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5-methylcytosine (m C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m C profiles of mammalian transcriptomes and suggests an essential role for m C modification in mRNA export and post-transcriptional regulation.

Background As a rate‐limiting enzyme of glycolysis, pyruvate kinase muscle isozyme M2 (PKM2) participates in tumor metabolism and growth. The regulatory network of PKM2 in cancer is complex and has not been fully studied in bladder cancer. The 5‐methylcytidine (m5C) modification in PKM2 mRNA might participate in the pathogenesis of bladder cancer and need to be further clarified. This study aimed to investigate the biological function and regulatory mechanism of PKM2 in bladder cancer. Methods The expression of PKM2 and Aly/REF export factor (ALYREF) was measured by Western blotting, qRT‐PCR, and immunohistochemistry. The bioprocesses of bladder cancer cells were demonstrated by a series of experiments in vitro and in vivo. RNA immunoprecipitation, RNA‐sequencing, and dual‐luciferase reporter assays were conducted to explore the potential regulatory mechanisms of PKM2 in bladder cancer. Results In bladder cancer, we first demonstrated that ALYREF stabilized PKM2 mRNA and bound to its m5C sites in 3′‐untranslated regions. Overexpression of ALYREF promoted bladder cancer cell proliferation by PKM2‐mediated glycolysis. Furthermore, high expression of PKM2 and ALYREF predicted poor survival in bladder cancer patients. Finally, we found that hypoxia‐inducible factor‐1alpha (HIF‐1α) indirectly up‐regulated the expression of PKM2 by activating ALYREF in addition to activating its transcription directly. Conclusions The m5C modification in PKM2 mRNA in the HIF‐1α/ALYREF/PKM2 axis may promote the glucose metabolism of bladder cancer, providing a new promising therapeutic target for bladder cancer. Our data suggested that m5C may play a critical role in hypoxia/glycolysis network and targeting HIF‐1α/ALYREF/PKM2 signaling may be a promising therapeutic strategy to treat bladder cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have emerged as key regulators of cancer development and progression, and as promising biomarkers for the diagnosis and prognosis of cancer. In this study, we identified a new lncRNA (LINC02159) that was upregulated in the tumor tissues and serum of non-small cell lung cancer (NSCLC) patients. We demonstrated that knockdown of LINC02159 inhibited NSCLC cell proliferation, migration, and invasion, but induced cell apoptosis and cell cycle arrest in vitro and retarded tumor growth in vivo, while overexpression of LINC02159 led to the opposite effect. We discovered that LINC02159 was highly correlated with cancer growth and metastasis-related pathways by using transcriptomic analysis and that YAP1 was a potential target gene of LINC02159. Mechanistically, LINC02159 bound to the Aly/REF export factor (ALYREF) to enhance the stability of YAP1 messenger RNA (mRNA) via m 5 C modification, which led to the overexpression of YAP1 and the activation of the Hippo and β-catenin signaling pathways in NSCLC cells. Rescue experiments showed that LINC01259 promoted NSCLC progression in a YAP1- and ALYREF-dependent manner. In conclusion, LINC02159 plays an oncogenic role in NSCLC progression by regulating ALYREF/YAP1 signaling, and it has the potential to be utilized as a diagnostic marker and therapeutic target for NSCLC.

Non-small-cell lung cancer (NSCLC) represents a highly prevalent form of malignancy. 5-methylcytosine (m 5 C) methylation functions as a key post-transcriptional regulatory mechanism linked to cancer progression. The persistent expression of PD-L1 in tumor cells plays a pivotal role in facilitating immune evasion and promoting T-cell exhaustion. However, the involvement of m 5 C in NSCLC immune evasion remains inadequately understood. This study seeks to explore the function of the m 5 C methyltransferase NSUN2 in modulating PD-L1 expression and facilitating immune evasion in NSCLC. Our findings indicate elevated levels of NSUN2 and ALYREF in NSCLC, and both promote the growth of NSCLC cells and the progression of lung cancer. Moreover, the expression of PD-L1 in NSCLC tissues positively correlates with NSUN2 and ALYREF expression. We then discovered that PD-L1 acts as a downstream target of NSUN2-mediated m 5 C modification in NSCLC cells. Knocking down NSUN2 significantly reduces m 5 C modification of PD-L1 mRNA, thereby decreasing its stability via the m 5 C reader ALYREF-dependent manner. Furthermore, inhibiting NSUN2 enhanced CD8 + T-cell activation and infiltration mediated by PD-L1, thereby boosting antitumor immunity, as confirmed in both in vitro and in vivo experiments. Collectively, these results suggested that NSUN2/ALYREF/PD-L1 axis plays a critical role in promoting NSCLC progression and tumor cell immune suppression, highlighting its potential as a novel therapeutic strategy for NSCLC immunotherapy.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1 , as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1 .

Background Diabetic retinopathy (DR) is the leading cause of blinding eye disease among working adults and is primarily attributed to the excessive proliferation of microvessels, which leads to vitreous hemorrhage and retinal traction, thereby significantly impairing patient vision. NSUN2-mediated RNA m 5 C methylation is implicated in various diseases, and in this investigation, we focused on elucidating the impact of NSUN2 on the regulation of the expression of the downstream gene MUC1, specifically through RNA m 5 C methylation, on the progression of DR. Method Utilizing Microarray analysis, we examined patient vitreous fluid to pinpoint potential therapeutic targets for DR. Differential expression of NSUN2 was validated through qRT-PCR, Western blot, and immunofluorescence in human tissue, animal tissue, and cell model of DR. The relationship between NSUN2 and DR was explored in vitro and in vivo through gene knockdown and overexpression. Various techniques, such as MeRIP-qPCR and dot blot, were applied to reveal the downstream targets and mechanism of action of NSUN2. Results The levels of both NSUN2 and RNA m 5 C methylation were significantly elevated in the DR model. Knockdown of NSUN2 mitigated DR lesion formation both in vitro and in vivo. Mechanistically, NSUN2 promoted MUC1 expression by binding to the RNA m 5 C reader ALYREF. Knockdown of ALYREF resulted in DR lesion alterations similar to those observed with NSUN2 knockdown. Moreover, MUC1 overexpression successfully reversed a series of DR alterations induced by NSUN2 silencing. Conclusions NSUN2 regulates the expression of MUC1 through ALYREF-mediated RNA m 5 C methylation, thereby regulating the progression of DR and providing a new option for the treatment of DR in the future.

5-Methylcytosine (m5C) is one of the most ubiquitous modifications of mRNA and contributes to cancer pathogenesis. Aly/REF export factor (ALYREF), an m5C reader, is associated with the prognosis of liver hepatocellular carcinoma (LIHC). However, the effects of ALYREF on the progression of LIHC and the underlying molecular mechanisms remains elusive. Through an analysis of an online database and 3 independent LIHC cohorts, we found that ALYREF was markedly elevated in human liver cancer tissues and was significantly correlated with LIHC clinicopathological parameters, including Ki67 cell rate, high-grade TNM stage, and poor prognosis. Several experiments were conducted to investigate the molecular basis and functional role of ALYREF-related progression in this study. ALYREF could enhance LIHC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) and tumor formation . Mechanistically, ALYREF promoted the progression of human LIHC through EGFR pathways. Furthermore, ALYREF could directly bind to the m5C modification site of EGFR 3' untranslated region (3' UTR) to stabilize EGFR mRNA. Collectively, ALYREF played a crucial oncogenic role in LIHC via the stabilization of EGFR mRNA and subsequent activation of the STAT3 signaling pathway. Our results may help to elucidate the potential mechanisms of ALYREF-induced m5C modification in the progression of human LIHC.

This study explores the regulatory function of BAP31 on exosomal miRNA and its impact on the EMT in CRC. Exosomes from BAP31-OE cells promoted recipient cell migration and triggered the EMT, as indicated by decreased E-cadherin and increased N-cadherin and Vimentin levels. By contrast, exosomes derived from shBAP31 cells were observed to inhibit cell migration and revert EMT markers. The administration of shBAP31 exosomes significantly inhibited tumor growth in vivo. miRNA profiling revealed 76 differentially expressed miRNAs in BAP31-OE exosomes. Six miRNA candidates associated with the EMT were identified in the GEO database, miR-423-3p was identified as a key mediator, the candidates from shBAP31 exosomes exhibited the opposite effect. EMT promotion by miR-423-3p was further evidenced by EMT marker expression, enhanced migratory capacity, and accelerated tumor growth. Sixteen potential target genes were identified through bioinformatics analysis. Bim exhibited significant downregulation by the miR-423-3p mimic. Luciferase reporter assays verified the direct interaction between miR-423-3p and the 3′UTR of Bim. Silencing Bim negated the effects of miR-423-3p. It was also revealed that BAP31 does not influence the total exosomal miRNA content but selectively regulates miR-423-3p, which contains an EXOmotif enriched in BAP31-OE exosomes. Mechanistic studies revealed that BAP31 enhances the expression of the RNA export adaptor Alyref, as validated by qRT-PCR and Western blot analyses. RNA immunoprecipitation assays verified that Alyref binds to miR-423-3p in BAP31-OE cells. Our results reveal that BAP31 facilitates the sorting of exosomal miR-423-3p via Alyref, thereby promoting EMT in CRC through the miR-423-3p/Bim signaling axis. This indicates that BAP31 could be a viable therapeutic target for managing the EMT in CRC.

Bladder cancer (BCa) presents a significant clinical challenge, with a pressing need for biomarkers to predict prognosis and guide immunotherapy. Aly/REF export factor (ALYREF) is a key regulator in various cancers, but its role in bladder cancer (BCa) remains unclear. This study aimed to investigate the clinical significance of ALYREF and its association with immunotherapy response and metastatic potential in BCa. We integrated pan-cancer bioinformatic analyses with clinical validation using RT-qPCR, immunohistochemistry, and Western blot on patient samples. We assessed ALYREF’s correlation with clinicopathological features, survival, and response to immune checkpoint inhibitors (ICIs). Circulating tumor cell (CTC) counts were analyzed to link ALYREF to metastatic potential. Functional roles in migration and invasion were validated in vitro using the T24 cell line. ALYREF was significantly upregulated in BCa tissues, correlating with higher tumor grade and poorer overall survival. Paradoxically, high ALYREF expression was also associated with a better response to ICI therapy. Furthermore, elevated ALYREF levels in tumors corresponded to increased CTC counts. In vitro experiments confirmed that ALYREF promotes BCa cell migration and invasion. ALYREF is a dual-role biomarker in BCa. Its overexpression signifies aggressive tumor biology and metastatic risk yet also predicts a favorable response to immunotherapy. ALYREF holds promise for refining patient stratification and personalizing BCa treatment.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m 7 G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.