RNase III influences microaerobic symbiotic pathways and RNA regulation in Sinorhizobium meliloti.

Innate lymphoid cells (ILCs) include T-bet-dependent NK and ILC1 cells, GATA-3-dependent ILC2 cells, and RORγt-dependent ILC3 cells. Their functional and developmental regulation at the posttranscriptional level remains elusive. The CCR4-NOT complex plays a central role in mRNA decay by mediating deadenylation. To explore the overall impact of mRNA decay on ILCs, we conditionally deleted Cnot3, an essential subunit of the CCR4-NOT complex. Loss of CNOT3 in ILC2 cells led to aberrant expression of T-bet and RORγt, accompanied by upregulation of type 1 and type 3 signature genes. Mechanistically, CNOT3 targeted the 3' untranslated regions of Tbx21 and Rorc mRNAs through interactions with Roquin and ZFP36L1, respectively. Elevated T-bet expression in CNOT3-deficient ILC2 cells suppressed GATA-3 levels, thereby impairing type 2 immune responses in models of airway allergy and helminth infection. Thus, our findings reveal that CNOT3 maintains ILC2 differentiation and function by restricting type 1 and type 3 transcriptional programs.

High-yield production of maltooligosaccharide-forming α-amylase in Bacillus subtilis.

Neural crest cells are a transient, multipotent population that gives rise to diverse structures during vertebrate embryonic development, including craniofacial cartilage and pigment cells. Although the transcriptional regulation of neural crest development is well characterized, the role of microRNAs remains less understood. Using a double-transgenic zebrafish model expressing fluorescent reporters under the sox10 promoter, combined with fluorescence-activated cell sorting and RNA sequencing, we identified microRNAs enriched in neural crest cells. We focused on miR-133a-3p and miR-338-3p, previously linked to tumor suppression, to explore their developmental roles. The overexpression of either microRNA led to craniofacial cartilage malformations and reduced melanophore number, accompanied by decreased expression of key regulators including sox9b, sox10, and runx3 Reporter assays confirmed direct targeting of the sox9b 3'untranslated region. In addition, miR-338-3p overexpression increased neural crest cell numbers, suggesting a role in proliferation. These findings uncover novel functions for miR-133a-3p and miR-338-3p in vertebrate craniofacial and pigment cell development, highlighting shared regulatory features between embryogenesis and tumorigenesis.

AEG-1 3'UTR functions as a ceRNA to facilitate Helicobacter pylori-induced gastric cancer EMT by regulating the miR-375-3p/JAK2 axis.

In mammals, the epididymis is composed of pseudostratified epithelium that forms the post-testicular milieu for spermatozoa. To date, the role of epithelial cells in shaping the luminal microenvironment for spermatozoa remains poorly understood. Here, we generated a conditional knockout mouse model of Nudt21 in the epididymis, the key component of mammalian cleavage factor I (CFIm) that participates in the 3'end processing of pre-mRNAs. We found that Nudt21 deletion occurred mainly in principal cells of the corpus and the cauda. The loss of NUDT21 in principal cells resulted in male infertility, owing to sperm abnormalities present in the cauda. Pseudotime analysis revealed that the differentiation of Nudt21-null principal cells was blocked, preventing them from exerting the physiological function. Amid transcripts with shortened 3' untranslated regions (3'UTRs) after Nudt21 deletion, Dicer1 transcripts not only had shortened 3'UTR, but also skipped the exon 1, resulting in down-regulation of full length DICER1 protein. Together, these results demonstrate the essential role of NUDT21-mediated 3'UTR dynamics in preserving sperm integrity, and provide insights into the intricate communication between the epididymal epithelium and spermatozoa.

Corynebacterium glutamicum is a crucial food-grade (GRAS) bacterial chassis widely utilized for the industrial production of amino acids and nutraceuticals. However, the efficient production of recombinant proteins and secondary metabolites in this host remains limited by context dependence and low translational efficiency. To overcome this, we introduce a 5'-end translationalization strategy. By repurposing passive 5' untranslated regions (5'UTRs) into actively translated fore-cistrons, we converted conventional monocistronic designs into context-independent, leaderless polycistronic designs (PCDs). This assembly of concatenated fore-cistrons functions as a translational amplifier, largely decoupling protein output from mRNA abundance. We validated this platform by optimizing two biomanufacturing paradigms: achieving a 4.07-fold enhanced secretion of OmlA, a porcine vaccine antigen, and boosting biosynthesis of the food-grade pigment indigoidine to 1.20 g/L (a 7.33-fold increase over baselines). Together, this framework establishes a versatile, portable toolkit to overcome translational bottlenecks, enabling robust hyperproduction of recombinant proteins and engineered metabolites in biotechnology.

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) severely limits clinical efficacy. Myeloid zinc finger 1 (MZF1) inhibited EGFR phosphorylation and internalization by maintaining zinc homeostasis, which suggested its potential as a therapeutic target for resensitization. The two MZF1 splice variants exhibited functional divergence. MZF1S demonstrated significantly reduced Zn2+-binding capacity compared with MZF1L owing to C2H2 domain deletion. Although MZF1S was specifically upregulated in resistant patients, MZF1L predominated in treatment-sensitive cohorts, the mechanisms regulating their differential expression remained uncharacterized. We identified NSUN7/YBX1-mediated RNA m5C methylation as a critical driver of resistance. This modification promoted competitive binding of SRSF1 over SRSF3 to MZF1 pre-mRNA and enhanced MZF1S production. Overlapping loci co-regulated by RNA m5C and DNA 5-methylcytosine methylation in the MZF1 5'-untranslated region suggested coordinated epigenetic control of alternative splicing. Characterization of these loci revealed a UHRF1/DNMT1-NSUN7/YBX1 axis that synergistically regulated splice variant selection. Targeting this axis suppressed MZF1S while restoring MZF1L expression, which resensitized resistant cells to EGFR-TKIs. Our findings established a dual-layer epigenetic mechanism governing alternative splicing in drug resistance and proposed methylation-regulated splice variants as biomarkers and therapeutic targets for overcoming EGFR-TKI resistance in non-small-cell lung cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and currently lacks defined therapeutic targets. Although miR-152-3p functions as a tumor suppressor in various cancers, its specific mechanism and regulatory network in TNBC remain poorly understood. To investigate the expression and tumor-suppressive function of miR-152-3p in TNBC cells and to elucidate its mechanism of targeting STAT3, RELA and ADCY6. miR-152-3p expression was compared between MDA-MB-231 and MCF-10A cells using qRT-PCR. MDA-MB-231 cells were transfected with miR-152-3p mimics or inhibitors and cell proliferation, apoptosis and invasion were assessed by MTT assay, flow cytometry and Transwell assay, respectively. Direct target interactions were validated by a dual-luciferase reporter assay, and protein levels of STAT3, RELA, and ADCY6 were examined by Western blot. Key findings were further validated in Hs 578T cells. miR-152-3p expression was significantly downregulated in TNBC cells. Overexpression of miR-152-3p markedly inhibited proliferation and invasion while promoting apoptosis in MDA-MB-231 cells. Dual-luciferase reporter assays confirmed that miR-152-3p directly binds to the 3'untranslated regions of STAT3, RELA and ADCY6. Overexpression of miR-152-3p significantly reduced STAT3 and RELA protein levels while upregulating ADCY6 expression. Rescue experiments demonstrated that restoration of STAT3 expression partially reversed the tumor-suppressive effects of miR-152-3p. These findings were recapitulated in Hs 578T cells, suggesting generalizability across TNBC subtypes. miR-152-3p suppresses TNBC progression by downregulating STAT3/RELA and upregulating ADCY6, thereby activating cAMP signaling. These findings provide a foundation for further investigation into the potential of miR-152-3p as a multi-target therapeutic strategy for TNBC.

Myotonic dystrophy type 1 is the most prevalent adult-onset muscular dystrophy and is characterized by progressive muscle weakness, myotonia, cardiac conduction defects, endocrine dysfunction, and central nervous system involvement. Myotonic dystrophy type 1is caused by an unstable CTG repeat expansion in the 3' untranslated region of the DMPK gene, which produces toxic CUG-expanded transcripts that sequester RNA-binding proteins such as Muscleblind-like, induce widespread alternative splicing defects, and drive an RNA gain-of-function mechanism rather than simple DMPK haploinsufficiency. Despite major advances in understanding the molecular pathogenesis of myotonic dystrophy type 1, there is still no approved cure or disease-modifying therapy. This review summarizes the molecular basis of myotonic dystrophy type 1 and provides an in-depth overview of emerging therapeutic strategies that directly target the underlying pathogenic cascade at the DNA and RNA levels. Gene therapy-based approaches, including CRISPR-mediated genome editing, aim to reduce or eliminate the expanded CTG repeats or expanded DMPK allele and its toxic transcripts. In parallel, a broad spectrum of RNA-directed interventions is being developed, encompassing antisense oligonucleotides, antibody-penetrating and cell-penetrating peptide-conjugated antisense oligonucleotides to enhance skeletal and cardiac muscle delivery, small interfering RNAs, and microRNA-based tools such as antagomiRs. Additional strategies exploit engineered RNA-binding proteins and peptide decoys to disrupt toxic ribonuclear aggregates, polyadenylation signal-driven premature transcriptional termination to selectively silence mutant DMPK, and small molecules that modulate RNA metabolism, dissolve CUG RNA foci, or correct downstream mis-splicing. By integrating data from preclinical models and ongoing clinical trials, including recent advances with muscle‑targeted antisense oligonucleotide conjugates and gene therapy, this review outlines the current status, strengths, and limitations of these mechanism-based therapies for myotonic dystrophy type 1. The discussion highlights key translational challenges such as efficient delivery to skeletal muscle, the heart, and brain, long-term safety, and robust pharmacodynamic biomarkers as well as opportunities for combination and next-generation approaches aimed at converting molecular correction into durable clinical benefit for patients with myotonic dystrophy type 1.

In case of emerging or re-emerging infections, vaccine platform technologies are needed to rapidly develop effective vaccines to aid public healthcare in pandemics. Besides mRNA vaccines, also viral platform technologies, that is, the adenovirus-derived vaccines Vaxzevria and JCOVDEN, have proven to be of immense value during the COVID-19 pandemic. For future pandemics, it is crucial to understand the factors in vector design that modulate immunogenicity. This knowledge allows the tailoring of vaccine vectors to fit specific target product profiles, for example, to build vectors which trigger an accentuated T cell or, alternatively, antibody response against an antigen of interest. Our study using the live-attenuated measles vaccine backbone as a promising example is therefore crucial in demonstrating that very minor differences in the vaccine backbone can alter the antigen expression profile of the vector-antigen system and impact the relative induction of T-cell or antibody responses against the added target antigen.

An LNP-encapsulated mRNA vaccine targeting the major capsid protein of largemouth bass virus confers protective immunity and reduces RIG-I pathway activation in Micropterus salmoides.

Ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2), an enzyme involved in extracellular nucleotide metabolism and purinergic signaling, has been linked to tumor-immune interactions, although its role in colorectal cancer (CRC) remains unclear. This study examined the expression pattern and regulatory context of ENTPD2 through integrative analysis of transcriptomic, proteomic, microRNA (miRNA), and single-cell transcriptomic datasets. Transcriptomic analyses showed that ENTPD2 mRNA levels are elevated in colorectal tumors compared with normal tissues and that higher expression is associated with shorter relapse-free survival. In contrast, proteomic analyses indicated reduced ENTPD2 protein abundance in tumor samples, suggesting a divergence between transcript and protein expression. Analysis of candidate miRNAs identified miR-708-5p as a potential post-transcriptional regulator, supported by its increased expression in CRC and a predicted binding site within the ENTPD2 3'-untranslated region (UTR). Single-cell transcriptomic datasets further indicated that ENTPD2 transcripts are mainly detected in malignant epithelial cells. We performed a functional validation using dual-luciferase reporter assays, qRT-PCR, and Western blot analysis in CRC cell lines. Experimental analyses demonstrated that miR-708-5p directly targets the ENTPD2 3'UTR in HCT116 cells and suppresses ENTPD2 expression in both HCT116 and HT-29 cells. These findings support a potential contribution of miR-708-5p to ENTPD2 regulation in CRC.

Selenocysteine (Sec) incorporation is a uniquely complex and essential form of ribosomal recoding that redefines in-frame UGA codons through an evolutionarily ancient apparatus. Eukaryotic selenoprotein biosynthesis requires a specialized tRNA as well as elongation and selenium-donor factors, and incorporation depends on a 3' untranslated region (UTR) RNA structure, the Sec insertion sequence (SECIS) element. The SECIS is recognized by SECIS-binding protein 2 (SECISBP2/SBP2), resulting in recruitment of the Sec-specific elongation factor eEFSec. Here, we delve into the evolutionary and biochemical basis for Sec incorporation in eukaryotes, summarizing the current understanding of the cis and trans determinants that tune this form of recoding.

mRNA-based therapeutics represent a highly promising platform for the prevention and treatment of diseases. However, further optimization is required to improve mRNA stability and translational efficiency. The 5' untranslated region (5' UTR) is a critical regulatory element that governs pre-initiation complex assembly and directly influences protein expression levels, making it an important target for rational design. The diverse challenges facing mRNA therapeutics necessitate the development of improved 5' UTR elements adaptable to specific applications. In this study, we evaluated the effects of five different 5' UTR variants, selected based on published data, on firefly luciferase (FLuc) production in cell culture and in an in vivo bioluminescence imaging model in animals. Subsequently, all variants were tested as components of mRNA vaccines against SARS-CoV-2. Our findings demonstrate that the H1.2-TISU and Synth 5' UTRs with mRNA constructs produce increased levels of reporter protein expression in mice, whereas mRNA constructs incorporating the H1.2-TISU 5' UTR demonstrated enhanced humoral immunogenicity compared to the 5' UTR derived from human alpha-globin (HBA) mRNA. Thus, the identified 5' UTRs (H1.2-TISU and Synth) represent promising, in vivo-validated candidates for incorporation into mRNA-based therapeutics and may substantially enhance their efficacy.

As the end-stage manifestation of cardiovascular diseases (CVDs), chronic heart failure (CHF) is associated with high morbidity and mortality. Our previous study showed that Running exercise could improve CHF by inducing autophagy, but the underlying mechanisms and applicability remain unclear. This study investigated the roles of NEAT1 and RNA-binding protein Quaking (QKI) in the cardioprotective effects of running exercise initiated during the early stage after abdominal aortic coarctation (AAC) surgery. NEAT1 and QKI were overexpressed in vivo or NEAT1 and QKI were knocked down in vitro. The effects of running exercise on the myocardial damage of CHF rats or Ang II-induced cardiomyocyte apoptosis, autophagic flux, autophagy-related protein expression, and the expression of the PI3K/ AKT pathway by regulating NEAT1 and QKI were assessed using echocardiography, tissue staining, immunofluorescence, western blotting, and confocal microscopy. Furthermore, RNA immunoprecipitation (RIP), RBPmap prediction, actinomycin D (ActD) chase experiments and Dual-luciferase reporter assays were performed to explore the post-transcriptional regulation of autophagy-related genes by QKI. Running exercise reduced myocardial tissue damage in CHF rats and up-regulated the expression of autophagy-related proteins. NEAT1 was up-regulated in the myocardial tissue of CHF rats, and Ang II-damaged cardiomyocytes. Whereas QKI expression showed the opposite trend. Additionally, running exercise inhibited NEAT1 while restoring QKI expression, particularly during weeks 2-3 of exercise intervention. Mechanistically, NEAT1 directly interacted with QKI and negatively regulated its expression. Simultaneously, QKI bound to the 3'untranslated regions (3'-UTRs) of Beclin1 and LC3B mRNA and enhances the stability of Beclin1 and LC3B transcripts, thereby promoting autophagy. Functionally, NEAT1 inhibition or QKI upregulation suppressed the PI3K/AKT/mTOR signaling pathway, and attenuated cardiomyocyte apoptosis under pathological conditions. Collectively, the current findings revealed the regulatory mechanism by which running exercise alleviates CHF, and clarified that running promotes cardiomyocyte autophagy and alleviates CHF through the NEAT1-QKI-Beclin1/LC3B regulatory axis, highlighting a novel mechanism underlying exercise-mediated cardioprotection. Given that exercise intervention was initiated during the early stage following AAC surgery, the observed benefits may include early remodeling-modifying and disease-progression-modifying effects, rather than solely therapeutic effects on established decompensated CHF.

Genetic variants in the 3' untranslated regions (3'-UTRs) of mRNAs can alter binding of RNA-binding proteins and microRNAs and thereby influence regulation by affecting RNA stability, localization, and translation. Despite their potential impact on the risk for complex traits, including alcohol use disorder, the contribution of 3'-UTR variants has not been systematically explored. We evaluate the impact of 3'-UTR variants within loci associated with substance use and neurological disorders using a massively parallel reporter assay (MPRA) in neuroblastoma and microglia cells. Of the 13,515 variants tested, 400 and 657 variants significantly alter gene expression in neuroblastoma and microglia cells, respectively. These functionally impactful variants account for more heritability of alcohol-related traits than non-functional variants. We develop a computational framework, MPRA-mediated Gene Expression Association (MGExA), that combines MPRA-derived variant effects with GWAS summary statistics and identify 31 genes whose expression changes may contribute to alcohol-related traits. CRISPR inhibition of 7 of these genes in neuronal cells leads to gene expression changes associated with neurodegenerative disorders and the oxidative phosphorylation pathway. Pharmacoepidemiological analysis of drugs that had similar effects on gene expression linked RBM14 and KANSL1 to risk for alcohol use disorder. We identify genetic variants in 3'-UTR regions that affect gene expression. By integrating these functional genomics data and pharmacoepidemiological assessment with GWAS analysis, we identify genes whose expression differences could contribute to alcohol related traits. This approach provides a framework for moving from GWAS data to identifying biologically and clinically relevant genes associated with complex disorders.

MiR-1281 downregulates LMX1B to inhibit gastric cancer development.

RNA structure is fundamental to its diverse biological functions. Current chemical probing methods, coupled with next-generation sequencing, offer insights into RNA secondary structure but are limited by indirect readouts and averaging of multiple molecules. Nanopore direct RNA sequencing (DRS) enables direct detection of modifications on long RNA reads, while accurately identifying probe-modified sites from DRS data remains challenging. Here, we present NanoRAPID (Nanopore RNA Structural Probe IDentification), a convolutional neural network (CNN)-based framework for analyzing RNA secondary structures using DRS data. By directly analyzing raw current signals, NanoRAPID achieves improved accuracy in probe-site identification. Validation with DRS datasets, including NAI-N3 [2-(azidomethyl)nicotinic acid imidazolide] and diethyl pyrocarbonate (DEPC) treatments, demonstrates the robustness and transferability of NanoRAPID. Transcriptome-wide analysis reveals distinct structural features across RNA categories, with mRNAs exhibiting variable structures in gene bodies and untranslated regions (UTRs), in contrast to the compact and uniform architecture of rRNAs. Notably, NanoRAPID identifies isoforms with distinct structural conformations, suggesting a dynamic equilibrium between conformational states. Furthermore, we observe a positive correlation between N6-methyladenosine (m6A) modification levels and 3' UTR accessibility, suggesting that RNA structural context may influence m6A deposition. NanoRAPID is a precise and versatile tool for detecting RNA structural probing signals from DRS data and is freely available on GitHub: https://github.com/luolab-sysu/NanoRAPID and BioCode: https://ngdc.cncb.ac.cn/biocode/tools/BT008087.

Maternal nutrition exerts long-term effects on offspring development, potentially mediated by epigenetic mechanisms. This study aimed to characterize the genome-wide DNA methylation profile of skeletal muscle in postnatal cattle and to determine the long-term impacts of maternal undernutrition on DNA methylation in offspring muscle. Wagyu cows were assigned to either nutritional-adequate control (CNT; n = 4, 120% of requirements) or nutritional-restricted (NR; n = 4, 60% of requirements) group from day 35 of gestation until parturition. After birth, all offspring received identical diets, and longissimus thoracis muscle (LM) biopsies were collected at 300 days of age for DNA methylation analysis using whole-genome bisulfite sequencing. Irrespective of maternal diet, DNA methylation levels in offspring muscle gradually decreased across the CpG from the upstream region toward the transcription start site, reached their lowest level at the transcription start site, and increased throughout the gene body. Compared with the CNT group, NR offspring LM exhibited 7076 hypomethylated and 6104 hypermethylated regions (|methylation difference| > 20%, Q < 0.05). Among genomic features, promoter and 5' untranslated regions exhibited the greatest susceptibility to methylation changes, with 0.96% and 1.09% of these regions being hypomethylated in NR offspring LM relative to CNT. Genes containing differentially methylated regions in distal upstream (1-5-kb upstream from transcription start site) regions or promoters were associated with fundamental biological processes such as gene expression regulation, protein function, cell and tissue development, cytoskeletal and contractile organization, and neurodevelopment (P < 0.05). An overlap-based integrative analysis of DNA methylation and gene expression data identified seven candidate epigenetically regulated genes, including neuronal precursor cell-expressed developmentally downregulated 4 (Entrez Gene ID: 507781) and solute carrier family 30 (zinc transporter) member 1 (Entrez Gene ID: 522265). Although only seven candidate genes were identified through integrative analysis, more than 13,000 differentially methylated regions were maintained in offspring muscle. These findings suggest that maternal undernutrition induces changes in DNA methylation patterns during the fetal stage that persist postnatally, and may contribute to long-term effects on muscle metabolism, growth efficiency, and meat quality.