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Without new transcription, gene expression across the oocyte-to-embryo transition (OET) relies instead on regulation of mRNA poly(A) tails to control translation. However, how tail dynamics shape translation across the OET in mammals remains unclear. We perform long-read RNA sequencing to uncover poly(A) tail lengths across the mouse OET and, incorporating published ribosome profiling data, provide an integrated, transcriptome-wide analysis of poly(A) tails and translation across the entire transition. We uncover an extended wave of global deadenylation during fertilization in which short-tailed, oocyte-deposited mRNAs are translationally activated without polyadenylation through resistance to deadenylation. Subsequently, in the embryo, mRNAs are readenylated and translated in a surge of global polyadenylation. We further identify regulation of poly(A) tail length at the isoform level and stage-specific enrichment of mRNA sequence motifs among regulated transcripts. These data provide insight into the stage-specific mechanisms of poly(A) tail regulation that orchestrate gene expression from oocyte to embryo in mammals.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host–microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1−/−) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1−/− mice. Furthermore, plasma IgA was increased in Bach1−/− mice, but fecal IgA was decreased, indicating that Bach1−/− mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, the overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression. NEW & NOTEWORTHY: The transcriptional repressor Bach1 has been implicated in diverse intestinal functions, but the effects of Bach1 on IgA-mediated mucosal immunity remain unclear. We demonstrate here that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen, although the proportions of IgA-producing cells were not altered. Furthermore, Bach1 regulates the expression of pIgR, which plays an important role in the transepithelial transport of IgA, at the posttranscriptional level. [ABSTRACT FROM AUTHOR]

Many viruses have evolved structured RNA elements that can influence transcript abundance and translational efficiency, and help evade host immune factors by hijacking cellular machinery during replication. Here, we evaluated the functional impact of sub-genomic flaviviral RNAs (sfRNAs) known to stall exoribonuclease activity, by incorporating these elements into recombinant adeno-associated viral (AAV) genome cassettes. Specifically, sfRNAs from Dengue, Zika, Japanese Encephalitis, Yellow Fever, Murray Valley Encephalitis, and West Nile viruses increased transcript stability and transgene expression compared to a conventional woodchuck hepatitis virus element (WPRE). Further dissection of engineered transcripts revealed that sfRNA elements (i) require incorporation in cis within the 3′ untranslated region (UTR) of AAV genomes, (ii) require minimal dumbbell structures to exert the observed effects, and (iii) can stabilize AAV transcripts independent of 5′−3′ exoribonuclease 1 (XRN1)-mediated decay. Additionally, preliminary in vivo assessment of AAV vectors bearing sfRNA elements in mice achieved increased transcript abundance and expression in cardiac tissue. Leveraging the functional versatility of engineered viral RNA elements may help improve the potency of AAV vector-based gene therapies. IMPORTANCE Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer. [ABSTRACT FROM AUTHOR]

Renal aging and the subsequent rise in kidney-related diseases are attributed to senescence in renal tubular epithelial cells (RTECs). Our study revealed that the abnormal expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a reader of RNA N6-methyladenosine, is critically involved in cisplatin-induced renal tubular senescence. In cisplatin-induced senescence of RTECs, the promoter activity and transcription of IGF2BP3 is markedly suppressed. It was due to the down regulation of MYC proto-oncogene (MYC), which regulates IGF2BP3 transcription by binding to the putative site at 1852–1863 of the IGF2BP3 promoter. Overexpression of IGF2BP3 ameliorated cisplatin-induced renal tubular senescence in vitro. Mechanistic studies revealed that IGF2BP3 inhibits cellular senescence in RTECs by enhancing cyclin-dependent kinase 6 (CDK6) mRNA stability and increasing its expression. The inhibition effect of IGF2BP3 on tubular senescence is partially reversed by the knockdown of CDK6. Further, IGF2BP3 recruits nuclear cap binding protein subunit 1 (NCBP1) and inhibits CDK6 mRNA decay, by recognizing m6A modification. Specifically, IGF2BP3 recognizes m6A motif "GGACU" at nucleotides 110–114 in the 5′ untranslated region (UTR) field of CDK6 mRNA. The involvement of IGF2BP3/CDK6 in alleviating tubular senescence was confirmed in a cisplatin-induced acute kidney injury (AKI)-to-chronic kidney disease (CKD) model. Clinical data also suggests an age-related decrease in IGF2BP3 and CDK6 levels in renal tissue or serum samples from patients. These findings suggest that IGF2BP3/CDK6 may be a promising target in cisplatin-induced tubular senescence and renal failure. [ABSTRACT FROM AUTHOR]

Abstract Background Alterations in epigenetic factors are recognized as key contributors to the emergence of human cancer. The active and reversible alteration of N6‐methyladenosine (m6A) RNA is crucial for controlling gene activity and determining cellular destiny. Even with these insights, the triggering of KIAA1429 (also called VIRMA) and its role in lung adenocarcinoma (LUAD) is mostly unclear. As a result, the objective of this study was to elucidate how KIAA1429 contributes to cancer development in LUAD. Methods This study utilized multiple methods for investigation, encompassing the in vitro functional examination of KIAA1429 in lung adenocarcinoma cells, transcriptome sequencing, methylation RNA immunoprecipitation sequencing (MeRIP‐seq), as well as RNA stability tests to ascertain the half‐life and stability of the target genes. Results The results indicated that modifying the expression of KIAA1429 regulated the proliferation and metastasis of LUAD. By employing transcriptome sequencing alongside MeRIP‐seq analysis, the research pinpointed genes affected by m6A alterations triggered by KIAA1429. In a more detailed manner, it was discovered that KIAA1429 plays a regulatory role in the expression of ARHGAP30. Suppressing KIAA1429 results in reduced m6A levels in the mRNA of the target gene ARHGAP30, boosting its stability and expression, thus inhibiting tumor proliferation and metastasis. Conclusion This study revealed the activation mechanism and pivotal function of KIAA1429 in LUAD tumor development, paving the way for molecular‐based interventions for LUAD.

Abstract Background KIAA1429, a regulatory subunit of the N6-methyladenosine (m6A) methyltransferase complex, has been implicated in the progression of various cancers. However, the role of KIAA1429 in gastric cancer (GC) and its underlying mechanisms remain elusive. This study aimed to investigate the role of KIAA1429 in GC and to elucidate the underlying mechanisms. Methods The expression patterns and clinical relevance of KIAA1429 in GC were assessed using quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and bioinformatic analysis. In vitro and in vivo loss- and gain-of-function assays, m6A dot blot assays, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, dual luciferase reporter assays, RNA stability assays, RNA immunoprecipitation (RIP) assays, and RNA pull-down assays were performed to investigate the biological functions and underlying molecular mechanisms of KIAA1429 in GC. Results Both the mRNA and protein expression of KIAA1429 were greater in GC tissues than in normal gastric tissues. High KIAA1429 expression correlated positively with poor prognosis in GC patients. KIAA1429 not only promoted GC cell proliferation, colony formation, G2/M cell cycle transition, migration, and invasion in vitro but also enhanced GC tumor growth and metastasis in vivo. Mechanistically, KIAA1429 increased the m6A level of RASD1 mRNA and enhanced its stability in an m6A-YTHDF2-dependent manner, thereby upregulating its expression. RASD1 knockdown partially rescued the KIAA1429 knockdown-induced impairment of pro‑oncogenic ability in GC cells. The expression levels of KIAA1429 and RASD1 were negatively correlated in GC tissues. Conclusions KIAA1429 plays a pro‑oncogenic role in GC by downregulating RASD1 expression through destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner. KIAA1429 may serve as a prognostic biomarker and therapeutic target for GC.

Background: Alterations in epigenetic factors are recognized as key contributors to the emergence of human cancer. The active and reversible alteration of N6‐methyladenosine (m6A) RNA is crucial for controlling gene activity and determining cellular destiny. Even with these insights, the triggering of KIAA1429 (also called VIRMA) and its role in lung adenocarcinoma (LUAD) is mostly unclear. As a result, the objective of this study was to elucidate how KIAA1429 contributes to cancer development in LUAD. Methods: This study utilized multiple methods for investigation, encompassing the in vitro functional examination of KIAA1429 in lung adenocarcinoma cells, transcriptome sequencing, methylation RNA immunoprecipitation sequencing (MeRIP‐seq), as well as RNA stability tests to ascertain the half‐life and stability of the target genes. Results: The results indicated that modifying the expression of KIAA1429 regulated the proliferation and metastasis of LUAD. By employing transcriptome sequencing alongside MeRIP‐seq analysis, the research pinpointed genes affected by m6A alterations triggered by KIAA1429. In a more detailed manner, it was discovered that KIAA1429 plays a regulatory role in the expression of ARHGAP30. Suppressing KIAA1429 results in reduced m6A levels in the mRNA of the target gene ARHGAP30, boosting its stability and expression, thus inhibiting tumor proliferation and metastasis. Conclusion: This study revealed the activation mechanism and pivotal function of KIAA1429 in LUAD tumor development, paving the way for molecular‐based interventions for LUAD. [ABSTRACT FROM AUTHOR]

Background: KIAA1429, a regulatory subunit of the N6-methyladenosine (m6A) methyltransferase complex, has been implicated in the progression of various cancers. However, the role of KIAA1429 in gastric cancer (GC) and its underlying mechanisms remain elusive. This study aimed to investigate the role of KIAA1429 in GC and to elucidate the underlying mechanisms. Methods: The expression patterns and clinical relevance of KIAA1429 in GC were assessed using quantitative real-time PCR (qRT-PCR), Western blotting, immunohistochemistry (IHC), and bioinformatic analysis. In vitro and in vivo loss- and gain-of-function assays, m6A dot blot assays, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA-seq, MeRIP-qPCR, dual luciferase reporter assays, RNA stability assays, RNA immunoprecipitation (RIP) assays, and RNA pull-down assays were performed to investigate the biological functions and underlying molecular mechanisms of KIAA1429 in GC. Results: Both the mRNA and protein expression of KIAA1429 were greater in GC tissues than in normal gastric tissues. High KIAA1429 expression correlated positively with poor prognosis in GC patients. KIAA1429 not only promoted GC cell proliferation, colony formation, G2/M cell cycle transition, migration, and invasion in vitro but also enhanced GC tumor growth and metastasis in vivo. Mechanistically, KIAA1429 increased the m6A level of RASD1 mRNA and enhanced its stability in an m6A-YTHDF2-dependent manner, thereby upregulating its expression. RASD1 knockdown partially rescued the KIAA1429 knockdown-induced impairment of pro‑oncogenic ability in GC cells. The expression levels of KIAA1429 and RASD1 were negatively correlated in GC tissues. Conclusions: KIAA1429 plays a pro‑oncogenic role in GC by downregulating RASD1 expression through destabilizing RASD1 mRNA in an m6A-YTHDF2-dependent manner. KIAA1429 may serve as a prognostic biomarker and therapeutic target for GC. [ABSTRACT FROM AUTHOR]

Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level. [ABSTRACT FROM AUTHOR]

Background: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. Methods: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. Results: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. Conclusions: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB. [ABSTRACT FROM AUTHOR]

As the most abundant form of methylation modification in messenger RNA (mRNA), the distribution of N6‐methyladenosine (m6A) has been preliminarily revealed in herbaceous plants under salt stress, but its function and mechanism in woody plants were still unknown. Here, we showed that global m6A levels increased during poplar response to salt stress. Methylated RNA immunoprecipitation sequencing (MeRIP‐seq) revealed that m6A significantly enriched in the coding sequence region and 3′‐untranslated regions in poplar, by recognising the conserved motifs, AGACU, GGACA and UGUAG. A large number of differential m6A transcripts have been identified, and some have been proved involving in salt response and plant growth and development. Further combined analysis of MeRIP‐seq and RNA‐seq revealed that the m6A hypermethylated and enrich in the CDS region preferred to positively regulate expression abundance. Writer inhibitor, 3‐deazaneplanocin A treatment increased the sensitivity of poplar to salt stress by reducing mRNA stability to regulate the expression of salt‐responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Furthermore, we verified that the methyltransferase PagFIP37 plays a positively role in the response of poplar to salt stress, overexpressed lines have stronger salt tolerance, while RNAi lines were more sensitive to salt, which relied on regulating mRNA stability in an m6A manner of salt‐responsive transcripts PagMYB48, PagGT2, PagNAC2, PagGPX8 and PagARF2. Collectively, these results revealed the regulatory role of m6A methylation in poplar response to salt stress, and revealed the importance and mechanism of m6A methylation in the response of woody plants to salt stress for the first time. Summary statement: We revealed the regulatory role of m6A methylation in poplar response to salt stress, and revealed the importance and mechanism of m6A methylation in the response of woody plants to salt stress for the first time. [ABSTRACT FROM AUTHOR]

Cellular metabolism is tightly regulated by growth factor signaling, which promotes metabolic rewiring to support growth and proliferation. While growth factor-induced transcriptional and post-translational modes of metabolic regulation have been well defined, whether post-transcriptional mechanisms impacting mRNA stability regulate this process is less clear. Here, we present the ZFP36/L1/L2 family of RNA-binding proteins and mRNA decay factors as key drivers of metabolic regulation downstream of acute growth factor signaling. We quantitatively catalog metabolic enzyme and nutrient transporter mRNAs directly bound by ZFP36 following growth factor stimulation-many of which encode rate-limiting steps in metabolic pathways. Further, we show that ZFP36 directly promotes the mRNA decay of Enolase 2 (Eno2), altering Eno2 protein expression and enzymatic activity, and provide evidence of a ZFP36/Eno2 axis during VEGF-stimulated developmental retinal angiogenesis. Thus, ZFP36-mediated mRNA decay serves as an important mode of metabolic regulation downstream of growth factor signaling within dynamic cell and tissue states.

Background: Post-ischemic angiogenesis is crucial for reestablishing blood flow in conditions such as peripheral artery disease (PAD). The role of insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP2) in post-transcriptional RNA metabolism and its involvement in post-ischemic angiogenesis remains unclear. Methods: Using a human GEO database and a hind-limb ischemia (HLI) mouse model, the predominant isoform IGF2BP2 in ischemic gastrocnemius tissue was identified. Adeno-associated virus with the Tie1 promoter induced IGF2BP2 overexpression in the HLI model, evaluating the expression of vascular structural proteins (CD31 and α-SMA) and blood flow recovery after HLI. In vitro experiments with human umbilical vein endothelial cells (HUVECs) demonstrated that lentivirus-mediated IGF2BP2 overexpression upregulates cell proliferation, migration, and tube formation. GeneCards, RNAct databases, and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) predicted IGF2BP2 interactions with fibroblast growth factor 2 (FGF2) mRNA, and actinomycin D treatment, binding site predictions and CLIP-seq data further confirmed this interaction. Furthermore, western blotting, enzyme-linked immunosorbent assay, and RNA immunoprecipitation followed by RT-qPCR were performed to validate IGF2BP2's interaction with FGF2 mRNA and to assess its role in stabilizing FGF2 mRNA, as well as its impact on FGF2 protein expression. Results: HLI reduced IGF2BP2 expression in the gastrocnemius tissue, which gradually increased during blood flow recovery. IGF2BP2 overexpression in HLI mice accelerated blood flow recovery and increased capillary and small artery densities. The overexpression of IGF2BP2 in HUVECs stimulated proliferation, migration, and tube formation by interacting with FGF2 mRNA to increase its stability. This interaction resulted in increased levels of FGF2 protein and secretion, ultimately promoting angiogenesis. Conclusions: IGF2BP2 contributes to blood flow restoration post-ischemia in vivo and promotes angiogenesis in HUVECs by enhancing FGF2 mRNA stability and FGF2 protein expression and secretion. These findings underscore IGF2BP2's therapeutic potential in ischemic conditions, such as PAD.

Abstract Codon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. Here we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Mathematical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly alter the effect of codon usage. This new mode of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression.

Abstract Background RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3′ untranslated regions (3′ UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3′ UTRs in gene expression in different organisms and/or contexts. Results Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3′ UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3′ UTR and, resultantly, a poorly structured 3′ UTR. RNA decay assays indicate that poorly structured 3′ UTRs could promote mRNA stability, whereas highly structured 3′ UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3′ UTRs’ RSS and transcript accumulation in the transcriptomes of Arabidopsis, rice, and even human. Mechanistically, transcripts with highly structured 3′ UTRs are preferentially degraded by 3′–5′ exoribonuclease SOV and 5′–3′ exoribonuclease XRN4, leading to decreased expression in Arabidopsis. Finally, we engineer different structured 3′ UTRs to an endogenous FT gene and alter the FT-regulated flowering time in Arabidopsis. Conclusions We conclude that highly structured 3′ UTRs typically cause reduced accumulation of the harbored transcripts in Arabidopsis. This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3′ UTRs’ RSS to modify plant traits in agricultural production and mRNA stability in biotechnology.

Abstract Background Several publications suggest that UTP11 may be a promising gene engaged for involvement of hepatocellular carcinoma (HCC) pathology. However, there are extremely limited biological, mechanistic and clinical studies of UTP11 in HCC. Methods To anayze the UTP11 mRNA expression in HCC and normal clinical samples and further investigate the correlation between UTP11 expression and pathology and clinical prognosis via the Cancer Tissue Gene Atlas (TCGA) database. The protein levels of UTP11 were checked using the Human Protein Atlas (HPA) database. GO-KEGG enrichment was performed from Cancer Cell Line Encyclopedia (CCLE) database and TCGA dataset. The levels of UTP11 were tested with qRT-PCR and western blotting assays. Cell viability, immunofluorescence and flow cytometry assays and animal models were used to explore the potential involvement of UTP11 in regulating HCC growth in vitro and in vivo. The correlation of UTP11 and tumor stemness scores and stemness-associated proteins from TCGA database. The mRNA stability was treated with Actinomycin D, followed by testing the mRNA expression using qRT-PCR assay. Results UTP11 was highly expressed in HCC samples compared to normal tissues from TCGA database. Similarly, UTP11 protein expression levels were obviously elevated in HCC tissue samples from HPA database. Furthermore, UTP11 levels were correlated with poor prognosis in HCC patient samples in TCGA dataset. In addition, the UTP11 mRNA levels was notably enhanced in different HCC cell lines than in normal liver cells and knocking down UTP11 was obviously reduced the viability and cell death of HCC cells. UTP11 knockdown suppressed the tumor growth of HCC in vivo experiment and extended the mice survival time. GO-KEEG analysis from CCLE and TCGA database suggested that UTP11 might involve in RNA splicing and the stability of mRNA. Further, UTP11 was positively correlated with tumor stemness scores and stemness-associated proteins from TCGA database. Knockdown of UTP11 was reduced the expression of stem cell-related genes and regulated the mRNA stability of Oct4. Conclusions UTP11 is potentially a diagnostic molecule and a therapeutic candidate for treatment of HCC.

The transcriptional regulation of skeletal muscle (SKM) development (myogenesis) has been documented for over 3 decades and served as a paradigm for tissue-specific cell type determination and differentiation. Myogenic stem cells (MuSC) in embryos and adult SKM are regulated by the transcription factors Pax3 and Pax7 for their stem cell characteristics, while their lineage determination and terminal differentiation are both dictated by the myogenic regulatory factors (MRF) that comprise Mrf4, Myf5, Myogenin, and MyoD. The myocyte enhancer factor Mef2c is activated by MRF during terminal differentiation and collaborates with them to promote myoblast fusion and differentiation. Recent studies have found critical regulation of these myogenic transcription factors at mRNA level, including subcellular localization, stability, and translational regulation. Therefore, the regulation of Pax3/7, MRFs and Mef2c mRNAs by RNA-binding factors and non-coding RNAs (ncRNA), including microRNAs and long non-coding RNAs (lncRNA), will be the focus of this review and the impact of this regulation on myogenesis will be further addressed. Interestingly, the stem cell characteristics of MuSC has been found to be critically regulated by ncRNAs, implying the involvement of ncRNAs in SKM homeostasis and regeneration. Current studies have further identified that some ncRNAs are implicated in the etiology of some SKM diseases and can serve as valuable tools/indicators for prediction of prognosis. The roles of ncRNAs in the MuSC biology and SKM disease etiology will also be discussed in this review. [ABSTRACT FROM AUTHOR]

Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3′UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer. [ABSTRACT FROM AUTHOR]

SUMMARY: Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery‐induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil‐grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light‐induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut‐off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript‐specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis. Significance Statement: We provide a method for administering the transcriptional inhibitor, cordycepin, to mature Arabidopsis leaves. We study changes in mRNA stability and translation during light stress and recovery. [ABSTRACT FROM AUTHOR]

Background: Several publications suggest that UTP11 may be a promising gene engaged for involvement of hepatocellular carcinoma (HCC) pathology. However, there are extremely limited biological, mechanistic and clinical studies of UTP11 in HCC. Methods: To anayze the UTP11 mRNA expression in HCC and normal clinical samples and further investigate the correlation between UTP11 expression and pathology and clinical prognosis via the Cancer Tissue Gene Atlas (TCGA) database. The protein levels of UTP11 were checked using the Human Protein Atlas (HPA) database. GO-KEGG enrichment was performed from Cancer Cell Line Encyclopedia (CCLE) database and TCGA dataset. The levels of UTP11 were tested with qRT-PCR and western blotting assays. Cell viability, immunofluorescence and flow cytometry assays and animal models were used to explore the potential involvement of UTP11 in regulating HCC growth in vitro and in vivo. The correlation of UTP11 and tumor stemness scores and stemness-associated proteins from TCGA database. The mRNA stability was treated with Actinomycin D, followed by testing the mRNA expression using qRT-PCR assay. Results: UTP11 was highly expressed in HCC samples compared to normal tissues from TCGA database. Similarly, UTP11 protein expression levels were obviously elevated in HCC tissue samples from HPA database. Furthermore, UTP11 levels were correlated with poor prognosis in HCC patient samples in TCGA dataset. In addition, the UTP11 mRNA levels was notably enhanced in different HCC cell lines than in normal liver cells and knocking down UTP11 was obviously reduced the viability and cell death of HCC cells. UTP11 knockdown suppressed the tumor growth of HCC in vivo experiment and extended the mice survival time. GO-KEEG analysis from CCLE and TCGA database suggested that UTP11 might involve in RNA splicing and the stability of mRNA. Further, UTP11 was positively correlated with tumor stemness scores and stemness-associated proteins from TCGA database. Knockdown of UTP11 was reduced the expression of stem cell-related genes and regulated the mRNA stability of Oct4. Conclusions: UTP11 is potentially a diagnostic molecule and a therapeutic candidate for treatment of HCC. [ABSTRACT FROM AUTHOR]

Simple Summary: RNA-binding proteins (RBPs) play a critical role in controlling gene expression post-transcriptionally and their dysregulation can lead to various diseases, including cancer. One such RBP is HuR, whose levels have been associated with a poor clinical prognosis in breast cancer. Nevertheless, the precise molecular mechanism underlying this connection remains incompletely characterized. Our study uncovers that HuR targets SOX9 mRNA in breast cancer cells and provides compelling evidence supporting HuR's involvement in cell migration and invasion. RNA-binding proteins play diverse roles in cancer, influencing various facets of the disease, including proliferation, apoptosis, angiogenesis, senescence, invasion, epithelial–mesenchymal transition (EMT), and metastasis. HuR, a known RBP, is recognized for stabilizing mRNAs containing AU-rich elements (AREs), although its complete repertoire of mRNA targets remains undefined. Through a bioinformatics analysis of the gene expression profile of the Hs578T basal-like triple-negative breast cancer cell line with silenced HuR, we have identified SOX9 as a potential HuR-regulated target. SOX9 is a transcription factor involved in promoting EMT, metastasis, survival, and the maintenance of cancer stem cells (CSCs) in triple-negative breast cancer. Ribonucleoprotein immunoprecipitation assays confirm a direct interaction between HuR and SOX9 mRNA. The half-life of SOX9 mRNA and the levels of SOX9 protein decreased in cells lacking HuR. Cells silenced for HuR exhibit reduced migration and invasion compared to control cells, a phenotype similar to that described for SOX9-silenced cells. [ABSTRACT FROM AUTHOR]

mRNA modifications impact the mRNA life cycle. Pseudouridine (Ψ) is the most prevalent uridine modification in mRNA. There have been recent developments in technologies to quantify and to study the functions of Ψ, particularly in mRNA. Ψ residues in mRNA have functions in splicing, translation, and stability. The presence and regulation of uridine modifications in mRNA urge future studies into their functions. The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA–protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review. The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA–protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review. [ABSTRACT FROM AUTHOR]

Background: Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. Objective: This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. Methods: Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24–26-month-old Sprague–Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated β-galactosidase (SA-β-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. Results: Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. Conclusions: HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer, with a highly aggressive phenotype and poor prognosis. RNA binding proteins (RBPs) play crucial roles in post-transcriptional gene regulation and have been implicated in tumorigenesis. RBPs have the potential to become a new therapeutic target for ccRCC. In this study, we screened and validated that insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) as an RBP, was down-regulated in ccRCC tissues and cell lines. Functionally, we verified that IGF2BP2 significantly suppressed the migration and invasion ability of ccRCC in vitro and in vivo. Mechanistically, RIP-seq and actinomycin D experiments results showed that IGF2BP2 enhanced the expression of Creatine Kinase B (CKB) by binding to CKB mRNA and enhancing its mRNA stability. Thus, IGF2BP2 inhibited ccRCC metastasis through enhancing the expression of CKB. Taken together, these finding suggests that IGF2BP2 is a novel metastasis suppressor of ccRCC and may serve as a potential therapeutic target.

Gut barrier dysfunction occurs commonly in patients with critical disorders, leading to the translocation of luminal toxic substances and bacteria to the bloodstream. Connexin 43 (Cx43) acts as a gap junction protein and is crucial for intercellular communication and the diffusion of nutrients. The levels of cellular Cx43 are tightly regulated by multiple factors, including polyamines, but the exact mechanism underlying the control of Cx43 expression remains largely unknown. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is involved in many aspects of intestinal epithelial pathobiology. Here we show that HuR directly bound to Cx43 mRNA via its 30-untranslated region in intestinal epithelial cells (IECs) and this interaction enhanced Cx43 expression by stabilizing Cx43 mRNA. Depletion of cellular polyamines inhibited the [HuR/Cx43 mRNA] complex and decreased the level of Cx43 protein by destabilizing its mRNA, but these changes were prevented by ectopic overexpression of HuR. Polyamine depletion caused intestinal epithelial barrier dysfunction, which was reversed by ectopic Cx43 overexpression. Moreover, overexpression of checkpoint kinase 2 in polyamine-deficient cells increased the [HuR/Cx43 mRNA] complex, elevated Cx43 levels, and promoted barrier function. These findings indicate that Cx43 mRNA is a novel target of HuR in IECs and that polyamines regulate Cx43 mRNA stability via HuR, thus playing a critical role in the maintenance of intestinal epithelial barrier function. NEW & NOTEWORTHY The current study shows that polyamines stabilize the Cx43 mRNA via HuR, thus enhancing the function of the Cx43-mediated gap junction. These findings suggest that induced Cx43 by HuR plays a critical role in the process by which polyamines regulate intestinal epithelial barrier. [ABSTRACT FROM AUTHOR]

Foxtail millet (Setaria italica), a vital drought‐resistant crop, plays a significant role in ensuring food and nutritional security. However, its drought resistance mechanism is not fully understood. N6‐methyladenosine (m6A) modification of RNA, a prevalent epi‐transcriptomic modification in eukaryotes, provides a binding site for m6A readers and affects plant growth and stress responses by regulating RNA metabolism. In this study, we unveiled that the YT521‐B homology (YTH) family gene SiYTH1 positively regulated the drought tolerance of foxtail millet. Notably, the siyth1 mutant exhibited reduced stomatal closure and augmented accumulation of excessive H2O2 under drought stress. Further investigations demonstrated that SiYTH1 positively regulated the transcripts harboring m6A modification related to stomatal closure and reactive oxygen species (ROS) scavenging under drought stress. SiYTH1 was uniformly distributed in the cytoplasm of SiYTH1‐GFP transgenic foxtail millet. It formed dynamic liquid‐like SiYTH1 cytosol condensates in response to drought stress. Moreover, the cytoplasmic protein SiYTH1 was identified as a distinct m6A reader, facilitating the stabilization of its directly bound SiARDP and ROS scavenging‐related transcripts under drought stress. Furthermore, natural variation analysis revealed SiYTH1AGTG as the dominant allele responsible for drought tolerance in foxtail millet. Collectively, this study provides novel insights into the intricate mechanism of m6A reader‐mediated drought tolerance and presents a valuable genetic resource for improving drought tolerance in foxtail millet breeding. [ABSTRACT FROM AUTHOR]

• METTL3 knockdown promoted inflammatory cytokine production and ROS generation. • METTL3 knockdown enhanced the activation of MAPKs and AKT signaling and upregulation of NOXs through Pyk2. • Mettl3 regulates the mRNA stability and expression of Pyk2 in a YTHDF2-dependent way. As one of the most prevalent modifications on RNA, N6-methyladenosine (m6A) has been recently found implicated in various pathological processes. Emerging studies have demonstrated the role of m6A and its writer Mettl3 in fine-tuning the immune response, which now becomes a research hotspot owing to its potential therapeutic value. However, the results are inconsistent and even contradictory, suggesting that there might be multiple Mettl3 target genes involved in different pathways. To delve deeper into the function of Mettl3 in the cellular inflammatory response, we first conducted bioinformatics analysis using RNA-seq in Mettl3 ablation macrophages, and found that Mettl3 might attenuate LPS-induced proinflammatory pathways and reactive oxygen species (ROS) generation process. Mettl3 knockdown significantly increased the LPS-induced IL-6, TNF-α, NOXs (Nox1, Nox2, Ncf1, and Ncf2) expression, ROS generation, and the phosphorylation of MAPKs and AKT signaling. Combining the results of RNA-seq and m6A mapping, we found that Pyk2 might be the target gene of Mettl3 affecting the inflammatory response. Mettl3 and Ythdf2 depletion increased the expression and mRNA stability of Pyk2, and RIP-PCR showed that Ythdf2 directly targeting Pyk2 was Mettl3 dependent. Moreover, the upregulated expression of TNF-α, IL-6, NOXs, ROS generation, and the phosphorylation of MAPKs and AKT signaling were downregulated by Pyk2 inhibitor in Mettl3 knockdown cells. All of these results suggest that Mettl3 regulates the mRNA stability and expression of Pyk2 in a Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS. Mettl3 suppression increases the mRNA stability and expression of Pyk2 in Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS. [Display omitted] [ABSTRACT FROM AUTHOR]

The 3′ untranslated regions (UTRs) of Ebola virus (EBOV) mRNAs are enriched in their AU content and therefore represent potential targets for RNA binding proteins targeting AU-rich elements (ARE-BPs). ARE-BPs are known to fine-tune RNA turnover and translational activity. We identified putative AREs within EBOV mRNA 3′ UTRs and assessed whether they might modulate mRNA stability. Using mammalian and zebrafish embryo reporter assays, we show a conserved, ARE-BP-mediated stabilizing effect and increased reporter activity with the tested EBOV 3′ UTRs. When coexpressed with the prototypic ARE-BP tristetraprolin (TTP, ZFP36) that mainly destabilizes its target mRNAs, the EBOV nucleoprotein (NP) 3′ UTR resulted in decreased reporter gene activity. Coexpression of NP with TTP led to reduced NP protein expression and diminished EBOV minigenome activity. In conclusion, the enrichment of AU residues in EBOV 3′ UTRs makes them possible targets for cellular ARE-BPs, leading to modulation of RNA stability and translational activity. [ABSTRACT FROM AUTHOR]

Objective: Apical periodontitis is the most frequently occurring pathological lesion. Fat mass and obesity‐associated protein (Fto) is the first identified RNA N6‐methyladenosine demethylase. However, whether Fto regulates apical periodontitis remains unclear. This study aimed to explore the mechanisms of Fto in the tumor necrosis factor‐α (TNF‐α)‐induced inflammatory response. Materials and Methods: We established an apical periodontitis model. An immortalized cementoblast cell line (OCCM‐30) cells were exposed to TNF‐α. Fto, Il6, Mcp1, and Mmp9 expressions were assessed by qRT‐PCR. We knocked down Fto using lentiviruses and detected TNF‐α‐induced inflammation‐related gene expressions and mRNA stability. Results: Mice with apical periodontitis showed downregulation of Fto expression. OCCM‐30 cells exposed to TNF‐α showed an upregulation of inflammation‐related genes with a decrease in Fto. Furthermore, knockdown of Fto promoted the expressions of Il6, Mcp1, and Mmp9 in TNF‐α‐treated OCCM‐30 cells as compared with negative control cells, whereas it did not affect the mRNA stability. Interestingly, Fto knockdown activated the p65, p38, and ERK1/2 pathways, and it slightly activated the JNK signaling pathway after TNF‐α administration in OCCM‐30 cells. Conclusion: A TNF‐α‐induced decrease in the expression of Fto might play a critical role in the inflammatory response in cementoblasts, and knockdown of Fto might upregulate the inflammatory response. [ABSTRACT FROM AUTHOR]

The role of fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) demethylase, in non-small cell lung cancer (NSCLC) has recently received widespread attention. However the underlying mechanisms of FTO-mediated autophagy regulation in NSCLC progression remain elusive. In this study, we found that FTO was significantly upregulated in NSCLC, and downregulation of FTO suppressed the growth, invasion and migration of NSCLC cells by inducing autophagy. FTO knockdown resulted in elevated m6A levels in NSCLC cells. Methylated RNA immunoprecipitation sequencing showed that sestrin 2 (SESN2) was involved in m6A regulation during autophagy in NSCLC cells. Interestingly, m6A modifications in exon 9 of SESN2 regulated its stability. FTO deficiency promoted the binding of insulin-like growth factor 2 mRNA-binding protein 1 to SESN2 mRNA, enhancing its stability and elevating its protein expression. FTO inhibited autophagic flux by downregulating SESN2, thereby promoting the growth, invasion and migration of NSCLC cells. Besides, the mechanism by which FTO blocked SESN2-mediated autophagy activation was associated with the AMPK-mTOR signaling pathway. Taken together, these findings uncover an essential role of the FTO–autophagy–SESN2 axis in NSCLC progression.

Background: Hepatocellular carcinoma (HCC) is a multistep process involving sophisticated genetic, epigenetic, and transcriptional changes. However, studies on microRNA (miRNA)'s regulatory effects of N6-methyladenosine (m6A) modifications on HCC progression are limited. Methods: Cell Counting Kit-8 (CCK-8), clone formation, and Transwell assays were used to investigate changes in cancer cell proliferation, invasion, and migration. RNA m6A levels were verified using methylated RNA immunoprecipitation. Luciferase reporter assay was used to study the potential binding between miRNAs and mRNA. A mouse tumor transplant model was established to study the changes in tumor progression. Results: Follistatin-like 5 (FSTL5) was significantly downregulated in HCC and inhibited its further progression. Additionally, methyltransferase-like 3 (METTL3) reduced FSTL5 mRNA stability in an m6A-YTH domain family 2(YTHDF2)-dependent manner. Functional experiments revealed that METTL3 downregulation inhibited HCC progression by upregulating FSTL5 in vitro and in vivo. Luciferase reporter assay verified that miR-186-5p directly targets METTL3. Additionally, miR-186-5p inhibits the proliferation, migration, and invasion of HCC cells by downregulating METTL3 expression. Conclusions: The miR-186-5p/METTL3/YTHDF2/FSTL5 axis may offer new directions for targeted HCC therapy.

AU-rich elements (AREs) are 3' UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3' UTR sequences that conform to these motifs affect their function. Here, we use functional annotation of sequences from 3' UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3' UTR sequence fragments from 4653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3' UTRs.

Lipids play a critical role in many cellular processes by serving as structural components of cell membranes or functioning as energy fuel and signaling molecules. The RNA-binding proteins RBM24 and RBM38 share an identical RNA-binding domain and thereby, regulate a group of same targets, such as p21. However, it is not certain whether RBM24 and RBM38 participates in lipid homeostasis. Here, lipidomic analysis showed that a deficiency in RBM24 or RBM38 leads to altered lipid metabolism, with more profound alteration by loss of RBM24 in MCF7 cells. We also showed that mice deficient in RBM24 were prone to chronic inflammation and liver steatosis, but not spontaneous tumors. These data let us speculate whether RBM24 regulates ferroptosis, a programmed cell death that links inflammation and liver steatosis via lipid peroxidation. Indeed, we found that over-expression of RBM24 protected, whereas knockout of RBM24 sensitized, cells to Erastin-induced ferroptosis by modulating the mRNA stability of SLC7A11, a ferroptosis inhibitor. Moreover, we showed that knockdown of SLC7A11 reversed the effect of RBM24 on ferroptosis. Together, our study revealed that RBM24 regulates lipid metabolism and SLC7A11 mRNA stability to modulate ferroptosis and inflammatory response.

Abstract As a frontline treatment for patients with advanced hepatocellular carcinoma (HCC), sorafenib is an effective drug approved by the Food and Drug Administration (FDA). Ferroptosis, a newly defined programmed cell death process with the hallmark of the accumulation of iron-dependent lipid peroxides, can be induced by sorafenib treatment. Our previous study identified oncogenic roles of long noncoding RNA (lncRNA) Cancer susceptibility candidate 11 (CASC11) in HCC progression. However, the relationship between CASC11 and sorafenib-induced ferroptosis in HCC remains unclear. In the present study, we aim to investigate the role of CASC11 in sorafenib-induced ferroptosis in HCC cell lines and determine the involved molecular mechanisms. Here, we demonstrated that sorafenib decreased CASCL11 expression. Knockdown of CASC11 enhanced sorafenib-induced ferroptosis, while overexpression of CASC11 exerted the opposite effect in HCC cells. Moreover, CASC11 led to the accumulation of intracellular malondialdehyde (MDA), lipid reactive oxygen species (ROS) and Fe2+ while depleting glutathione (GSH), thereby suppressing sorafenib-induced ferroptosis and cell death. Ferrostatin-1 (Ferr-1), a ferroptosis inhibitor, reversed the enhanced anticancer effect of sorafenib caused by the silence of CASC11 in HCC cells. Mechanistically, CASC11 upregulated the expression of solute carrier family 7 member 11 (SLC7A11) which is critical for ferroptosis inhibition. CASC11 associated with and stabilized SLC7A11 mRNA. In summary, our data revealed, for the first time, that CASC11 inhibits the sorafenib-induced ferroptosis in HCC cells via regulating SLC7A11, providing a new basis for clinical therapeutic strategies for patients with HCC.

Mevalonate metabolism plays an important role in regulating tumor growth and progression; however, its role in immune evasion and immune checkpoint modulation remains unclear. Here, we found that non-small cell lung cancer (NSCLC) patients with higher plasma mevalonate response better to anti-PD-(L)1 therapy, as indicated by prolonged progression-free survival and overall survival. Plasma mevalonate levels were positively correlated with programmed death ligand-1 (PD-L1) expression in tumor tissues. In NSCLC cell lines and patient-derived cells, supplementation of mevalonate significantly up-regulated the expression of PD-L1, whereas deprivation of mevalonate reduced PD-L1 expression. Mevalonate increased CD274 mRNA level but did not affect CD274 transcription. Further, we confirmed that mevalonate improved CD274 mRNA stability. Mevalonate promoted the affinity of the AU-rich element-binding protein HuR to the 3′-UTR regions of CD274 mRNA and thereby stabilized CD274 mRNA. By in vivo study, we further confirmed that mevalonate addition enhanced the anti-tumor effect of anti-PD-L1, increased the infiltration of CD8+ T cells, and improved cytotoxic function of T cells. Collectively, our findings discovered plasma mevalonate levels positively correlated with the therapeutic efficacy of anti-PD-(L)1 antibody, and provided the evidence that mevalonate supplementation could be an immunosensitizer in NSCLC.

Abstract Ovarian cancer (OC) represents one of the most detrimental gynecological malignancies. RNA‐binding protein eukaryotic translation initiation factor 4A isoform 3 (EIF4A3) is well‐regarded as a definitive oncogene that contributes to the development of multiple malignant tumors. This study sought to elucidate the molecular mechanism of EIF4A3 in OC growth and aerobic glycolysis by regulation of pyruvate dehydrogenase kinase 4 (PDK4) mRNA stability. We determined the EIF4A3 and PDK4 expression levels in OC cell lines and normal ovarian epithelial cells, and subsequently evaluated the cell viability and colony formation by cell counting kit‐8 and colony formation assays. The degree of cell aerobic glycolysis was evaluated by measurements of lactic acid production, glucose intake, adenosine triphosphate level, extracellular oxygen consumption, and protein levels of pyruvate kinase isozymes M2 and hexokinase‐2. Afterwards, we verified the binding of EIF4A3 and PDK4 mRNA via RNA immunoprecipitation, and determined the mRNA stability after actinomycin D treatment. Finally, a series of rescue experiments was performed with pcDNA3.1‐PDK4. EIF4A3 and PDK4 were upregulated in OC cells. Silencing EIF4A3 obstructed cell proliferation and aerobic glycolysis, while the same was annulled by EIF4A3 overexpression. Mechanically, EIF4A3 could bind to PDK4 mRNA to stabilize its mRNA and upregulate its protein levels. PDK4 overexpression inverted the inhibitory role of silencing EIF4A3 in proliferation and aerobic glycolysis. Overall, our findings highlighted that EIF4A3 induced OC progression by stabilizing PDK4 mRNA.

Abstract Background Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized. Results Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa −/− mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs. Conclusions Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

Abstract Breast cancer is the most common malignancy among women and the leading cause of cancer deaths, with complicated pathogenesis that is largely unknown. In this study, we identified a novel long non-coding RNA (lncRNA) as a critical driver of breast cancer tumorigenesis. RUNX1 intronic transcript 1 (RUNX1-IT1) was notably overexpressed in human breast cancer tissues, and knockdown of RUNX1-IT1 inhibited breast cancer cell viability and invasion, as well as tumor growth in orthotopic transplantation model. Further, RUNX1-IT1 repressed ferroptosis, a novel iron-dependent form of regulated cell death, via increasing glutathione peroxidase 4 (GPX4) expression. Specifically, RUNX1-IT1 directly bound to N6-methyladenosine m6A reader IGF2BP1 and promoted the formation of (insulin like growth factor 2 mRNA binding protein 1) IGF2BP1 liquid-liquid phase separation (LLPS) biomolecular condensates, resulting in more IGF2BP1 occupation on GPX4 mRNA, increasing GPX4 mRNA stability. Moreover, high RUNX1-IT1 was linked to poor prognosis, and a strong positive correlation between RUNX1-IT1 and GPX4 was observed in clinical breast cancer tissues. Taken together, our data reveal that RUNX1-IT1 promotes breast cancer carcinogenesis through blocking ferroptosis via elevating GPX4, targeting of the previously unappreciated regulatory axis of RUNX1-IT1/IGF2BP1/GPX4 may be a promising treatment for patient with breast cancer.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5–7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3–7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature. [ABSTRACT FROM AUTHOR]

Directly injecting naked or lipid nanoparticle (LNP)-encapsulated modified mRNA (modRNA) allows rapid and efficient protein expression. This non-viral technology has been used successfully in modRNA vaccines against SARS-CoV-2. The main challenges in using modRNA vaccines were the initial requirement for an ultra-cold storage to preserve their integrity and concerns regarding unwanted side effects from this new technology. Here, we showed that naked modRNA maintains its integrity when stored up to 7 days at 4 °C, and LNP-encapsulated modRNA for up to 7 days at room temperature. Naked modRNA is predominantly expressed at the site of injection when delivered into cardiac or skeletal muscle. In comparison, LNP-encapsulated modRNA granted superior protein expression but also additional protein expression beyond the cardiac or skeletal muscle injection site. To overcome this challenge, we developed a skeletal-muscle-specific modRNA translation system (skeletal muscle SMRTs) for LNP-encapsulated modRNA. This system allows controlled protein translation predominantly at the site of injection to prevent potentially detrimental leakage and expression in major organs. Our study revealed the potential of the SMRTs platform for controlled expression of mRNA payload delivered intramuscularly. To conclude, our SMRTs platform for LNP-encapsulated modRNA can provide safe, stable, efficient and targeted gene expression at the site of injection. [ABSTRACT FROM AUTHOR]

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability. [ABSTRACT FROM AUTHOR]

As a frontline treatment for patients with advanced hepatocellular carcinoma (HCC), sorafenib is an effective drug approved by the Food and Drug Administration (FDA). Ferroptosis, a newly defined programmed cell death process with the hallmark of the accumulation of iron-dependent lipid peroxides, can be induced by sorafenib treatment. Our previous study identified oncogenic roles of long noncoding RNA (lncRNA) Cancer susceptibility candidate 11 (CASC11) in HCC progression. However, the relationship between CASC11 and sorafenib-induced ferroptosis in HCC remains unclear. In the present study, we aim to investigate the role of CASC11 in sorafenib-induced ferroptosis in HCC cell lines and determine the involved molecular mechanisms. Here, we demonstrated that sorafenib decreased CASCL11 expression. Knockdown of CASC11 enhanced sorafenib-induced ferroptosis, while overexpression of CASC11 exerted the opposite effect in HCC cells. Moreover, CASC11 led to the accumulation of intracellular malondialdehyde (MDA), lipid reactive oxygen species (ROS) and Fe2+ while depleting glutathione (GSH), thereby suppressing sorafenib-induced ferroptosis and cell death. Ferrostatin-1 (Ferr-1), a ferroptosis inhibitor, reversed the enhanced anticancer effect of sorafenib caused by the silence of CASC11 in HCC cells. Mechanistically, CASC11 upregulated the expression of solute carrier family 7 member 11 (SLC7A11) which is critical for ferroptosis inhibition. CASC11 associated with and stabilized SLC7A11 mRNA. In summary, our data revealed, for the first time, that CASC11 inhibits the sorafenib-induced ferroptosis in HCC cells via regulating SLC7A11, providing a new basis for clinical therapeutic strategies for patients with HCC. [ABSTRACT FROM AUTHOR]

Global estimates suggest that over 300 million individuals of all ages are affected by serious fungal infections every year, culminating in about 1.7 million deaths. The societal and economic burden on the public health sector due to opportunistic fungal pathogens is quite significant, especially among immunocompromised patients. Despite the high clinical significance of these infectious agents, treatment options are limited with only three major classes of antifungal drugs approved for use. Clinical management of fungal diseases is further compromised by the emergence of antifungal resistant strains. Transcriptional and genetic mechanisms that control drug resistance in human fungal pathogens are well-studied and include drug target alteration, upregulation of drug efflux pumps as well as changes in drug affinity and abundance of target proteins. In this review, we highlight several recently discovered novel post-transcriptional mechanisms that control antifungal resistance, which involve regulation at the translational, post-translational, epigenetic, and mRNA stability levels. The discovery of many of these novel mechanisms has opened new avenues for the development of more effective antifungal treatment strategies and new insights, perspectives, and future directions that will facilitate this process are discussed. [ABSTRACT FROM AUTHOR]

The accumulation and deposition of beta-amyloid (Aß) are key neuropathological hallmarks of Alzheimer's disease (AD). PARP16, a Poly(ADP-ribose) polymerase, is a known tail-anchored endoplasmic reticulum (ER) transmembrane protein that transduces ER stress during pathological processes. Here, we found that PARP16 was significantly increased in the hippocampi and cortices of APPswe/PS1dE9 (APP/PS1) mice and hippocampal neuronal HT22 cells exposed to Aß, suggesting a positive correlation between the progression of AD pathology and the overexpression of PARP16. To define the effect of PARP16 on AD progression, adeno-associated virus mediated-PARP16 knockdown was used in APP/PS1 mice to investigate the role of PARP16 in spatial memory, amyloid burden, and neuroinflammation. Knockdown of PARP16 partly attenuated impaired spatial memory, as indicated by the Morris water maze test, and decreased amyloid deposition, neuronal apoptosis, and the production of inflammatory cytokines in the brains of APP/PS1 mice. In vitro experiments demonstrated that the knockdown of PARP16 expression rescued neuronal damage and ER stress triggered by Aß. Furthermore, we discovered that intracellular PARP16 acts as an RNA-binding protein that regulates the mRNA stability of amyloid precursor protein (APP) and protects targeted APP from degradation, thereby increasing APP levels and AD pathology. Our findings revealed an unanticipated role of PARP16 in the pathogenesis of AD, and at least in part, its association with increased APP mRNA stability. [ABSTRACT FROM AUTHOR]

Salt is one of the most important environmental factors in crop growth and development. N6-methyladenosine (m6A) is an epigenetic modification that regulates plant–environment interaction at transcriptional and translational levels. Sugar beet is a salt-tolerant sugar-yielding crop, but how m6A modification affects its response to salt stress remains unknown. In this study, m6A-seq was used to explore the role of m6A modification in response to salt stress in sugar beet (Beta vulgaris). Transcriptome-wide m6A methylation profiles and physiological responses to high salinity were investigated in beet roots. After treatment with 300 mM NaCl, the activities of peroxidase and catalase, the root activity, and the contents of Na+, K+, and Ca2+ in the roots were significantly affected by salt stress. Compared with the control plants, 6904 differentially expressed genes (DEGs) and 566 differentially methylated peaks (DMPs) were identified. Association analysis revealed that 243 DEGs contained DMP, and 80% of these DEGs had expression patterns that were negatively correlated with the extent of m6A modification. Further analysis verified that m6A methylation may regulate the expression of some genes by controlling their mRNA stability. Functional analysis revealed that m6A modifications primarily affect the expression of genes involved in energy metabolism, transport, signal transduction, transcription factors, and cell wall organization. This study provides evidence that a post-transcriptional regulatory mechanism mediates gene expression during salt stress by affecting the stability of mRNA in the root. [ABSTRACT FROM AUTHOR]

The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein named damage suppressor (Dsup), which was discovered to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup-transfected human cells suggests that Dsup mediates an overall response in DNA damage signaling, DNA repair, and cell cycle regulation, resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells under basal conditions and at 24 h recovery after exposure to UV-C. We demonstrate via enrichment and network analyses, for the first time, that even in the absence of external stimuli, and more significantly, after stress, Dsup activates mechanisms involved with the unfolded protein response, the mRNA processing and stability, cytoplasmic stress granules, the DNA damage response, and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms and increases our knowledge of the molecular machineries of extraordinary protection against UV-C stress. [ABSTRACT FROM AUTHOR]

Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N6-methyladenosine (m6A) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution m6A mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains m6A, and increased m6A modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via m6A RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers.