by GO and KEGG enrichment and patient’s survival correlation analyses. Next, 40 DE-miRNAs, each of which potentially bound a pair of DE-circRNA and hub gene, were discovered. Together with 201 circRNAs and 24 hub genes potentially bound by these miRNAs, 1151 ceRNA networks were constructed. Among them, 75 ceRNA networks consisting of 24 circRNAs, 28 miRNAs and 17 hub genes showed a positive circRNA–hub gene correlation. For validation, we carried out experiments for 4 randomly selected circRNAs regulating 19 potential ceRNA networks and verified 5 of them. This study represents a powerful strategy to identify essential gene networks and provides insights into designing effective therapeutic strategies.

Alternative splicing of the BCL2 precursor mRNA (pre-mRNA) generate two protein-coding transcript isoforms, BCL2α and BCL2β. Numerous reports indicate that BCL2, namely BCL2α, contributes to cancer development through promoting cancer cell survival, while the mechanism underlying BCL2 pre-mRNA alternative splicing and the functional role of the BCL2β in oncogenesis remain unknown. Here, we discovered an RNA G-quadruplex (rG4) motif and three m6A RNA methylation motifs downstream rG4's proximity at the 5'-end of BCL2 pre-mRNA exon 3. In vitro and in cellulo assays verified rG4 structure formation in this region. The methylation status at these m6A motifs was also demonstrated in breast cancer cells. rG4 structure favored BCL2β splicing, but the underlying mechanisms in triple negative breast cancer (TNBC) and non-TNBC cells were different. In TNBC cells, rG4 stabilization promoted m6A modification that decreased both overall BCL2 mRNA levels and BCL2α splicing, but enhanced BCL2β production; conversely, in non-TNBC cells, rG4 structure reduced m6A modification that increased both overall BCL2 mRNA and BCL2α isoform levels, but reduced BCL2β splicing. When exploring splicing factors involved in BCL2 pre-mRNA splicing, we identified SRSF1 as a novel rG4 binding protein that resolved rG4 structure, subsequently promoting BCL2α splicing and reducing BCL2β isoform. Further, BCL2α exhibited oncogenic activities as previously reported, but BCL2β reduced breast cancer cell survivability and migration, and sensitized cells to chemotherapeutics-induced apoptosis. Overall, in this study, we revealed the mechanisms regulating BCL2 pre-mRNA alternative splicing through rG4 structure, m6A modification and SRSF1 binding, and discovered antiproliferative and proapoptotic activity of BCL2β.

Cyclin-dependent kinase 1 (CDK1) is the pivotal kinase responsible for initiating cell division. Its activation is dependent on binding to regulatory cyclins, such as CCNB1. Our research demonstrates that copper binding to both CDK1 and CCNB1 is essential for activating CDK1 in cells. Mutations in the copper-binding amino acids of either CDK1 or CCNB1 do not disrupt their interaction but are unable to activate CDK1. We also reveal that CCNB1 facilitates the transfer of copper from ATOX1 to CDK1, consequently activating its kinase function. Disruption of copper transfer through the ATOX1-CCNB1-CDK1 pathway can impede CDK1 activation and halt cell cycle progression. In summary, our findings elucidate a mechanism through which copper promotes CDK1 activation and the G2/M transition in the cell cycle. These results could provide insight into the acquisition of proliferative properties associated with increased copper levels in cancer and offer targets for cancer therapy.

Backgroundβ-caryophyllene (BCP) is a naturally occurring bicyclic sesquiterpene extracted from various plants, and widely used as a medicinal agent for various diseases. During hepatocellular carcinoma (HCC) development, cancer cells generally exhibit increased cell proliferation due to mutations or aberrant expression of key regulatory genes. The current study determines the cytotoxic effects of BCP alone or in combination with doxorubicin (DOX) and cisplatin (DDP) on HCC cells, and elucidates the underlying mechanism of BCP to exert its anticancer activities.Materials and methodsHepG2, SMMC-7721 HCC cells, and HL-7702 normal liver cells were treated with BCP, DOX, and DDP individually or combinatorially. Cell proliferation assay, flow cytometric assay, and Western blot were employed to evaluate the cytotoxic effects of these treatments. Transwell assays were used to examine BCP’s effects on HCC cell migration and invasion. RNA-seq analysis was used to determine BCP’s primary target genes in HepG2 cells. Integrative analysis of differentially expressed genes (DEGs) of RNA-seq data with an HCC TCGA dataset identified BCP-targeted genes that were verified by RT-qPCR analysis. Ectopic gene expression, cell viability, and colony formation assay were performed to validate the primary targets of BCP.ResultsBCP selectively inhibited HCC cell proliferation while exhibited relatively low toxicity in normal liver cells; however, DOX and DDP showed higher toxicity in normal cells than that in HCC cells. In combinatorial treatments, BCP synergistically enhanced cytotoxicity of DOX and DDP in HCC cells but this effect was markedly reduced in HL-7702 cells. BCP treatment reduced migration and invasion of HCC cells. Furthermore, RNA-seq analyses of BCP-treated HepG2 cells identified 433 protein-coding DEGs. Integrative analyses revealed five BCP-targeted DEGs regulating the MAPK signaling pathway. Among these five genes, three displayed a significantly positive correlation of their expression with the overall survival of HCC patients. As a primary target, PGF was significantly downregulated by BCP treatment, and its exogenous expression desensitized HCC cells to BCP-mediated inhibition.DiscussionBCP inhibits malignant properties of HCC and synergistically sensitizes the anticancer activity of DOX and DDP. In HCC cells, BCP primarily targets the PGF gene and MAPK signaling pathway.

Neurodegenerative diseases are the leading cause of human disability and immensely reduce patients’ life span and quality. The diseases are characterized by the functional loss of neuronal cells and share several common pathogenic mechanisms involving the malfunction, structural distortion, or aggregation of multiple key regulatory proteins. Cellular phase separation is the formation of biomolecular condensates that regulate numerous biological processes, including neuronal development and synaptic signaling transduction. Aberrant phase separation may cause protein aggregation that is a general phenomenon in the neuronal cells of patients suffering neurodegenerative diseases. In this review, we summarize the pathological causes of common neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, among others. We discuss the regulation of key amyloidogenic proteins with an emphasis of their aberrant phase separation and aggregation. We also introduce the approaches as potential therapeutic strategies to ameliorate neurodegenerative diseases through intervening protein aggregation. Overall, this review consolidates the research findings of phase separation and aggregation caused by misfolded proteins in a context of neurodegenerative diseases.

Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms remain enigmatic. Here, we demonstrate that G4s in the CCND1 promoter propel the motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is a responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation.

Biosensors based on allosteric transcription factors have been widely used in synthetic biology. In this study, we utilized the Acinetobacter ADP1 transcription factor PobR to develop a biosensor activating the PpobA promoter when bound to its natural ligand, 4-hydroxybenzoic acid (4HB). To screen for PobR mutants responsive to 4-hydroxyphenylpyruvate(HPP), we developed a dual selection system in E. coli. The positive selection of this system was used to enrich PobR mutants that identified the required ligands. The following negative selection eliminated or weakened PobR mutants that still responded to 4HB. Directed evolution of the PobR library resulted in a variant where PobRW177R was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobRWT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.

Abstract Experimental and epidemiologic evidence suggests that dysregulation of proteins involved in iron metabolism plays a critical role in cancer. The mechanisms by which cancer cells alter homeostatic iron regulation are just beginning to be understood. Here, we demonstrate that iron regulatory protein 2 (IRP2) plays a key role in iron accumulation in breast cancer. Although both IRP1 and IRP2 are overexpressed in breast cancer, the overexpression of IRP2, but not IRP1, is associated with decreased ferritin H and increased transferrin receptor 1 (TfR1). Knockdown of IRP2 in triple-negative MDA-MB-231 human breast cancer cells increases ferritin H expression and decreases TfR1 expression, resulting in a decrease in the labile iron pool. Further, IRP2 knockdown reduces growth of MDA-MB-231 cells in the mouse mammary fat pad. Gene expression microarray profiles of patients with breast cancer demonstrate that increased IRP2 expression is associated with high-grade cancer. Increased IRP2 expression is observed in luminal A, luminal B, and basal breast cancer subtypes, but not in breast tumors of the ERBB2 molecular subtype. These results suggest that dysregulation of IRP2 is an early nodal point underlying altered iron metabolism in breast cancer and may contribute to poor outcome of some patients with breast cancer. Cancer Res; 74(2); 497–507. ©2013 AACR.

Biochemistry and Molecular Biology are the cornerstone courses of talent training in the field of life science. Taking these course as an example, this study explored reconstructing the knowledge framework, developing teaching cases, sharing teaching resources, innovating teaching means and establishing ideological education patterns. Supported by the scientific research achievements with discipline characteristics and online teaching platform, this research explored and practiced an integrated curriculum reform mode. This mode is guided by scientific research and education, based on the course development, and driven by communication and cooperation. A shared space of "exchange, practice, openness and informatization" was developed to achieve free and independent integration of undergraduate and graduate teaching motivated by learning knowledge, resulting in an effective student training.

Supplementary Table Legends 1-7, Figure Legends 1-9 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D3 in Prostate Progenitor/Stem Cells

Supplementary Figures 1-9 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D3 in Prostate Progenitor/Stem Cells

PDF file - 1MB, Hemizygous deletion of MAP3K7 in human prostatic tumorigenic cell lines.

PDF file - 514K, Supplemental Figures 1-5. Supplemental Fig 1: IRP2, TfR1 and Ferritin H transcript levels in a series of breast cancer cell lines examined by real-time qPCR. Supplemental Fig 2. IRP2, TfR1 and Ferritin H transcript levels in IRP2 knockdown MDA-MB-231 cells. Supplemental Fig 3. Growth of MCF7 cells following IRP2 knockdown. Supplemental Fig 4. IRP2 overexpression is not sufficient to increase proliferation of MCF10A cells. Supplemental Fig 5. Bioluminescent images of tumor xenografts on day 32.

Supplementary Tables 1-4 from Interleukin-1α Mediates the Antiproliferative Effects of 1,25-Dihydroxyvitamin D₃ in Prostate Progenitor/Stem Cells