The metamorphosis of flatfishes involves a significant transformation from a pelagic larval stage to a benthic juvenile stage. This process is driven by thyroid hormone signaling and involves extensive morphological and functional remodeling. While the regulation of gene expression is central to this process, the epigenetic mechanisms coordinating these transitions remain poorly understood. Although DNA methylation has been implicated in vertebrate metamorphosis, its role in coordinating the epigenetic regulation of flatfish brain metamorphosis remains largely unexplored. In this study, we examine the epigenetic and transcriptional dynamics of the brain, a critical regulator of metamorphosis, by analyzing chromatin accessibility, DNA methylation and transcriptomic profiles across three developmental stages (pre-metamorphosis, metamorphic climax and post-metamorphosis) in turbot (Scophthalmus maximus). We identify widespread DNA methylation remodeling during metamorphosis, characterized by dynamic changes in DNA methylation. Differentially methylated regions (DMRs) exhibit a bimodal distribution at the pre-metamorphic stage, shift toward intermediate methylation levels at the metamorphic climax, and return to a bimodal pattern following metamorphosis. Notably, DMRs are predominantly associated with open chromatin regions and are significantly enriched at CpG islands. Furthermore, DNA methylation levels near transcription start sites are inversely related to gene expression, suggesting a regulatory role in transcriptional control. Collectively, our findings reveal dynamic epigenetic remodeling in the brain during flatfish metamorphosis, provide insights into how DNA methylation contributes to the coordination of developmental transitions, and extend current knowledge of vertebrate developmental epigenetics by providing the first integrative analysis of DNA methylation, chromatin accessibility, and transcriptional regulation during flatfish brain metamorphosis.
A large portion of the genome is transcribed into noncoding RNAs (ncRNAs). Although initially thought to be functionally insignificant, many ncRNAs are now recognized as important regulators of gene expression. Among these, long non-coding RNAs (lncRNAs), transcripts longer than 200 bases with almost no coding potential, and enhancer RNAs (eRNAs), which are transcribed from regulatory regions on the genome, can regulate nearby genes through diverse cis-acting mechanisms. This review discusses six primary transcription-dependent regulatory mechanisms: transcription-dependent activation, chromatin remodeling, R-loop formation, transcriptional condensate formation, DNA looping, and transcriptional interference. Throughout the review, multiple examples of lncRNAs and eRNAs are presented to illustrate their diverse regulatory functions. lncRNAs often function through the act of transcription itself or by recruiting chromatin-modifying complexes, whereas eRNAs more commonly require their RNA transcript for condensate formation or enhancer-promoter looping. However, the contexts determining the preferred mechanisms remain unclear, and individual transcripts may function through multiple mechanisms. Further advances are needed to distinguish transcription-mediated from RNA-mediated gene regulation.
Chronic inflammation is a hallmark of cancer, driving initiation, progression, and metastasis through sustained pro-inflammatory signaling and immune microenvironment remodeling. Physical exercise reduces systemic low-grade inflammation and improves cancer outcomes, yet the molecular conduits linking transient exercise stress to durable anti-inflammatory effects remain poorly defined. Epigenetic mechanisms-DNA methylation, histone modifications, and non-coding RNAs-translate environmental stimuli into stable gene expression changes. In this review, we dissect the tripartite interplay between physical exercise, epigenetic regulation, and cancer-associated inflammation. We first outline how chronic inflammatory signaling aberrantly reprograms the cancer epigenome, silencing tumor-suppressor and pro-resolution genes via promoter hypermethylation and repressive histone marks, while activating oncogenic and pro-inflammatory mediators through permissive chromatin states. We then synthesize evidence that structured exercise counteracts this corruption by modulating DNA methyltransferases, TET dioxygenases, and histone deacetylases, thereby reversing pathological methylation and acetylation patterns at inflammatory loci. We further examine how exercise-induced circulating microRNAs and exosomal cargo propagate these epigenetic signals systemically to distant tumor niches. A mechanistic model is proposed wherein exercise-dependent epigenetic reprogramming attenuates NF-κB-driven inflammatory circuits and restores immune surveillance. Finally, we identify critical knowledge gaps-tissue-specificity, dose-response relationships, and durability of exercise-induced epigenetic modifications-that must be addressed to translate the exercise-epigenetics-inflammation axis into personalized cancer prevention and therapy.
The efficacy of chemotherapeutic drugs is often limited by cancer cells' adaptive pro-survival mechanisms that attenuate treatment response. Here, we observed marked downregulation of miR-203a-3p in OSCC cells upon exposure to cisplatin, which, upon restoration, enhanced cisplatin sensitivity by increasing DNA double-stranded breaks, promoting apoptotic cell death through decreased Bcl-2 and caspase-9/3 activation, elevating reactive oxygen species, and inducing mitochondrial stress responses by suppressing the RAN-RRM2 axis. Concurrently, OSCC cells responded to cisplatin by upregulating and nuclear-localizing RRM2, thereby promoting pro-survival signaling. Functionally, RAN, a small GTPase, was critical for RRM2 nuclear localization and RRM2-mediated DNA repair, as demonstrated by GTP/GDP-locked mutants and RAN knockdown. Furthermore, rescue experiments using RRM2 and RAN constructs lacking miR-203a-3p-responsive 3'UTR sequences alleviate the chemosensitizing effects of miR-203a-3p, validating RRM2 and RAN as key downstream mediators. Altogether, our study demonstrates a miR-203a-3p/RRM2/RAN regulatory axis that modulates cisplatin response and provides insight into the molecular mechanisms underlying this process in OSCC.
Background Cardiac hypertrophy is a pivotal pathological process leading to heart failure, driven by chronic stress, pressure overload, and neurohormonal stimulation, such as Angiotensin II (Ang II). Although METTL3-mediated m6A modification has been implicated in cardiovascular diseases, the precise role and underlying mechanism of the KLF5 in Ang II-induced myocardial hypertrophy remain poorly understood. Methods H9C2 and AC16 cardiomyocytes were treated with Ang II to establish an in vitro hypertrophy model. The expression of METTL3, KLF5, and hypertrophic markers (ANP, BNP, and β-MHC) was quantified using RT-qPCR and Western blot. Functional validation was performed via lentiviral-mediated knockdown and overexpression. The direct interaction and m6A modification of KLF5 mRNA were validated using RIP-qPCR and MeRIP-qPCR assays. Cardiomyocyte surface area and α-actinin distribution were assessed by immunofluorescence. mRNA stability was determined by Actinomycin D assays. Results Ang II stimulation significantly upregulated the expression of METTL3 and KLF5 in a dose-dependent manner. Silencing of either METTL3 or KLF5 effectively attenuated Ang II-induced cardiomyocyte enlargement and suppressed the fetal gene program. Mechanistically, METTL3 was found to promote KLF5 expression by increasing its m6A modification levels and enhancing its mRNA stability. Furthermore, rescue experiments demonstrated that the inhibitory effect of METTL3 knockdown on cardiomyocyte hypertrophy was substantially reversed by KLF5 overexpression, confirming that KLF5 is a functional downstream effector of METTL3. Conclusions METTL3 facilitates Ang II-induced cardiac hypertrophy by modulating KLF5 expression through an m6A-dependent mechanism. Targeting the METTL3/KLF5 axis may offer a novel therapeutic strategy for the treatment of pathological myocardial hypertrophy.
Recent evidence implicates epigenetic mechanisms in vitamin metabolism, yet most studies remain focused on individual vitamins. In this mini-review, we extend beyond single-vitamin paradigms to propose the Vitamin Rheostat: a unifying model in which vitamins influence regulatory signaling networks that shape their own uptake, utilization, and degradation. By integrating fat-soluble vitamins (nuclear receptor-mediated signaling) and water-soluble vitamins (methyl-donor and enzymatic cofactor systems), this model provides a predictive and experimentally testable framework that may help explain inter-individual variability in response to vitamin intake. It repositions epigenetics as an active mediator in vitamin homeostasis and supports the development of chromatin-informed approaches to precision nutrition.
Long non-coding RNAs (lncRNAs) are classified by sequence, genomic context, and subcellular localization, yet RNA stability remains an underexplored axis of functional classification. Genome-wide profiling by BRIC-seq identified hundreds of lncRNAs with half-lives below 4 h, termed Short-Lived noncoding Transcripts (SLiTs), while Long-Lived RNAs (LL-RNAs) that persist for years in post-mitotic cells define the opposite extreme. We argue that stability is an underappreciated determinant of lncRNA function: short-lived lncRNAs act as dynamic molecular sensors enabling rapid transcriptional responses, whereas long-lived lncRNAs may serve as structural scaffolds maintaining chromatin architecture in post-mitotic cells. We review the molecular mechanisms of lncRNA turnover, the functional consequences of stability in disease, and propose RNA half-life as a candidate additional axis—a working “fifth code”—complementing sequence, structure, modification, and localization for predicting non-coding RNA function. We emphasize that this proposal currently rests largely on correlative associations between half-life and functional class, that the “fifth code” is best understood as an integrated readout of upstream regulatory inputs rather than a primary independent determinant, and that the manuscript explicitly distinguishes experimentally established observations from hypothesis-driven interpretations throughout.
In recent years, circular RNAs (circRNAs), a class of RNA molecules characterized by a covalently closed circular structure, have emerged as a complex family of eukaryotic transcripts with distinctive biological features. Beyond their unusual structure, which generally enhances stability by eliminating free ends, circRNAs have attracted attention because their expression is often cell- and tissue-specific and many are conserved across species. These characteristics support diverse molecular functions, including regulation through interactions with microRNAs and RNA-binding proteins (RBP), and in selected contexts, protein translation. The recognition of circRNAs has therefore added an additional layer of post-transcriptional regulation and expanded our understanding of how gene expression programs are organized across cellular states. Notably, an increasing number of studies links circRNA dynamics to differentiation and development, implicating circRNAs in the regulatory logic that governs stemness maintenance and lineage commitment. In this Review, we summarize current knowledge of circRNA biogenesis and function, highlight emerging connections between circRNAs and cell fate decisions, and discuss how these insights can inform the rational development of bioengineered circRNAs as stable yet programmable platforms to steer differentiation toward targeted cell types and support future RNA-based therapeutic strategies.
Deinococcus radiodurans exhibits exceptional resilience to extreme stressors like ionizing radiation, oxidative damage, and desiccation. This extraordinary resistance stems from a sophisticated regulatory network that enables survival in environments lethal to most organisms. Within this regulatory framework, small RNAs (sRNA) have emerged as key regulators of the stress response. Acting primarily through post-transcriptional regulation, sRNA modulates gene expression by binding target mRNAs to influence their stability or translation, enabling rapid cellular adaptation. Despite significant advances in understanding D. radiodurans' DNA repair capabilities, studies of its RNA-based regulatory pathways are relatively limited. Recent studies have revealed the critical role of sRNAs in D. radiodurans, particularly through advanced RNA sequencing techniques that identified numerous previously unknown sRNAs. These sRNAs act as global regulators, altering the expression of genes involved in the stress response, DNA repair, and metal homeostasis. Mn2+ homeostasis is an important adaptation that enables D. radiodurans to alleviate oxidative stress, a major risk in radiation-rich environments. The role of sRNA in regulating D. radiodurans is more complex than simply responding to the stress. In addition, sRNA controls other bacterial systems, such as quorum sensing and biofilm formation, though these systems are still poorly understood in D. radiodurans. This review focuses on the sRNAs of D. radiodurans and their responses to various stresses. We highlight these riboregulators for their role in one of the most remarkable bacterial survival mechanisms.
The MAGOH and MAGOHB paralogs are core components of the Exon Junction Complex (EJC). Previous studies have established that their simultaneous depletion is lethal, underscoring their essentiality. However, their potential redundancy and individual roles remain poorly defined. To dissect their specific functions, we generated MAGOH or MAGOHB knockout cell lines. We demonstrate that either paralog alone is sufficient to maintain core EJC functions, but their redundancy does not extend to cellular proliferation, as individual MAGOH and MAGOHB knockouts exhibit significant growth defects. Underlying this, proteomic analysis revealed distinct, paralog-specific dysregulation. MAGOH loss uniquely downregulated the mitochondrial ADP/ATP carrier SLC25A4, whereas MAGOHB loss specifically impaired PI3K-Akt signalling. This study reveals a critical non-redundancy, where MAGOH and MAGOHB buffer core EJC function but perform specialised roles in regulating mitochondrial metabolism and proliferative signalling to maintain cellular fitness.
Interferon-induced STAT1 (signal transducer and activator of transcription 1) signal transduction constitutes a dynamic network of concurrent activation and inactivation steps that directly transmits extracellular information from the plasma membrane to the nucleus. To elucidate how nuclear retention of tyrosine-phosphorylated STAT1 as a fundamental principle of the STAT1 network directs signal transduction, we have developed a mathematical rate equation model for STAT1 nucleocytoplasmic shuttling. Based on an interplay between experimental data on the behavior of transport variants with altered nucleocytoplasmic translocation and computational simulations, we calculated the mean residence time of phosphorylated STAT1 in the nucleus to be 13.1 min (5%-95%-CI = 1.6-48.4 min), indicating the highly dynamic nature of the STAT1 signal network. However, when we assessed the time course of unphosphorylated STAT1 from nuclear entry to its subsequent cytoplasmic exit, we found that the half-life of nuclear STAT1 in resting cells was even shorter (5.0 min, 5%-95%-CI = 0.3-15.0 min). Our data reveal that the STAT1 pathway has not evolved to achieve maximal signal amplification, but rather allows for a compromise between rapid termination and efficient signal output. Decoupling of nuclear export from its prior dephosphorylation, such that also phospho-STAT1 can unphysiologically exit the nucleus, results in an overall lower nuclear concentration of activated STAT1 throughout the entire stimulation period, demonstrating the physiological significance of the nuclear export block of phosphorylated STAT1 for cytokine-driven signal propagation. Thus, the complex dynamics of the unique STAT1 network accounts for both efficient and flexible signal transduction, particularly in the course of transient stimulation.
RNA helicases are fundamental ATP-dependent enzymes that remodel RNA structures and ribonucleoprotein complexes. Recent studies reveal that RNA helicases are critical regulators of circular RNA networks, where circRNAs function as stable regulators of gene expression with important implications in disease. This study integrates structural, mechanistic, and functional insights into how RNA helicase families regulate circRNA biogenesis, stability, and function. Accumulating evidence demonstrates that RNA helicases can suppress circRNA biogenesis by remodeling intronic structures, such as DHX9 suppression, or promote biogenesis through DDX5/DDX17, which resolve inhibitory intronic structures by recruiting splicing factors (QKI, SRSF1) and stabilizing spliceosome assembly. We also highlight helicase-mediated control of circRNA stability and decay, such as EIF4A3, DDX5, and DDX17 protecting circRNAs from degradation by blocking decay factors, as well as reciprocal regulation in which circRNAs scaffold to modulate translation. In this review, we summarize RNA helicase families structures and circRNA biogenesis mechanisms, explore their extensive roles in circRNA regulation, and propose RNA helicases as central regulators and promising therapeutic targets, offering new avenues for biomarker development and RNA-based therapies.
Innate immunity is a multilevel system, where each stage plays a key role in ensuring effective immune defense. At the level of chromatin, transcription factors and coactivator protein complexes play a significant role in regulating the immune response. Here, we demonstrate that the conserved transcriptional coactivator SAYP, a component of the Brahma chromatin remodeling complex, participates in the immune response of Drosophila melanogaster. Using immunoprecipitation, we detected an interaction between SAYP and the transcription factor DEAF1 and localized the domains within these proteins that mediate their association. Our data show that SAYP and DEAF1 are recruited to various regions of antimicrobial peptide (AMP) genes in a gene- and pathogen-specific manner following an immune challenge by Escherichia coli or Micrococcus luteus in S2 cell culture. SAYP knockdown significantly reduces the activation of multiple AMP genes, whereas DEAF1 knockdown has a more limited effect. In vivo analysis revealed that loss of full SAYP activity in a hypomorphic mutant significantly impairs the induction of several AMP genes in response to E. coli infection. Furthermore, we found that SAYP cooperates with Relish and DEAF1 in some regulatory regions of AMP genes. Consistently, flies harboring a hypomorphic SAYP mutation exhibited reduced survival following infection by Gram-positive and Gram-negative bacteria. Overall, these findings reveal that SAYP participates in a complex regulatory mechanism with Relish and DEAF1 that controls the innate immune response at the molecular and organismal levels.
Epigenetic marks induced by the environment play a key role in phenotypic plasticity and stress tolerance. We examined phenotypic responses in European sea bass D. labrax following a two-week exposure to fresh water (FW). FW transfer resulted in two distinct tolerance phenotypes: a tolerant (FWt) and an intolerant (FWi) group. FWi showed severe ion imbalance and pronounced stress. Using a genome-wide approach, we compared gill transcriptomic and DNA methylation profiles between phenotypes. RNA-seq analysis identified 7953 differentially expressed genes. Although ion transport pathways were not significantly altered, FWi showed profoundly modulated expression of genes involved in protein synthesis and quality control. These changes were associated with enhanced metabolic and biosynthetic processes, likely reflecting active gill remodelling under osmotic stress. In contrast, genes involved in tissue integrity, epithelial barrier function, and cell adhesion were downregulated. DNA methylation analysis revealed 5320 differentially methylated regions (DMRs) between phenotypes, with over 80% hypomethylated in FWi, suggestive of accelerated biological aging. Hypomethylation in promoters and first exons/introns was mainly linked to genes involved in cell structure and cell-cell interactions. Concordant hypomethylation and upregulation were observed for genes involved in the sphingolipid pathway, cytoskeleton organization, and cell signalling. Downregulated, hypermethylated genes were associated with GTPase activity and immune defence. Overall, our results exclude gill dysfunction as the cause of osmoregulatory failure. However, they also suggest that FW exposure may accelerate aging and reduce lifespan in FWi, potentially due to reduced plasticity and/or genetic variability.
In bacteria, flagellar filaments consist of thousands of flagellin subunits, making their assembly energetically costly. Flagellin synthesis is tightly regulated and in alpha-proteobacteria, this is achieved through a posttranscriptional mechanism mediated by the FlbT and FlaF proteins, although, their specific roles appear to vary among different species. In this study, we show that in Cereibacter sphaeroides in addition to FlaF and FlbT, a third regulatory protein, FlbR, posttranscriptionally regulate the FlaA flagellin synthesis. In the absence of any of these proteins, a severe reduction of FlaA is observed compared to the wild-type strain. This effect does not correlate with the expression of a reporter gene under the control of the flaA promoter but a marked decrease in flaA mRNA levels was observed. Because FlaA synthesis was restored by deleting the 5'-UTR of the flaA mRNA, we propose that this region is presumably the target of FlaF, FlbT and FlbR to control flaA mRNA stability. Furthermore, we show that FlbT by itself binds the flaA 5'-UTR. This posttranscriptional regulation is likely relevant during flagellar morphogenesis, given that preventing the assembly of early flagellar structures results in a reduction of FlaA, which is reversed upon deletion of the 5'-UTR. However, the negative role of the 5'-UTR was not reproduced when this region was placed upstream of a reporter gene, indicating that other regions of the flaA mRNA may also contribute to its regulation.
Plants are immobile organisms, constantly exposed to a wide variety of environmental challenges, among these, drought, salinity and extreme temperatures are the most detrimental factors that significantly affect growth and productivity, leading to substantial annual crop losses worldwide. To respond and survive such challenges, plants have developed complex adaptive mechanisms involving gene regulation at multiple levels. Epigenetic modifications, such as DNA methylation, histone modification, RNA modification, and non-coding RNAs, play a significant role in modulating gene expressions without altering underlying DNA sequences. These modifications facilitate stress-induced gene activation or repression and contribute to the establishment of "stress memory", allowing plants to respond more effectively to repeated stresses. Recent advancements in gene editing technologies, particularly CRISPR/dCas9-based systems, have enabled researchers to target these epigenetic modifications with high precision. By fusing dCas9 (deactivated or dead Cas9) with effectors such as DNA methyltransferases or demethylases or histone acetyltransferases, researchers can modify epigenetic marks that influence gene expression at specific loci, enhancing stress resilience. Most promising techniques like SunTag system have further improved the efficiency of these tools. This review explores how environmental factors influence epigenetic changes and how the knowledge gained could be used for the development of stress resilient crops. These approaches offer a promising avenue for improving desirable traits and ensuring stable crop yields, thereby supporting global food security in a rapidly changing climate.
BACKGROUND:Diabetic kidney disease (DKD) is a major microvascular complication of diabetes, characterized by abnormal mesangial cell (MC) proliferation, yet the underlying mechanisms remain unclear. METHODS:Using RNA-seq and qRT-PCR, Mov10 was identified as the most highly expressed splicing factor (SF) in high-glucose MCs among seven candidates (Mov10, Ssb, Rbm25, Fastk, Dek, Nsrp1, Cwc15). Functional assays (EdU, CCK-8) assessed MC proliferation, while qRT-PCR and Western blot measured inflammatory and fibrotic markers. RIP-seq, RNA-seq, and bioinformatics analyses were used to identify MOV10-binding sites, target genes, pathways, and alternative splicing (AS) events. RESULTS:Transcriptome analysis revealed extensive AS in DKD, with exon skipping being the most frequent. MOV10 knockdown suppressed MC proliferation and reduced inflammation and fibrosis, whereas MOV10 overexpression had opposite effects. RIP-seq showed MOV10 preferentially bound the ACGACG motif within CDS regions. MOV10 regulated AS of proliferation-related genes, including Tead1, Dcbld2, Slc9a5, Akap13, Tfdp2, and Trp53bp1, primarily via exon skipping. CONCLUSIONS:This study identifies MOV10 as a novel DKD-associated splicing factor. By modulating AS of proliferation-related genes, MOV10 promotes mesangial proliferation, inflammation, and fibrosis, highlighting a potential therapeutic target in DKD.
Ten-eleven translocation 2 (Tet2) catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, facilitating active DNA demethylation. Although implicated in adipogenesis, its precise role remains controversial. Here, we investigated Tet2 function during early 3T3-L1 adipocyte differentiation, focusing on regulation of the imprinted gene Pref-1. Differentiation time-course experiments revealed that Tet2 protein, but not mRNA, was progressively reduced via proteasome-dependent degradation, contrasting with previous reports of Tet2 mRNA upregulation. Tet2 overexpression delayed lipid accumulation and reduced adipogenic marker expression, whereas knockdown had the opposite effect. Mechanistically, Tet2 positively regulated Pref-1 transcription by demethylating specific differentially methylated regions in its promoter and exon 6. Loss of Tet2 increased DNA methylation and decreased hydroxymethylation at these loci, leading to Pref-1 repression and derepression of C/EBPα and PPARγ. This stage-specific, proteasome-mediated downregulation of Tet2 integrates epigenetic and post-translational control to regulate adipocyte commitment. The Tet2-Pref-1 axis offers mechanistic insight into adipose tissue expansion and may represent a therapeutic target for obesity and imprinting-related disorders.
Hepatocellular carcinoma (HCC) is a highly lethal malignancy in which acquired resistance to sorafenib remains a major therapeutic challenge. To investigate mechanisms of resistance, we performed transcriptomic profiling of sorafenib-resistant HCC cells, which revealed enrichment of processes related to tumor progression, including mitotic regulation, chromatin remodeling, and apoptotic signaling. Notably, these cells displayed marked downregulation of tissue inhibitor of metalloproteinases-3 (TIMP3), a tumor suppressor known to regulate invasion and epithelial-mesenchymal transition (EMT). Functional studies showed that TIMP3 overexpression (OV) suppressed cell viability, stemness-associated proteins, and a key cell cycle regulator, while upregulating apoptosis-related proteins. Conversely, TIMP3 knockdown (KD) enhanced proliferation, stemness, and EMT. EMT markers were reduced in TIMP3-OV cells but increased with TIMP3-KD, consistent with spheroid sprouting assays, highlighting TIMP3 as a brake on aggressiveness. To explore upstream regulators, we integrated in silico predictions with validation, identifying miR-149-3p as a novel repressor of TIMP3. miR-149-3p overexpression reduced TIMP3 levels and promoted EMT and invasion, whereas inhibition of miR-149-3p restored TIMP3 expression and suppressed migration. Clinical analyses using TCGA datasets and HCC tissue microarrays confirmed significantly lower TIMP3 expression in tumors compared with normal liver tissue, and showed inverse correlations between TIMP3 and proliferative or stemness markers, including Ki67 and CD44. Collectively, these findings establish a mechanistic axis in which miR-149-3p-mediated suppression of TIMP3 promotes sorafenib resistance, EMT, and stemness in HCC. This work identifies TIMP3 as a pivotal determinant of tumor aggressiveness and suggests restoring TIMP3 or targeting downstream pathways as strategies to overcome resistance.
The phage Φ24B-encoded small RNA (sRNA), named UpRoi1, was identified following the prophage induction. The UpRoi1 molecule is encoded in the antirepressor region of the phage genome and is notably small, consisting of only 30 nucleotides. This sRNA is comparable in size to microRNAs found in eukaryotic cells, and thus, unusual for prokaryotic sRNAs. Bioinformatic analysis revealed that UpRoi1 has multiple interaction sites within the phage and host genomes, suggesting its complex regulatory role. In turn, RNA-seq analyses confirmed that the UpRoi1 molecule has a broad impact on the host's gene expression and influences numerous biological processes, particularly those involved in bacterial motility. Furthermore, binding assays revealed that the UpRoi1 molecule may directly interact with the 5' UTR of the phage antirepressor Roi mRNA as well as a bacterial transcript of the flgL gene encoding a protein associated with the structural unit of the bacterial flagellum. This affects both phage and host development, facilitating the switch of the phage to the lytic cycle. Besides, the RNA chaperone protein ProQ has been shown to bind the investigated sRNA with its target transcripts. Based on these results, we have found many similarities between UpRoi1 and herpesviral microRNAs, which is of high importance, because it justifies the occurrence of microRNA-type molecules in Shiga toxin-converting phages.
