Emerging roles of RNA-binding proteins in fatty liver disease.

A rampant and urgent global health issue of the 21st century is the emergence and progression of fatty liver disease (FLD), including alcoholic fatty liver disease and the more heterogenous metabolism-associated (or non-alcoholic) fatty liver disease (MAFLD/NAFLD) phenotypes. These conditions manifest as disease spectra, progressing from benign hepatic steatosis to symptomatic steatohepatitis, cirrhosis, and, ultimately, hepatocellular carcinoma. With numerous intricately regulated molecular pathways implicated in its pathophysiology, recent data have emphasized the critical roles of RNA-binding proteins (RBPs) in the onset and development of FLD. They regulate gene transcription and post-transcriptional processes, including pre-mRNA splicing, capping, and polyadenylation, as well as mature mRNA transport, stability, and translation. RBP dysfunction at every point along the mRNA life cycle has been associated with altered lipid metabolism and cellular stress response, resulting in hepatic inflammation and fibrosis. Here, we discuss the current understanding of the role of RBPs in the post-transcriptional processes associated with FLD and highlight the possible and emerging therapeutic strategies leveraging RBP function for FLD treatment. This article is categorized under: RNA in Disease and Development > RNA in Disease.

YTHDF2 promotes gastric cancer progression and enhances chemoradiotherapy resistance.

The role of YTHDF2 in gastric cancer (GC) is controversial. Due to the limitations of technical difficulty and experimental period, research on completely knocking out YTHDF2 is rare. Therefore, further investigations are still needed to clarify the YTHDF2's clinical significance and biological function in GC. To carry out the investigation, an analysis was performed on the expression levels of YTHDF2 in both publicly available databases and samples obtained from patients with gastric cancer. Based on the complete knockout of YTHDF2 using the CRISPR-Cas9 system, in vivo and in vitro experiments were conducted to analyze the effects of YTHDF2 on tumor formation, radiotherapy and chemoradiotherapy resistance in GC. Our investigation revealed an increase in YTHDF2 levels in GC tissues, which was found to be associated with a negative prognosis. Under hypoxic conditions, high expression of YTHDF2 enhanced the invasion of gastric cancer cells, and high expression of YTHDF2 was associated with HIF-1a. YTHDF2 facilitated gastric cancer cell growth in vitro and in vivo. Moreover, the results of the present study demonstrated that YTHDF2 mediated the expression of CyclinD1 and stability of CyclinD1 mRNA. CyclinD1 knockdown inhibited YTHDF2-mediated GC cell proliferation whereas CyclinD1 overexpression ameliorated YTHDF2 knockdown-induced inhibition of GC progression. Furthermore, YTHDF2 also promoted resistance to DDP and CTX chemotherapy, along with radiotherapy treatment for GC cells. The findings suggested that YTHDF2 expression accelerated GC progression through a potential mechanism involving CyclinD1 expression, and enhanced chemoradiotherapy resistance. This indicated that YTHDF2 could be a promising prognostic biomarker and therapeutic target for individuals diagnosed with GC.

Understanding YTHDF2-mediated mRNA degradation by m6A-BERT-Deg.

N6-methyladenosine (m6A) is the most abundant mRNA modification within mammalian cells, holding pivotal significance in the regulation of mRNA stability, translation and splicing. Furthermore, it plays a critical role in the regulation of RNA degradation by primarily recruiting the YTHDF2 reader protein. However, the selective regulation of mRNA decay of the m6A-methylated mRNA through YTHDF2 binding is poorly understood. To improve our understanding, we developed m6A-BERT-Deg, a BERT model adapted for predicting YTHDF2-mediated degradation of m6A-methylated mRNAs. We meticulously assembled a high-quality training dataset by integrating multiple data sources for the HeLa cell line. To overcome the limitation of small training samples, we employed a pre-training-fine-tuning strategy by first performing a self-supervised pre-training of the model on 427 760 unlabeled m6A site sequences. The test results demonstrated the importance of this pre-training strategy in enabling m6A-BERT-Deg to outperform other benchmark models. We further conducted a comprehensive model interpretation and revealed a surprising finding that the presence of co-factors in proximity to m6A sites may disrupt YTHDF2-mediated mRNA degradation, subsequently enhancing mRNA stability. We also extended our analyses to the HEK293 cell line, shedding light on the context-dependent YTHDF2-mediated mRNA degradation.

Clinical phenotype of a Kallmann syndrome patient with IL17RD and CPEB4 variants.

Background: This study aimed to characterize the clinical phenotype and genetic variations in patients with Kallmann syndrome (KS). Methods: This study involved the collection and analysis of clinical data from an individual with sporadic KS. Following this, peripheral blood samples were obtained from the patient and his parents. Genomic deoxyribonucleic acid was extracted and subjected to whole-exome sequencing and genomic copy number variation (CNV) detection. Finally, Sanger sequencing was performed to validate the suspected pathogenic variants. Results: Whole-exome sequencing confirmed that the child carried both the IL17RD variant (c.2101G>A, p.Gly701Ser) inherited from the mother and the new CPEB4 variant (c.1414C>T, p.Arg472*). No pathogenic CNVs were identified in CNV testing. Conclusion: Bioinformatics analysis shows that the IL17RD protein undergoing Gly701Ser mutation and is speculated to be phosphorylated and modified, thereby disrupting fibroblast growth factor signaling. This study also suggested that the CPEB4 might play a crucial role in the key signaling process affecting olfactory bulb morphogenesis. Overall, the findings of this study broaden the gene expression profile of KS-related pathogenic genes. This offers a new avenue for exploring the pathogenic mechanism of KS and provides valuable insights for precise clinical diagnosis and treatment strategies for this condition.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Unraveling the Significance of DGCR8 and miRNAs in Thyroid Carcinoma.

MicroRNAs (miRNAs) act as negative regulators for protein-coding gene expression impacting cell proliferation, differentiation, and survival. These miRNAs are frequently dysregulated in cancer and constitute classes of blood-based biomarkers useful for cancer detection and prognosis definition. In thyroid cancer (TC), the miRNA biogenesis pathway plays a pivotal role in thyroid gland formation, ensuring proper follicle development and hormone production. Several alterations in the miRNA biogenesis genes are reported as a causality for miRNA dysregulation. Mutations in microprocessor component genes are linked to an increased risk of developing TC; in particular, a recurrent mutation affecting DGCR8, the E518K. In this review, we explore these novel findings and resume the current state-of-the-art in miRNAs in thyroid carcinomas.

A1CF Binding to the p65 Interaction Site on NKRF Decreased IFN-ß Expression and p65 Phosphorylation (Ser536) in Renal Carcinoma Cells.

Apobec-1 complementation factor (A1CF) functions as an RNA-binding cofactor for APO-BEC1-mediated C-to-U conversion during RNA editing and as a hepatocyte-specific regulator in the alternative pre-mRNA splicing of metabolic enzymes. Its role in RNA editing has not been clearly established. Western blot, co-immunoprecipitation (Co-IP), immunofluorescence (IF), methyl thiazolyl tetrazolium (MTT), and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to examine the role of A1CF beyond RNA editing in renal carcinoma cells. We demonstrated that A1CF interacts with NKRF, independent of RNA and DNA, without affecting its expression or nuclear translocation; however, it modulates p65(Ser536) phosphorylation and IFN-ß levels. Truncation of A1CF or deletion on NKRF revealed that the RRM1 domain of A1CF and the p65 binding motif of NKRF are required for their interaction. Deletion of RRM1 on A1CF abrogates NKRF binding, and the decrease in IFN-ß expression and p65(Ser536) phosphorylation was induced by A1CF. Moreover, full-length A1CF, but not an RRM1 deletion mutant, promoted cell proliferation in renal carcinoma cells. Perturbation of A1CF levels in renal carcinoma cells altered anchorage-independent growth and tumor progression in nude mice. Moreover, p65(Ser536) phosphorylation and IFN-ß expression were lower, but ki67 was higher in A1CF-overexpressing tumor tissues of a xenograft mouse model. Notably, primary and metastatic samples from renal cancer patients exhibited high A1CF expression, low p65(Ser536) phosphorylation, and decreased IFN-ß levels in renal carcinoma tissues compared with the corresponding paracancerous tissues. Our results indicate that A1CF-decreased p65(Ser536) phosphorylation and IFN-ß levels may be caused by A1CF competitive binding to the p65-combined site on NKRF and demonstrate the direct binding of A1CF independent of RNA or DNA in signal pathway regulation and tumor promotion in renal carcinoma cells.

m6A-dependent upregulation of DDX21 by super-enhancer-driven IGF2BP2 and IGF2BP3 facilitates progression of acute myeloid leukaemia.

BACKGROUND: Acute myeloid leukaemia (AML) is a haematological malignancy with unfavourable prognosis. Despite the effectiveness of chemotherapy and targeted therapy, relapse or drug resistance remains a major threat to AML patients. N6-methyladenosine (m6A) RNA methylation and super-enhancers (SEs) are extensively involved in the leukaemogenesis of AML. However, the potential relationship between m6A and SEs in AML has not been elaborated. METHODS: Chromatin immunoprecipitation (ChIP) sequencing data from Gene Expression Omnibus (GEO) cohort were analysed to search SE-related genes. The mechanisms of m6 A-binding proteins IGF2BP2 and IGF2BP3 on DDX21 were explored via methylated RNA immunoprecipitation (MeRIP) assays, RNA immunoprecipitation (RIP) assays and luciferase reporter assays. Then we elucidated the roles of DDX21 in AML through functional assays in vitro and in vivo. Finally, co-immunoprecipitation (Co-IP) assays, RNA sequencing and ChIP assays were performed to investigate the downstream mechanisms of DDX21. RESULTS: We identified two SE-associated transcripts IGF2BP2 and IGF2BP3 in AML. High enrichment of H3K27ac, H3K4me1 and BRD4 was observed in IGF2BP2 and IGF2BP3, whose expression were driven by SE machinery. Then IGF2BP2 and IGF2BP3 enhanced the stability of DDX21 mRNA in an m6A-dependent manner. DDX21 was highly expressed in AML patients, which indicated a poor survival. Functionally, knockdown of DDX21 inhibited cell proliferation, promoted cell apoptosis and led to cell cycle arrest. Mechanistically, DDX21 recruited transcription factor YBX1 to cooperatively trigger ULK1 expression. Moreover, silencing of ULK1 could reverse the promoting effects of DDX21 overexpression in AML cells. CONCLUSIONS: Dysregulation of SE-IGF2BP2/IGF2BP3-DDX21 axis facilitated the progression of AML. Our findings provide new insights into the link between SEs and m6A modification, elucidate the regulatory mechanisms of IGF2BP2 and IGF2BP3 on DDX21, and reveal the underlying roles of DDX21 in AML.

Mex-3 RNA binding family member A (MEX3A)/circMPP6 complex promotes colorectal cancer progression by inhibiting autophagy.

RNA-binding proteins (RBPs)-RNA networks have contributed to cancer development. Circular RNAs (circRNAs) are considered as protein recruiters; nevertheless, the patterns of circRNA-protein interactions in colorectal cancer (CRC) are still lacking. Processing bodies (PBs) formed through liquid-liquid phase separation (LLPS) are membrane-less organelles (MLOs) consisting of RBPs and RNA. Previous evidence suggests a connection between PBs dynamics and cancer progression. Despite the increasingly acknowledged crucial role of RBPs and RNA in the accumulation and maintenance of MLOs, there remains a lack of specific research on the interactions between PBs-related RBPs and circRNAs in CRC. Herein, we identify that MEX-3 RNA binding family member A (MEX3A), frequently upregulated in CRC tissues, predicts poorer patient survival. Elevated MEX3A accelerates malignance and inhibits autophagy of CRC cells. Importantly, MEX3A undergoes intrinsically disordered regions (IDRs)-dependent LLPS in the cytoplasm. Specifically, circMPP6 acts as a scaffold to facilitate the interaction between MEX3A and PBs proteins. The MEX3A/circMPP6 complex modulates PBs dynamic and promotes UPF-mediated phosphodiesterase 5A (PDE5A) mRNA degradation, consequently leading to the aggressive properties of CRC cells. Clinically, CRC patients exhibiting high MEX3A expression and low PDE5A expression have the poorest overall survival. Our findings reveal a collaboration between MEX3A and circMPP6 in the regulation of mRNA decay through triggering the PBs aggregation, which provides prognostic markers and/or therapeutic targets for CRC.

LIN28B induced PCAT5 promotes endometrial cancer progression and glycolysis via IGF2BP3 deubiquitination.

Endometrial cancer (EC) cells exhibit abnormal glucose metabolism, characterized by increased aerobic glycolysis and decreased oxidative phosphorylation. Targeting cellular glucose metabolism in these cells could be an effective therapeutic approach for EC. This study aimed to assess the roles of LIN28B, PCAT5, and IGF2BP3 in the glucose metabolism, proliferation, migration, and invasion of EC cells. LIN28B highly expressed in EC, binds and stabilizes PCAT5. PCAT5, overexpressed in EC, and its 1485-2288nt region can bind to the KH1-2 domain of IGF2BP3 to prevent MKRN2 from binding to the K294 ubiquitination site of IGF2BP3, thus stabilizing IGF2BP3. Finally, IGF2BP3 promotes the aerobic glycolysis, proliferation, migration and invasion of EC cells by stabilizing the key enzymes of glucose metabolism HK2 and PKM2. Taken together, our data reveal that the LIN28B/PCAT5/IGF2BP3 axis is critical for glucose reprogramming and malignant biological behavior in EC cells. Therefore, targeting this axis may contribute to the development of a novel therapeutic strategy for EC metabolism.

Systematic analysis of RNA-binding proteins identifies targetable therapeutic vulnerabilities in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor with a strong tendency to metastasize, limiting the prognosis of affected patients. Genomic, epigenomic and transcriptomic analyses have demonstrated the exquisite molecular complexity of this tumor, but have not sufficiently defined the underlying mechanisms or identified promising therapeutic targets. To systematically explore RNA-protein interactions relevant to OS, we define the RNA interactomes together with the full proteome and the transcriptome of cells from five malignant bone tumors (four osteosarcomata and one malignant giant cell tumor of the bone) and from normal mesenchymal stem cells and osteoblasts. These analyses uncover both systematic changes of the RNA-binding activities of defined RNA-binding proteins common to all osteosarcomata and individual alterations that are observed in only a subset of tumors. Functional analyses reveal a particular vulnerability of these tumors to translation inhibition and a positive feedback loop involving the RBP IGF2BP3 and the transcription factor Myc which affects cellular translation and OS cell viability. Our results thus provide insight into potentially clinically relevant RNA-binding protein-dependent mechanisms of osteosarcoma.

[High LINC00626 expression promotes esophagogastric junction adenocarcinoma metastasis: the mediating role of the JAK1/STAT3/KHSRP axis].

OBJECTIVE: To investigate the role of JAK1/STAT3/KHSRP axis in mediating the regulatory effect of LINC00626 on progression of esophagogastric junction adenocarcinoma. METHODS: We collected surgical tumor and adjacent tissue specimens from 64 patients with esophagogastric junction adenocarcinoma and examined the expression levels of LINC00626 and KHSRP. qRT-PCR was used to detect the expressions of LINC00626 and KHSRP in 6 esophageal adenocarcinoma cell lines (OE-19, TE-7, Bic-1, Flo-1, SK-GT-4, and BE-3) and a normal esophageal epithelial cell line (HET-1A). OE-19 and TE-7 cell lines with stable LINC00626 knockdown and FLO-1 and SK-GT-4 cells stably overexpressing LINC00626 were constructed by lentiviral transfection, and the changes in proliferation, migration and invasion of the cells were evaluated using Cell Counting Kit-8 (CCK-8) assay and Transwell migration/invasion assay. The expressions of KHSRP and JAK/STAT pathway proteins in the transfected cells were detected with Western blotting. The effects of LINC006266 knockdown and overexpression on subcutaneous tumor formation and lung metastasis of OE-19 and FLO-1 cell xenografts were tested in nude mice. RESULTS: The expression levels of LINC00626 and KHSRP were significantly increased in esophagogastric junction adenocarcinoma tissues and in esophageal adenocarcinoma cells. LINC00626 knockdown obviously inhibited the proliferation, migration and invasion of esophageal adenocarcinoma cells in vitro and decreased their tumor formation and lung metastasis abilities in nude mice, while overexpression of LINC00626 produced the opposite effects. In esophageal adenocarcinoma cells, LINC0626 knockdown significantly decreased and LINC00626 overexpression strongly enhanced the phosphorylation of JAK1 and STAT3. CONCLUSION: High LINC00626 expression promotes esophageal-gastric junction adenocarcinoma metastasis by activating the JAK1/STAT3/KHSRP signal axis.

LLPS of FXR proteins drives replication organelle clustering for ß-coronaviral proliferation.

ß-Coronaviruses remodel host endomembranes to form double-membrane vesicles (DMVs) as replication organelles (ROs) that provide a shielded microenvironment for viral RNA synthesis in infected cells. DMVs are clustered, but the molecular underpinnings and pathophysiological functions remain unknown. Here, we reveal that host fragile X-related (FXR) family proteins (FXR1/FXR2/FMR1) are required for DMV clustering induced by expression of viral non-structural proteins (Nsps) Nsp3 and Nsp4. Depleting FXRs results in DMV dispersion in the cytoplasm. FXR1/2 and FMR1 are recruited to DMV sites via specific interaction with Nsp3. FXRs form condensates driven by liquid-liquid phase separation, which is required for DMV clustering. FXR1 liquid droplets concentrate Nsp3 and Nsp3-decorated liposomes in vitro. FXR droplets facilitate recruitment of translation machinery for efficient translation surrounding DMVs. In cells depleted of FXRs, SARS-CoV-2 replication is significantly attenuated. Thus, SARS-CoV-2 exploits host FXR proteins to cluster viral DMVs via phase separation for efficient viral replication.

HIV-1 gp120 amplifies astrocyte elevated gene-1 activity to compromise the integrity of the outer blood-retinal barrier.

OBJECTIVE: This study aims to investigate the functions and mechanistic pathways of Astrocyte Elevated Gene-1 (AEG-1) in the disruption of the blood-retinal barrier (BRB) caused by the HIV-1 envelope glycoprotein gp120. DESIGN: We utilized ARPE-19 cells challenged with gp120 as our model system. METHODS: Several analytical techniques were employed to decipher the intricate interactions at play. These included PCR, Western blot, and immunofluorescence assays for the molecular characterization, and transendothelial electrical resistance (TEER) measurements to evaluate barrier integrity. RESULTS: We observed that AEG-1 expression was elevated, whereas the expression levels of tight junction proteins ZO-1, Occludin, and Claudin5 were downregulated in gp120-challenged cells. TEER measurements corroborated these findings, indicating barrier dysfunction. Additional mechanistic studies revealed that the activation of NFκB and MMP2/9 pathways mediated the AEG-1-induced barrier destabilization. Through the use of lentiviral vectors, we engineered cell lines with modulated AEG-1 expression levels. Silencing AEG-1 alleviated gp120-induced downregulation of tight junction proteins and barrier impairment while concurrently inhibiting the NFκB and MMP2/9 pathways. Conversely, overexpression of AEG-1 exacerbated these pathological changes, further compromising the integrity of the BRB. CONCLUSION: Gp120 upregulates the expression of AEG-1 and activates the NFκB and MMP2/9 pathways. This in turn leads to the downregulation of tight junction proteins, resulting in the disruption of barrier function.

Comparison of Brn3a and RBPMS Labeling to Assess Retinal Ganglion Cell Loss During Aging and in a Model of Optic Neuropathy.

Purpose: Retinal ganglion cell (RGC) loss provides the basis for diagnosis and stage determination of many optic neuropathies, and quantification of RGC survival is a critical outcome measure in models of optic neuropathy. This study examines the accuracy of manual RGC counting using two selective markers, Brn3a and RBPMS. Methods: Retinal flat mounts from 1- to 18-month-old C57BL/6 mice, and from mice after microbead (MB)-induced intraocular pressure (IOP) elevation, are immunostained with Brn3a and/or RBPMS antibodies. Four individuals masked to the experimental conditions manually counted labeled RGCs in three copies of five images, and inter- and intra-person reliability was evaluated by the intraclass correlation coefficient (ICC). Results: A larger population (approximately 10% higher) of RGCs are labeled with RBPMS than Brn3a antibody up to 6 months of age, but differences decrease to approximately 1% at older ages. Both RGC-labeled populations significantly decrease with age. MB-induced IOP elevation is associated with a significant decrease of both Brn3a- and RBPMS-positive RGCs. Notably, RGC labeling with Brn3a provides more consistent cell counts than RBPMS in interpersonal (ICC = 0.87 to 0.11, respectively) and intra-personal reliability (ICC = 0.97 to 0.66, respectively). Conclusions: Brn3a and RBPMS markers are independently capable of detecting significant decreases of RGC number with age and in response to IOP elevation despite RPBMS detecting a larger number of RGCs up to 6 months of age. Brn3a labeling is less prone to manual cell counting variability than RBPMS labeling. Overall, either marker can be used as a single marker to detect significant changes in RGC survival, each offering distinct advantages.

An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress.

Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.

Extracellular Vesicles Contribute to Viral-Induced Hepatocellular Carcinoma: Understanding Their Involvement in Viral Hepatitis and Their Potential as Biomarkers for Early Hepatocellular Carcinoma Detection.

The high global prevalence of hepatitis B and hepatitis C and the poor prognosis of hepatitis B and hepatitis C-associated hepatocellular carcinoma (HCC), necessitates the early diagnosis and treatment of the disease. Recent studies show that cell-to-cell communication via extracellular vesicles (EVs) is involved in the HCC progression. The objective of the following study was to explore the role of EVs in the progression of viral-induced HCC and investigate their potential for the early diagnosis of cancer. First, the mRNA derived from EVs of HCC patients was compared to the mRNA derived from EVs from the healthy controls. Expression analysis of ANGPTL3, SH3BGRL3, and IFITM3 genes from the EVs was done. Afterward, to confirm whether hepatocytes can uptake EVs, HuH7 cells were exposed to EVs, and the expression analysis of downstream target genes (AKT, TNF-α, and MMP-9) in Huh7 cells was done. Transcriptional analysis showed that in the EVs from HCC patients, the expression levels of ANGPTL3, SH3BGRL3, and IFITM3 were significantly increased by 2.62-, 4.3-, and 9.03-folds, respectively. The downstream targets, AKT, TNF-α, and MMP-9, also showed a considerable change of 4.1-, 1.46-, and 5.05-folds, respectively, in Huh7 cells exposed to HCC EVs. In conclusion, the following study corroborates the role of EVs in HCC progression. Furthermore, the significant alteration in mRNA levels of the selected genes demonstrates their potential to be used as possible biomarkers for the early diagnosis of HCC.

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic ß cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells. Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Results: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression. Conclusion: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

Regulation by the RNA-binding protein Unkempt at its effector interface.

How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP-effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of 'dual-purpose' peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.