Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of in vivo interactions by sequencing of the resulting chimeric RNAs. Modifications are incorporated to increase the capacity for processing low-abundance samples and permit cell-type-selective profiling of sncRNA:target RNA interactions, as demonstrated in mouse brain cortex. CIMERA-seq represents a cohesive and optimized method for unambiguously characterizing the in vivo network of sncRNA:target RNA interactions in numerous biological contexts and even subcellular fractions. Genome-wide and cell-type-selective CIMERA-seq enhances researchers\' ability to study gene regulation by sncRNAs in diverse model systems and tissue types.

Periodontitis, with its persistent nature, causes significant distress for most sufferers. Current treatments, such as mechanical cleaning and surgery, often fail to fully address the underlying overactivation of fibroblasts that drives this degradation. Targeting the post-transcriptional regulation of fibroblasts, particularly at the 3\'-untranslated regions (3\'UTR) of pathogenic genes, offers a therapeutic strategy for periodontitis. Herein, we developed a DNA nanorobot for this purpose. This system uses a dynamic DNA nanoframework to incorporate therapeutic microRNAs through molecular recognition and covalent bonds, facilitated by DNA monomers modified with disulfide bonds. The assembled-DNA nanoframework is encapsulated in a cell membrane embedded with a fibroblast-targeting peptide. By analyzing the 3\'UTR regions of pathogenic fibroblast genes FOSB and JUND, we identified the therapeutic microRNA as miR-1-3p and integrated it into this system. As expected, the DNA nanorobot delivered the internal components to fibroblasts by the targeting peptide and outer membrane that responsively releases miR-1-3p under intracellular glutathione. It resulted in a precise reduction of mRNA and suppression of protein function in pathogenic genes, effectively reprogramming fibroblast behavior. Our results confirm that this approach not only mitigates the inflammation but also promotes tissue regeneration in periodontal models, offering a promising therapeutic avenue for periodontitis.

Assisted reproduction technology (ART) procedures are often impacted by post-ovulatory aging (POA), which can lead to reduced fertilization rates and impaired embryo development. This study used RNA sequencing analysis and experimental validation to study the similarities and differences between in vivo- and vitro-matured porcine oocytes before and after POA. Differentially expressed genes (DEGs) between fresh in vivo-matured oocyte (F_vivo) and aged in vivo-matured oocyte (A_vivo) and DEGs between fresh in vitro-matured oocyte (F_vitro) and aged in vitro-matured oocyte (A_vitro) were intersected to explore the co-effects of POA. It was found that \"organelles\", especially \"mitochondria\", were significantly enriched Gene Ontology (GO) terms. The expression of genes related to the \"electron transport chain\" and \"cell redox homeostasis\" pathways related to mitochondrial function significantly showed low expression patterns in both A_vivo and A_vitro groups. Weighted correlation network analysis was carried out to explore gene expression modules specific to A_vivo. Trait-module association analysis showed that the red modules were most associated with in vivo aging. There are 959 genes in the red module, mainly enriched in \"RNA binding\", \"mRNA metabolic process\", etc., as well as in GO terms, and \"spliceosome\" and \"nucleotide excision repair\" pathways. DNAJC7, IK, and DDX18 were at the hub of the gene regulatory network. Subsequently, the functions of DDX18 and DNAJC7 were verified by knocking down their expression at the germinal vesicle (GV) and Metaphase II (MII) stages, respectively. Knockdown at the GV stage caused cell cycle disorders and increase the rate of abnormal spindle. Knockdown at the MII stage resulted in the inefficiency of the antioxidant melatonin, increasing the level of intracellular oxidative stress, and in mitochondrial dysfunction. In summary, POA affects the organelle function of oocytes. A_vivo oocytes have some unique gene expression patterns. These genes may be potential anti-aging targets. This study provides a better understanding of the detailed mechanism of POA and potential strategies for improving the success rates of assisted reproductive technologies in pigs and other mammalian species.

Tigecycline and the newly Food and Drug Administration-approved tetracyclines, including eravacycline and omadacycline, are regarded as last-resort treatments for multidrug-resistant Enterobacterales. However, tigecycline resistance in Klebsiella pneumoniae has increased, especially the underlying mechanism of heteroresistance is unclear. This study aimed to elucidate the mechanisms underlying tigecycline resistance and heteroresistance in clinical K. pneumoniae isolates. A total of 153 clinical K. pneumoniae isolates were collected, and identified 15 tigecycline-resistant and three tigecycline-heteroresistant isolates using broth microdilution and population analysis profile methods, respectively. Total RNAs from K. pneumoniae ATCC13883 and the laboratory-induced tigecycline-resistant strain were extracted and sequenced on an Illumina platform. Differentially expressed genes and regulatory small RNAs (sRNAs) were analyzed and validated in clinical isolates of K. pneumoniae using quantitative real-time PCR. RNA sequencing results showed that mdtABC efflux pump genes were significantly upregulated in the tigecycline-resistant strains. Overexpression of mdtABC was observed in a clinical K. pneumoniae isolate, which increased tigecycline minimum inhibitory concentrations (MICs) and was involved in tigecycline heteroresistance. Sequencing analysis of sRNA demonstrated that candidate sRNA-120 directly interacted with the mdtABC operon and was downregulated in tigecycline-resistant strains. We generated an sRNA-120 deletion mutation strain and a complemented strain of K. pneumoniae. The sRNA-120 deletion strain displayed increased mRNA levels of mdtA, mdtB, and mdtC and an increase in MICs of tigecycline. The complemented strain of sRNA-120 restored the mRNA levels of these genes and the susceptibility to tigecycline. RNA antisense purification and parallel reaction monitoring mass spectrometry were performed to verify the interactions between sRNA-120 and mdtABC. Collectively, our study highlights that the post-transcriptional repression of mdtABC through sRNA-120 may provide an additional layer of efflux pump gene expression control, which is important for resistance and heteroresistance in clinical K. pneumoniae isolates.

Excessive reactive oxygen species stress due to salinity poses a significant threat to the growth of Glycyrrhiza uralensis Fisch. To adapt to salt stress, G. uralensis engages in alternative splicing (AS) to generate a variety of proteins that help it withstand the effects of salt stress. While several studies have investigated the impact of alternative splicing on plants stress responses, the mechanisms by which AS interacts with transcriptional regulation to modulate the salt stress response in G. uralensis remain poorly understood. In this study, we utilized high-throughput RNA sequencing data to perform a comprehensive analysis of AS events at various time points in G. uralensis under salt stress, with exon skipping (SE) being the predominant AS type. KEGG enrichment analysis was performed on the different splicing genes (DSG), and pathways associated with AS were significantly enriched, including RNA transport, mRNA surveillance, and spliceosome. This indicated splicing regulation of genes, resulting in AS events under salt stress conditions. Moreover, plant response to salt stress pathways were also enriched, such as mitogen-activated protein kinase signaling pathway - plant, flavonoid biosynthesis, and oxidative phosphorylation. We focused on four differentially significant genes in the MAPK pathway by AS and qRT-PCR analysis. The alternative splicing type of MPK4 and SnRK2 was skipped exon (SE). ETR2 and RbohD were retained intron (RI) and alternative 5\'splice site (A5SS), respectively. The expression levels of isoform1 of these four genes displayed different but significant increases in different tissue sites and salt stress treatment times. These findings suggest that MPK4, SnRK2, ETR2, and RbohD in G. uralensis activate the expression of isoform1, leading to the production of more isoform1 protein and thereby enhancing resistance to salt stress. These findings suggest that salt-responsive AS directly and indirectly governs G. uralensis salt response. Further investigations into AS function and mechanism during abiotic stresses may offer novel references for bolstering plant stress tolerance.

Hematopoiesis represents a meticulously regulated and dynamic biological process. Genetic aberrations affecting blood cells, induced by various factors, frequently give rise to hematological tumors. These instances are often accompanied by a multitude of abnormal post-transcriptional regulatory events, including RNA alternative splicing, RNA localization, RNA degradation, and storage. Notably, post-transcriptional regulation plays a pivotal role in preserving hematopoietic homeostasis. The DEAD-Box RNA helicase genes emerge as crucial post-transcriptional regulatory factors, intricately involved in sustaining normal hematopoiesis through diverse mechanisms such as RNA alternative splicing, RNA modification, and ribosome assembly. This review consolidates the existing knowledge on the role of DEAD-box RNA helicases in regulating normal hematopoiesis and underscores the pathogenicity of mutant DEAD-Box RNA helicases in malignant hematopoiesis. Emphasis is placed on elucidating both the positive and negative contributions of DEAD-box RNA helicases within the hematopoietic system.

Pre-mRNA splicing is a significant step for post-transcriptional modifications and functions in a wide range of physiological processes in plants. Human NHP2L binds to U4 snRNA during spliceosome assembly; it is involved in RNA splicing and mediates the development of human tumors. However, no ortholog has yet been identified in plants. Therefore, we report At4g12600 encoding the ortholog NHP2L protein, and AtSNU13 associates with the component of the spliceosome complex; the atsnu13 mutant showed compromised resistance in disease resistance, indicating that AtSNU13 is a positive regulator of plant immunity. Compared to wild-type plants, the atsnu13 mutation resulted in altered splicing patterns for defense-related genes and decreased expression of defense-related genes, such as RBOHD and ALD1. Further investigation shows that AtSNU13 promotes the interaction between U4/U6.U5 tri-snRNP-specific 27 K and the motif in target mRNAs to regulate the RNA splicing. Our study highlights the role of AtSNU13 in regulating plant immunity by affecting the pre-mRNA splicing of defense-related genes.

As the most prevalent and reversible internal epigenetic modification in eukaryotic mRNAs, N 6-methyladenosine (m6A) post-transcriptionally regulates the processing and metabolism of mRNAs involved in diverse biological processes. m6A modification is regulated by m6A writers, erasers, and readers. Emerging evidence suggests that m6A modification plays essential roles in modulating the cell-fate transition of embryonic stem cells. Mechanistic investigation of embryonic stem cell maintenance and differentiation is critical for understanding early embryonic development, which is also the premise for the application of embryonic stem cells in regenerative medicine. This review highlights the current knowledge of m6A modification and its essential regulatory contribution to the cell fate transition of mouse and human embryonic stem cells.

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3\' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.

LncRNAs have been demonstrated to regulate biological processes in malignant tumors. In our previous study, we identified the immune-related LncRNA RNF144A-AS1 as a potential regulator in SKCM. However, its precise function and regulatory mechanism remain unclear. In this study, we observed upregulation of RNF144A-AS1 in SKCM and found that knockdown of RNF144A-AS1 suppressed proliferation, migration, invasion, and epithelial-mesenchymal transition abilities of melanoma cells. Mechanistically, as a high-risk prognostic factor, RNF144A-AS1 regulated biological processes of SKCM by interacting with TAF15 through an RNA-binding protein-dependent (RBP-dependent) manner. Furthermore, we confirmed that TAF15 activated downstream transcriptional regulation of YAP1 to modulate malignant behaviors in melanoma cells. In vivo experiments revealed that knockdown of RNF144A-AS1 inhibited tumorigenic capacity of melanoma cells and exhibited promising therapeutic effects. Collectively, these findings highlight the significance of the RNF144A-AS1/TAF15/YAP1 axis in promoting malignant behaviors in SKCM and provide novel insights into potential prognostic biomarkers and therapeutic targets for this disease.