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Massive numbers of modified bases in mRNAs sculpt the epitranscriptome and play vital roles in RNA metabolism. The only known acetylated RNA modification, N‐4‐acetylcytidine (ac4C), is highly conserved across cell types and among species. Although the GCN5‐related acetyltransferase 10 (NAT10) functions as an ac4C writer, the mechanism underlying the acetylation process is largely unknown. In this study, the NAT10/PCBP/TDP43 complex mediated mRNA ac4C formation in mammalian cells is identified. RNA‐binding proteins (RBPs) are identified, affiliated with two different families, poly(rC)‐binding protein 1/2 (PCBP1/2) and TAR DNA binding protein 43 (TDP43), as NAT10 adaptors for mRNA tethering and substrate selection. Knockdown of the adaptors resulted in decreased mRNA acetylation abundance in HEK293T cells and ablated cytidine‐rich ac4C motifs. The adaptors also affect the ac4C sites by recruiting NAT10 to their binding sequences. The presence of the NAT10/PCBP/TDP43 complex in mouse testes highlights its potential physiological functions in vivo. These findings reveal the composition of the mRNA ac4C writer complex in mammalian cells and expand the knowledge of mRNA acetylation and ac4C site preferences. [ABSTRACT FROM AUTHOR]

Summary Plant pigments are diverse natural molecules involved in numerous biological functions such as development, growth, and metabolism. As plants age, not only new organs will be formed, but also, they will acquire the necessary pigments in response to the environment and endogenous programming in order to achieve reproductive success. Among the endogenous cues, the small RNAs (sRNAs), an endogenous group of ubiquitous regulatory molecules, may regulate the pigments‐associated biosynthetic pathways at posttranscriptional level. Although plant pigments and sRNAs have been comprehensively studied in several processes throughout the entire plant cycle in model and nonmodel species, connections among these central players must be revised. Studying these complex networks allow us not only to know the progress that has been made in this area, but also generate research questions to be explored in order to unravel novel mechanisms for improving plant yield; therefore, in this review we have summarized the emerging roles of sRNAs‐regulated nodes in mediating plant pigmentation‐associated biosynthetic pathways, focused on chlorophylls, flavonoids, carotenoids, and betalains. In addition, we discuss perspectives related to the manipulation of those genes associated with plant pigments for obtaining genetically improved plants. [ABSTRACT FROM AUTHOR]

CD8+ T cells kill target cells by releasing cytotoxic molecules and proinflammatory cytokines, such as TNF and IFN‐γ. The magnitude and duration of cytokine production are defined by posttranscriptional regulation, and critical regulator herein are RNA‐binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production are not well understood. Previously, we showed that the RBP ZFP36L2 blocks the translation of preformed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a time‐dependent manner. T cell‐specific deletion of ZFP36L2 (CD4‐cre) had no effect on T‐cell development or cytokine production during early time points (2–6 h) of T‐cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN‐γ during prolonged T‐cell activation (20–48 h). ZFP36L2 deficiency also resulted in increased production of IFN‐γ production in tumor‐infiltrating T cells that are chronically exposed to antigens. Mechanistically, ZFP36L2 regulates IFN‐γ production at late time points of activation by destabilizing Ifng mRNA in an AU‐rich element‐dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production. [ABSTRACT FROM AUTHOR]

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here, we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium. NEW & NOTEWORTHY: Our results indicate that intestinal epithelial tissue-specific transgenic miR-195 expression decreases the levels of SOX9 expression, along with reduced numbers of Paneth cells. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restores Paneth cell development ex vivo. miR-195 inhibits SOX9 translation by preventing binding of HuR to Sox9 mRNA. These findings suggest that interaction between miR-195 and HuR controls Paneth cell function via SOX9 in the intestinal epithelium. [ABSTRACT FROM AUTHOR]

SUMMARY: Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. In this study, we investigate the expression of gfp reporter gene in tobacco chloroplasts, guided by variants of the plastid atpH 5′ UTR, which harbors a binding site for PPR10, a protein that activates atpH at the posttranscriptional level. Our findings reveal that endogenous tobacco PPR10 confers distinct levels of reporter activation when coupled with the tobacco and maize atpH 5′ UTRs in different design contexts. Notably, high GFP expression was not coupled to the stabilization of monocistronic gfp transcripts in dicistronic reporter lines, adding to the evidence that PPR10 activates translation via a mechanism that is independent of its stabilization of monocistronic transcripts. Furthermore, the incorporation of a tRNA upstream of the UTR nearly abolishes gfp mRNA (and GFP protein), presumably by promoting such rapid RNA cleavage and 5′ exonucleolytic degradation that PPR10 had insufficient time to bind and protect gfp RNA, resulting in a substantial reduction in GFP accumulation. When combined with a mutant atpH 5′ UTR, the tRNA leads to an exceptionally low level of transgene expression. Collectively, this approach allows for tuning of reporter gene expression across a wide range, spanning from a mere 0.02–25% of the total soluble cellular protein. These findings highlight the potential of employing cis‐elements from heterologous species and expand the toolbox available for plastid synthetic biology applications requiring multigene expression at varying levels. Significance Statement: Achieving optimally balanced gene expression within synthetic operons requires regulatory elements capable of providing a spectrum of expression levels. We report posttranscriptional tools to tune gene expression in synthetic Nicotiana tabacum (tobacco) chloroplast operons over the range of 0.02–25% of total soluble cellular protein. [ABSTRACT FROM AUTHOR]

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Type I toxin-antitoxin systems (T1TAs) are bipartite bacterial loci encoding a growth-inhibitory toxin and an antitoxin small RNA (sRNA). In many of these systems, the transcribed toxin mRNA is translationally inactive, but becomes translation-competent upon ribonucleolytic processing. The antitoxin sRNA targets the processed mRNA to inhibit its translation. This two-level control mechanism prevents cotranscriptional translation of the toxin and allows its synthesis only when the antitoxin is absent. Contrary to this, we found that the timP mRNA of the timPR T1TA locus does not undergo enzymatic processing. Instead, the full-length timP transcript is both translationally active and can be targeted by the antitoxin TimR. Thus, tight control in this system relies on a noncanonical mechanism. Based on the results from in vitro binding assays, RNA structure probing, and cell-free translation experiments, we suggest that timP mRNA adopts mutually exclusive structural conformations. The active form uniquely possesses an RNA pseudoknot structure which is essential for translation initiation. TimR preferentially binds to the active conformation, which leads to pseudoknot destabilization and inhibited translation. Based on this, we propose a model in which "structural processing" of timP mRNA enables tight inhibition by TimR in nonpermissive conditions, and TimP synthesis only upon TimR depletion. [ABSTRACT FROM AUTHOR]

Objective Preeclampsia (PE) is a pregnancy-specific condition featured by high blood pressure, edema, and proteinuria. Research about the role of microRNA (miR)-203 in PE remains insufficient. This experiment is designed to investigate the specific role of miR-203 in trophoblasts in PE. Study Design miR-203 expression in placenta tissues of normal pregnant women and PE patients was examined to analyze the relevance between miR-203 and PE diagnostic efficiency and between miR-203 and blood pressure (systolic pressure and diastolic pressure) and proteinuria of PE patients. miR-203 expression was downregulated in hypoxia-cultured trophoblasts using miR-203 inhibitor to assess matrix metalloproteinase-9 (MMP-9) level. Then, the angiogenesis of trophoblasts with different treatments was determined. Subsequently, the target relation between miR-203 and insulin-like growth factor receptor 1 (IGF-1R) was predicted and verified. Additionally, the effect of IGF-1R in the mechanism of miR-203 modulating trophoblast vascular remodeling was detected. Results miR-203 was overexpressed in the placenta of PE patients and it acted as a promising diagnostic indicator for PE. Moreover, miR-203 was positively associated with blood pressure (systolic pressure and diastolic pressure) and proteinuria of PE patients. miR-203 silencing in hypoxia-cultured trophoblasts enhanced trophoblast vascular remodeling. Mechanically, miR-203 bound to IGF-1R to suppress its transcription. IGF-1R downregulation counteracted the promotive effect of miR-203 silencing on trophoblast vascular remodeling. Conclusion miR-203 was overexpressed in PE, and it targeted IGF-1R to limit trophoblast vascular remodeling. Key Points miR-203 is overexpressed in the placenta of PE patients. miR-203 acts as a potential diagnostic marker for PE. miR-203 targets IGF-1R to reduce trophoblast vascular remodeling in PE. [ABSTRACT FROM AUTHOR]

ABSTRACT The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways.IMPORTANCENumerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins’ mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Chronic obstructive pulmonary disease (COPD) represents an independent risk factor for lung cancer development. Accelerated cell senescence, induced by oxidative stress and inflammation, is a common pathogenic determinant of both COPD and lung cancer. The post transcriptional regulation of genes involved in these processes is finely regulated by RNA-binding proteins (RBPs), which regulate mRNA turnover, subcellular localization, splicing and translation. Multiple pro-inflammatory mediators (including cytokines, chemokines, proteins, growth factors and others), responsible of lung microenvironment alteration, are regulated by RBPs. Several mouse models have shown the implication of RBPs in multiple mechanisms that sustain chronic inflammation and neoplastic transformation. However, further studies are required to clarify the role of RBPs in the pathogenic mechanisms shared by lung cancer and COPD, in order to identify novel biomarkers and therapeutic targets. This review will therefore focus on the studies collectively indicating the role of RBPs in oxidative stress and chronic inflammation as common pathogenic mechanisms shared by lung cancer and COPD.

Development is a chain reaction in which one event leads to another until the completion of a life cycle. Phase transitions are milestone events in the cycle of life. LEAFY COTYLEDON1 (LEC1), ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), and LEC2 proteins, collectively known as LAFL, are master transcription factors (TFs) regulating seed and other developmental processes. Since the initial characterization of the LAFL genes, more than three decades of active research has generated tremendous amounts of knowledge about these TFs, whose roles in seed development and germination have been comprehensively reviewed. Recent advances in cell biology with genetic and genomic tools have allowed the characterization of the LAFL regulatory networks in previously challenging tissues at a higher throughput and resolution in reference species and crops. In this review, we provide a holistic perspective by integrating advances at the epigenetic, transcriptional, posttranscriptional, and protein levels to exemplify the spatiotemporal regulation of the LAFL networks in Arabidopsis seed development and phase transitions, and we briefly discuss the evolution of these TF networks. [ABSTRACT FROM AUTHOR]

MKT1 is a pleiotropic stress response gene identified by several quantitative trait studies with MKT189G as a causal variant, contributing to growth advantage in multiple stress environments. MKT1 has been shown to regulate HO endonuclease posttranscriptionally via the Pbp1–Pab1 complex. RNA‐binding protein Puf3 modulates a set of nuclear‐encoded mitochondrial transcripts whose expression was found to be affected by MKT1 alleles. This study attempts to relate the MKT1 allele‐derived growth advantage with the stability of Puf3 targets during stress and elucidate the roles of Pbp1 and Puf3 in this mechanism. Our results showed that the growth advantage of the MKT189G allele in cycloheximide and H2O2 was PBP1‐dependent, whereas in 4‐nitroquinoline 1‐oxide, the growth advantage was dependent on both PUF3 and PBP1. We compared the messenger RNA decay kinetics of a set of Puf3 targets in multiple stress environments to understand the allele‐specific regulation by MKT1. In oxidative stress, the MKT189G allele modulated the differential expression of nuclear‐encoded mitochondrial genes in a PBP1‐ and PUF3‐dependent manner. Additionally, MKT189G stabilised Puf3 targets, namely, COX17, MRS1 and RDL2, in an allele and stress‐specific manner. Our results showed that COX17, MRS1 and RDL2 had a stress‐specific response in stress environments, with the MKT189G allele contributing to better growth; this response was both PBP1‐ and PUF3‐dependent. Our results indicate that the common allele, MKT189G, regulates stress responses by differentially stabilising Puf3‐target mitochondrial genes, which allows for the strain's better growth in stress environments. Take‐away: MKT1 alleles vary stress responses by posttranscriptional modulation of Puf3 targets.PBP1 and PUF3 influence the regulation of Puf3 target stability under stress.Nuclear‐encoded mitochondrial Puf3 targets, COX17, MRS1 and RDL2, contribute to the growth advantage of MKT189G allele in oxidative stress. [ABSTRACT FROM AUTHOR]

N6-Methyladenosine (m6A) plays key roles in the regulation of biological functions and cellular mechanisms for ischaemia reperfusion (IR) injury in different organs. However, little is known about the underlying mechanisms of m6A-modified mRNAs in hepatic IR injury. In mouse models, liver samples were subjected to methylated RNA immunoprecipitation with high-throughput sequencing (MeRIP-seq) and RNA sequencing (RNA-seq). In total, 16917 m6A peaks associated with 4098 genes were detected in the sham group, whereas 21,557 m6A peaks associated with 5322 genes were detected in the IR group. There were 909 differentially expressed m6A peaks, 863 differentially methylated transcripts and 516 differentially m6A modification genes determined in both groups. The distribution of m6A peaks was especially enriched in the coding sequence and 3‘UTR. Furthermore, we identified a relationship between differentially m6A methylated genes (fold change≥1.5/≤ 0.667, p value≤0.05) and differentially expressed genes (fold change≥1.5 and p value≤0.05) to obtain three overlapping predicted target genes (Fnip2, Phldb2, and Pcf11). Our study revealed a transcriptome-wide map of m6A mRNAs in hepatic IR injury and might provide a theoretical basis for future research in terms of molecular mechanisms.

Post-transcriptional regulation, by small RNAs (sRNAs) as well as the global Carbon Storage Regulator A (CsrA) protein, play critical roles in bacterial metabolic control and stress responses. The CsrA protein affects selective sRNA-mRNA networks, in addition to regulating transcription factors and sigma factors, providing additional avenues of cross talk between other stress-response regulators. Here, we expand the known set of sRNA-CsrA interactions and study their regulatory effects. In vitro binding assays confirm novel CsrA interactions with ten sRNAs, many of which are previously recognized as key regulatory nodes. Of those 10 sRNA, we identify that McaS, FnrS, SgrS, MicL, and Spot42 interact directly with CsrA in vivo. We find that the presence of CsrA impacts the downstream regulation of mRNA targets of the respective sRNA. In vivo evidence supports enhanced CsrA McaS-csgD mRNA repression and showcases CsrA-dependent repression of the fucP mRNA via the Spot42 sRNA. We additionally identify SgrS and FnrS as potential new sRNA sponges of CsrA. Overall, our results further support the expanding impact of the Csr system on cellular physiology via CsrA impact on the regulatory roles of these sRNAs.

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B. subtilis are still not fully explored. Here, a bottom-up approach is proposed for designing artificial trans-acting sRNAs. By engineering the intrinsic sRNA SR6, a minimized core scaffold structure consisting of an 8 bp stem, a 4 nt loop, and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity (83%). Moreover, we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli (above 80%). A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences, which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence. By applying these auto-designed sRNAs, we rapidly modified the cell morphology and biofilm formation, and regulated metabolic flux toward acetoin biosynthesis. This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B. subtilis and E. coli but also enhanced our understanding of trans-acting sRNAs.

As the only oxygenic phototrophs among prokaryotes, cyanobacteria employ intricate mechanisms to regulate common metabolic pathways. These mechanisms include small protein inhibitors exerting their function by protein–protein interaction with key metabolic enzymes and regulatory small RNAs (sRNAs). Here we show that the sRNA NsiR4, which is highly expressed under nitrogen limiting conditions, interacts with the mRNA of the recently described small protein PirA in the model strain Synechocystis sp. PCC 6803. In particular, NsiR4 targets the pirA 5ʹUTR close to the ribosome binding site. Heterologous reporter assays confirmed that this interaction interferes with pirA translation. PirA negatively impacts arginine synthesis under ammonium excess by competing with the central carbon/nitrogen regulator PII that binds to and thereby activates the key enzyme of arginine synthesis, N-acetyl-L-glutamate-kinase (NAGK). Consistently, ectopic nsiR4 expression in Synechocystis resulted in lowered PirA accumulation in response to ammonium upshifts, which also affected intracellular arginine pools. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and conveys additional metabolic flexibility under highly fluctuating nitrogen regimes. Pairs of small protein inhibitors and of sRNAs that control the abundance of these enzyme effectors at the post-transcriptional level appear as fundamental building blocks in the regulation of primary metabolism in cyanobacteria.

Benzopyrene is a high-molecular-weight polycyclic aromatic hydrocarbon that is highly recalcitrant and induces carcinogenic effects. CsrA is a conserved regulatory protein that controls the translation and stability of its target transcripts, having negative or positive effects depending on the target mRNAs. It is known that Bacillus licheniformis M2-7 has the ability to grow and survive in certain concentrations of hydrocarbons such as benzopyrene, prompted in part by CsrA, as is present in gasoline. However, there are a few studies that reveal the genes involved in that process. To identify the genes involved in the Bacillus licheniformis M2-7 degradation pathway, the plasmid pCAT-sp containing a mutation in the catE gene was constructed and used to transform B. licheniformis M2-7 and generate a CAT1 strain. We determined the capacity of the mutant B. licheniformis (CAT1) to grow in the presence of glucose or benzopyrene as a carbon source. We observed that the CAT1 strain presented increased growth in the presence of glucose but a statistically considerable decrease in the presence of benzopyrene compared with the wild-type parental strain. Additionally, we demonstrated that the Csr system positively regulates its expression since it was observed that the expression of the gene in the mutant strain LYA12 (M2-7 csrA:: Sp, SpR) was considerably lower than that in the wild-type strain. We were thus able to propose a putative regulation model for catE gene in B. licheniformis M2-7 strain by CsrA regulator in the presence of benzopyrene. [ABSTRACT FROM AUTHOR]

Chemical modifications on mRNA represent a critical layer of gene expression regulation. Research in this area has continued to accelerate over the last decade, as more modifications are being characterized with increasing depth and breadth. mRNA modifications have been demonstrated to influence nearly every step from the early phases of transcript synthesis in the nucleus through to their decay in the cytoplasm, but in many cases, the molecular mechanisms involved in these processes remain mysterious. Here, we highlight recent work that has elucidated the roles of mRNA modifications throughout the mRNA life cycle, describe gaps in our understanding and remaining open questions, and offer some forward-looking perspective on future directions in the field. [ABSTRACT FROM AUTHOR]

In Type 2 diabetes (T2D), elevated lipid levels have been suggested to contribute to insulin resistance and β‐cell dysfunction. We previously reported that the expression of the PGE2 receptor EP3 is elevated in islets of T2D individuals and is preferentially stimulated by palmitate, leading to β‐cell failure. The mouse EP3 receptor generates three isoforms by alternative splicing which differ in their C‐terminal domain and are referred to as mEP3α, mEP3β, and mEP3γ. We bring evidence that the expression of the mEP3γ isoform is elevated in islets of diabetic db/db mice and is selectively upregulated by palmitate. Specific knockdown of the mEP3γ isoform restores the expression of β‐cell‐specific genes and rescues MIN6 cells from palmitate‐induced dysfunction and apoptosis. This study indicates that palmitate stimulates the expression of the mEP3γ by a posttranscriptional mechanism, compared to the other spliced isoforms, and that the de novo synthesized ceramide plays an important role in FFA‐induced mEP3γ expression in β‐cells. Moreover, induced levels of mEP3γ mRNA by palmitate or ceramide depend on p38 MAPK activation. Our findings suggest that mEP3γ gene expression is regulated at the posttranscriptional level and defines the EP3 signaling axis as an important pathway mediating β‐cell‐impaired function and demise. [ABSTRACT FROM AUTHOR]

Light-independent functions of carotenoids in photosynthetic organisms are poorly understood. Here, we investigated the growth properties of microalga, Euglena gracilis, under altered light and temperature using norflurazon-treated carotenoid-deficient cells and genetically modified strains, including nonphotosynthetic SM-ZK and colorless cl4 . Norflurazon treatment decreased carotenoid and chlorophyll contents, causing cell bleaching. SM-ZK strain had lower carotenoid content than wild-type ( WT ) strain, and it was below the detectable level in the cl4 strain. Norflurazon treatment decreased phytoene synthase EgCrtB levels, although EgcrtB was transcriptionally induced. Carotenoid deficiency in norflurazon-treated cells and the cl4 strain caused similar extents of delayed growth under light and dark conditions at 25 °C, indicating that carotenoids promote growth in darkness. Both WT and SM-ZK strains exhibited similar growth rates. Dark conditions at 20 °C enhanced the growth delay of norflurazon-treated cells and the cl4 strain. These results indicate that carotenoids impart environmental stress tolerance to E. gracilis in light-dependent and light-independent manners. [ABSTRACT FROM AUTHOR]

N6-Methyladenosine (m6A) is among the most abundant modifications of eukaryotic mRNAs. mRNA methylation regulates many biological processes including playing an essential role in meiosis. During meiosis in the budding yeast, Saccharomyces cerevisiae, m6A levels peak early, before the initiation of the meiotic divisions. High-throughput studies suggested, and this work confirms that the uncharacterized protein Ygl036wp interacts with Kar4p, a component of the mRNA m6A-methyltransferase complex. Protein structure programs predict that Ygl036wp folds like VIRMA/Virilizer/VIR, which is involved in mRNA m6A-methylation in higher eukaryotes. In addition, Ygl036wp contains conserved motifs shared with VIRMA/Virilizer/VIR. Accordingly, we propose the name VIR1 for budding yeast ortholog of VIRMA/Virilizer/VIR 1. Vir1p interacts with all other members of the yeast methyltransferase complex and is itself required for mRNA m6A methylation and meiosis. In the absence of Vir1p proteins comprising the methyltransferase complex become unstable, suggesting that Vir1p acts as a scaffold for the complex. The vir1Δ/Δ mutant is defective for the premeiotic S-phase, which is suppressed by overexpression of the early meiotic transcription factor IME1; additional overexpression of the translational regulator RIM4 is required for sporulation. The vir1Δ/Δ mutant exhibits reduced levels of IME1 mRNA, as well as transcripts within Ime1p's regulon. Suppression by IME1 revealed an additional defect in the expression of the middle meiotic transcription factor, Ndt80p (and genes in its regulon), which is rescued by overexpression of RIM4. Together, these data suggest that Vir1p is required for cells to initiate the meiotic program and for progression through the meiotic divisions and spore formation. [ABSTRACT FROM AUTHOR]

Introduction: STAT4 is a transcriptional regulator that has been reported to have oncogenic activities in various cancers. In our study, the posttranscriptional regulatory effect of miR-200a-3p on STAT4 and the prognostic significance of miR-200a-3p and STAT4 were evaluated in bladder cancer (BCa). Material and methods: Proliferation and apoptosis of BCa cell lines were monitored using CCK-8 and Annexin V-FITC assays, respectively. Gene and protein expression levels in BCa tissues and cells were detected using RTqPCR and western blotting, respectively. Results: Significant downregulation of miR-200a-3p and upregulation of STAT4 were observed in BCa tissues and cells compared with the corresponding non-tumor adjacent tissues. Both STAT4 and miR-200a-3p were validated as independent prognostic indicators in sixty-nine BCa patients for predicting overall survival and disease-free survival. In vitro experimental analyses revealed that knockdown of STAT4 repressed BCa cell growth and elevated cell apoptosis. Molecular interactive analysis revealed STAT4 as a direct target of miR-200a-3p, which could suppress STAT4 protein expression by posttranscriptional repression. Cotransfection of miR-200a-3p mimics and STAT4 overexpression plasmids into BCa cells demonstrated that the antineoplastic activities of miR-200a-3p in vitro were neutralized by overexpressed STAT4. Conclusions: The miR-200a-3p/STAT4 signaling cascade plays an important role in the progression of BCa, which provides a new promising target for targeted BCa therapies. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) belong to non-coding small RNAs which have been shown to take a regulatory function at the posttranscriptional level in plant growth development and response to abiotic stress. Hemerocallis fulva is an herbaceous perennial plant with fleshy roots, wide distribution, and strong adaptability. However, salt stress is one of the most serious abiotic stresses to limit the growth and production of Hemerocallis fulva. To identify the miRNAs and their targets involved in the salt stress resistance, the salt-tolerant H. fulva with and without NaCl treatment were used as materials, and the expression differences of miRNAs–mRNAs related to salt-tolerance were explored and the cleavage sites between miRNAs and targets were also identified by using degradome sequencing technology. In this study, twenty and three significantly differential expression miRNAs (p-value < 0.05) were identified in the roots and leaves of H. fulva separately. Additionally, 12,691 and 1538 differentially expressed genes (DEGs) were also obtained, respectively, in roots and leaves. Moreover, 222 target genes of 61 family miRNAs were validated by degradome sequencing. Among the DE miRNAs, 29 pairs of miRNA targets displayed negatively correlated expression profiles. The qRT-PCR results also showed that the trends of miRNA and DEG expression were consistent with those of RNA-seq. A gene ontology (GO) enrichment analysis of these targets revealed that the calcium ion pathway, oxidative defense response, microtubule cytoskeleton organization, and DNA binding transcription factor responded to NaCl stress. Five miRNAs, miR156, miR160, miR393, miR166, and miR396, and several hub genes, squamosa promoter-binding-like protein (SPL), auxin response factor 12 (ARF), transport inhibitor response 1-like protein (TIR1), calmodulin-like proteins (CML), and growth-regulating factor 4 (GRF4), might play central roles in the regulation of NaCl-responsive genes. These results indicate that non-coding small RNAs and their target genes that are related to phytohormone signaling, Ca2+ signaling, and oxidative defense signaling pathways are involved in H. fulva's response to NaCl stress. [ABSTRACT FROM AUTHOR]

Aims The posttranscriptional regulator CsrA regulates many cellular processes, including stress responses in diverse bacteria. However, the role of CsrA in multidrug resistance (MDR) and biocontrol activity in Lysobacter enzymogenes strain C3 (LeC3) remains unknown. Methods and results In this study, we demonstrated that deletion of the csrA gene resulted in the initial slow growth of LeC3 and reduced its resistance to multiple antibiotics, including nalidixic acid (NAL), rifampicin (RIF), kanamycin (Km), and nitrofurantoin (NIT). Loss of the csrA gene also reduced its ability in inhibiting hypha growth of Sclerotium sclerotiorum and influenced its extracellular cellulase and protease activities. Two putative small noncoding regulatory RNAs (sRNAs), referred to as csrB and csrC , were also revealed in the genome of LeC3. Double deletion of csrB and csrC in LeC3 led to increased resistance to NAL, RIF, Km, and NIT. However, no difference was observed between LeC3 and the csrB/csrC double mutant in their suppression of S. sclerotiorum hypha growth and production of extracellular enzymes. Conclusion These results suggest that CsrA in LeC3 not only conferred its intrinsic MDR, but also contributed to its biocontrol activity. [ABSTRACT FROM AUTHOR]

Vault RNAs (vtRNAs) are small noncoding RNAs and highly expressed in many eukaryotes. Here, we identified vtRNA2‐1 as a novel regulator of the intestinal barrier via interaction with RNA‐binding protein HuR. Intestinal mucosal tissues from patients with inflammatory bowel diseases and from mice with colitis or sepsis express increased levels of vtRNAs relative to controls. Ectopically expressed vtRNA2‐1 decreases the levels of intercellular junction (IJ) proteins claudin 1, occludin, and E‐cadherin and causes intestinal epithelial barrier dysfunction in vitro, whereas vtRNA2‐1 silencing promotes barrier function. Increased vtRNA2‐1 also decreases IJs in intestinal organoid, inhibits epithelial renewal, and causes Paneth cell defects ex vivo. Elevating the levels of tissue vtRNA2‐1 in the intestinal mucosa increases the vulnerability of the gut barrier to septic stress in mice. vtRNA2‐1 interacts with HuR and prevents HuR binding to claudin 1 and occludin mRNAs, thus decreasing their translation. These results indicate that vtRNA2‐1 impairs intestinal barrier function by repressing HuR‐facilitated translation of claudin 1 and occludin. Synopsis: Vault RNAs (vtRNAs) are small noncoding RNAs, and their levels increase in intestinal mucosa from patients with IBD. vtRNA2‐1 disrupts intestinal epithelial barrier function by decreasing intercellular junctions via interaction with HuR. vtRNA2‐1 inhibits expression of claudin 1, occludin, and E‐cadherin posttranscriptionally.vtRNA2‐1 impairs intestinal epithelial renewal and causes Paneth cell defects ex vivo.Elevating tissue vtRNA2‐1 levels increases vulnerability of gut barrier to septic stress in mice.vtRNA2‐1 interacts with HuR and prevents HuR binding to mRNAs. [ABSTRACT FROM AUTHOR]

Argonautes (AGOs) interact with microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), which can posttranscriptionally regulate the expression of targeted genes. To date, however, the AGOs and their miRNA triggers remain elusive in rapeseed (Brassica napus). Here, we systematically performed a phylogenetic analysis and examined the collinear relationships of the AGOs among four Brassicaceae species. Their physicochemical properties, gene structures, and expression patterns among 81 tissues from multiple materials and developmental stages were further analyzed. Additionally, their posttranscriptional regulation was analyzed using psRNATarget prediction, miRNA-/mRNA-Seq analyses, and a qRT-PCR verification. We finally identified 10 AtAGOs, 13 BolAGOs, 11 BraAGOs, and 24 BnaAGOs. An expression analysis of the BnaAGOs in the B. napus cultivar ZS11, as well as genotypes with extreme phenotypes in various yield-related traits, revealed the conservation and diversity of these genes. Furthermore, we speculated the posttranscriptional regulation of the B. napus miR168a–AGO1s and miR403–AGO2s modules. Combining miRNA-Seq and mRNA-Seq analyses, we found that the B. napus miR168a–AGO1s module may play an essential role in negatively regulating yield traits, whereas the miR403–AGO2s module positively impacts yield. This is the first attempt to comprehensively analyze the AGOs and their miRNA triggers in B. napus and provides a theoretical basis for breeding high-yielding varieties through the manipulation of the miRNA–AGOs modules. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) represents an independent risk factor for lung cancer development. Accelerated cell senescence, induced by oxidative stress and inflammation, is a common pathogenic determinant of both COPD and lung cancer. The post transcriptional regulation of genes involved in these processes is finely regulated by RNA-binding proteins (RBPs), which regulate mRNA turnover, subcellular localization, splicing and translation. Multiple pro-inflammatory mediators (including cytokines, chemokines, proteins, growth factors and others), responsible of lung microenvironment alteration, are regulated by RBPs. Several mouse models have shown the implication of RBPs in multiple mechanisms that sustain chronic inflammation and neoplastic transformation. However, further studies are required to clarify the role of RBPs in the pathogenic mechanisms shared by lung cancer and COPD, in order to identify novel biomarkers and therapeutic targets. This review will therefore focus on the studies collectively indicating the role of RBPs in oxidative stress and chronic inflammation as common pathogenic mechanisms shared by lung cancer and COPD. [ABSTRACT FROM AUTHOR]

Abstract Background Mammalian sex chromosomes provide dosage compensation, but avian lack a global mechanism of dose compensation. Herein, we employed nanopore sequencing to investigate the genetic basis of gene expression and gene dosage effects in avian Z chromosomes at the posttranscriptional level. Results In this study, the gonad and head skin of female and male duck samples (n = 4) were collected at 16 weeks of age for Oxford nanopore sequencing. Our results revealed a dosage effect and local regulation of duck Z chromosome gene expression. Additionally, AS and APA achieve tissue-specific gene expression, and male-biased lncRNA regulates its Z-linked target genes, with a positive regulatory role for gene dosage effects on the duck Z chromosome. In addition, GO enrichment and KEGG pathway analysis showed that the dosage effects of Z-linked genes were mainly associated with the cellular response to hormone stimulus, melanin biosynthetic, metabolic pathways, and melanogenesis, resulting in sex differences. Conclusions Our data suggested that post transcriptional regulation (AS, APA and lncRNA) has a potential impact on the gene expression effects of avian Z chromosomes. Our study provides a new view of gene regulation underlying the dose effects in avian Z chromosomes at the RNA post transcriptional level.

ABSTRACT Small bacterial regulatory RNAs (sRNAs) have been implicated in the regulation of numerous metabolic pathways. In most of these studies, sRNA-dependent regulation of mRNAs or proteins of enzymes in metabolic pathways has been predicted to affect the metabolism of these bacteria. However, only in a very few cases has the role in metabolism been demonstrated. Here, we performed a combined transcriptome and metabolome analysis to define the regulon of the sibling sRNAs NgncR_162 and NgncR_163 (NgncR_162/163) and their impact on the metabolism of Neisseria gonorrhoeae. These sRNAs have been reported to control genes of the citric acid and methylcitric acid cycles by posttranscriptional negative regulation. By transcriptome analysis, we now expand the NgncR_162/163 regulon by several new members and provide evidence that the sibling sRNAs act as both negative and positive regulators of target gene expression. Newly identified NgncR_162/163 targets are mostly involved in transport processes, especially in the uptake of glycine, phenylalanine, and branched-chain amino acids. NgncR_162/163 also play key roles in the control of serine-glycine metabolism and, hence, probably affect biosyntheses of nucleotides, vitamins, and other amino acids via the supply of one-carbon (C1) units. Indeed, these roles were confirmed by metabolomics and metabolic flux analysis, which revealed a bipartite metabolic network with glucose degradation for the supply of anabolic pathways and the usage of amino acids via the citric acid cycle for energy metabolism. Thus, by combined deep RNA sequencing (RNA-seq) and metabolomics, we significantly extended the regulon of NgncR_162/163 and demonstrated the role of NgncR_162/163 in the regulation of central metabolic pathways of the gonococcus. IMPORTANCE Neisseria gonorrhoeae is a major human pathogen which infects more than 100 million people every year. An alarming development is the emergence of gonococcal strains that are resistant against virtually all antibiotics used for their treatment. Despite the medical importance and the vanishing treatment options of gonococcal infections, the bacterial metabolism and its regulation have been only weakly defined until today. Using RNA-seq, metabolomics, and 13C-guided metabolic flux analysis, we here investigated the gonococcal metabolism and its regulation by the previously studied sibling sRNAs NgncR_162/163. The results demonstrate the regulation of transport processes and metabolic pathways involved in the biosynthesis of nucleotides, vitamins, and amino acids by NgncR_162/163. In particular, the combination of transcriptome and metabolic flux analyses provides a heretofore unreached depth of understanding the core metabolic pathways and their regulation by the neisserial sibling sRNAs. This integrative approach may therefore also be suitable for the functional analysis of a growing number of other bacterial metabolic sRNA regulators.

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

N6-Methyladenosine (m6A) plays key roles in the regulation of biological functions and cellular mechanisms for ischaemia reperfusion (IR) injury in different organs. However, little is known about the underlying mechanisms of m6A-modified mRNAs in hepatic IR injury. In mouse models, liver samples were subjected to methylated RNA immunoprecipitation with high-throughput sequencing (MeRIP-seq) and RNA sequencing (RNA-seq). In total, 16917 m6A peaks associated with 4098 genes were detected in the sham group, whereas 21,557 m6A peaks associated with 5322 genes were detected in the IR group. There were 909 differentially expressed m6A peaks, 863 differentially methylated transcripts and 516 differentially m6A modification genes determined in both groups. The distribution of m6A peaks was especially enriched in the coding sequence and 3'UTR. Furthermore, we identified a relationship between differentially m6A methylated genes (fold change=1.5/= 0.667, p value=0.05) and differentially expressed genes (fold change=1.5 and p value=0.05) to obtain three overlapping predicted target genes (Fnip2, Phldb2, and Pcf11). Our study revealed a transcriptome-wide map of m6A mRNAs in hepatic IR injury and might provide a theoretical basis for future research in terms of molecular mechanisms. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS), a common and frustrating syndrome in women of reproductive age, is characterized by symptoms including hyperandrogenemia, ovulation dysfunction, and polycystic ovaries. The role of competitive endogenous RNA (ceRNA) networks is receiving increasing attention and has been reported in multiple complicated diseases, such as various carcinomas, endometriosis, and tubal factor infertility. However, the association of ceRNA networks with the pathogenesis of PCOS remains unclear. This study aimed to construct a ceRNA network orchestrated by exosomal lnRNA and circRNA in PCOS. We screened RNA data of 34 samples from the Gene Expression Omnibus (GEO) database for differentially expressed lncRNAs (DELs), miRNAs (DEMs), mRNAs (DEGs), and circRNA associated with the progression of PCOS (PCOS, n = 17 vs. normal, n = 17). A protein-protein interaction (PPI) network, gene set enrichment analysis (GSEA), and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted. Importantly, the function of the ceRNA network was explored using GO and KEGG enrichment analyses. We identified 46 DELs (25 upregulated and 21 downregulated), 31 DEMs (20 upregulated and 11 downregulated), 165 DEGs (52 upregulated and 113 downregulated), and 1 differentially expressed circRNA. The PPI network had 79 nodes and 112 edges. The GSEA results showed that these genes were mainly related to oxidative phosphorylation; TNF signaling pathways; and valine, leucine, and isoleucine degradation. GO and KEGG analyses revealed that the DEGs were significantly enriched in lipid metabolism, peroxisome proliferatoractivated receptor (PPAR) signaling pathways, and fatty acid metabolism. Additionally, we constructed a novel PCOS-associated lncRNA-miRNA-mRNA ceRNA triple network and a circRNA-related network. Thereafter, we described the potential roles played by follicular fluid exosomes in PCOS. Our present study describes the molecular pathogenesis of PCOS in human ovarian granulosa cells at the posttranscriptional level, which provides new insights for the clinical diagnosis and treatment of PCOS and further scientific research. [ABSTRACT FROM AUTHOR]

Circadian rhythms enable organisms to anticipate and adjust their physiology to periodic environmental changes. These rhythms are controlled by biological clocks that consist of a set of clock genes that regulate each other's expression. Circadian oscillations in messenger RNA (mRNA) levels require the regulation of mRNA production and degradation. While transcription factors controlling clock function have been well characterized from cyanobacteria to humans, the role of factors controlling mRNA decay is largely unknown. Here, we show that mutations in SM-LIKE PROTEIN 1 (LSM1) and exoribonucleases 4 (XRN4), components of the 5ʹ-3ʹ mRNA decay pathway, alter clock function in Arabidopsis. We found that lsm1 and xrn4 mutants display long-period phenotypes for clock gene expression. In xrn4 , these circadian defects were associated with changes in circadian phases of expression, but not overall mRNA levels, of several core-clock genes. We then used noninvasive transcriptome-wide mRNA stability analysis to identify genes and pathways regulated by XRN4. Among genes affected in the xrn4 mutant at the transcriptional and posttranscriptional level, we found an enrichment in genes involved in auxin, ethylene and drought recovery. Large effects were not observed for canonical core-clock genes, although the mRNAs of several auxiliary clock genes that control the pace of the clock were stabilized in xrn4 mutants. Our results establish that the 5ʹ-3ʹ mRNA decay pathway constitutes a novel posttranscriptional regulatory layer of the circadian gene network, which probably acts through a combination of small effects on mRNA stability of several auxiliary and some core-clock genes. [ABSTRACT FROM AUTHOR]

Mesenchymal glioma stem cells (MES-GSCs) are a major subtype of GSCs that reside within glioma tissues and contribute to metastasis, therapy resistance, and glioma recurrence. However, the precise molecular mechanisms governing MES-GSC functions remain elusive. Our findings revealed that expression levels of miR-505-5p/-3p are elevated in MES-GSCs compared with those in proneural (PN)-GSCs, glioma cell lines, and normal brain tissue and that miR-505-5p/-3p expression levels are decreased in differentiated MES-GSCs. We assumed that miR-505-5p/-3p would play distinctive roles in MES-GSCs and performed loss-of-function experiments targeting miR-505-5p/-3p. Knockdown of miR-505-5p/-3p increased proliferation and promoted differentiation in MES-GSCs while suppressing invasion, temozolomide resistance, and enhancing apoptosis in MES-GSCs. Mechanistically, miR-505-5p/-3p directly targets the 3' UTR of AUF1, leading to its repression in MES-GSCs. Notably, AUF1 expression levels were significantly lower in MES-GSCs compared with those in PN-GSCs, glioma cell lines, and normal brain tissues. Co-inhibition of AUF1 expression with miR-505-5p/-3p knockdown ameliorated the observed cellular phenotypes caused by miR-505-5p/-3p knockdown in MES-GSCs. These results suggest that miR-505-5p/-3p exerts MES-GSC-specific roles in regulating proliferation, differentiation, invasion, apoptosis, and temozolomide resistance by repressing AUF1 expression., (© 2024 Wiley Periodicals LLC.)

Abstract Background Although numerous risk loci for ulcerative colitis (UC) have been identified in the human genome, the pathogenesis of UC remains unclear. Recently, multiple transcriptomic analyses have shown that aberrant gene expression in the colon tissues of UC patients is associated with disease progression. A pioneering study also demonstrated that altered post-transcriptional regulation is involved in the progression of UC. Here, we provide a genome-wide analysis of alternative splicing (AS) signatures in UC patients. We analyzed three datasets containing 74 tissue samples from UC patients and identified over 2000 significant AS events. Results Skipped exon and alternative first exon were the two most significantly altered AS events in UC patients. The immune response-related pathways were remarkably enriched in the UC-related AS events. Genes with significant AS events were more likely to be dysregulated at the expression level. Conclusions We present a genomic landscape of AS events in UC patients based on a combined analysis of two cohorts. Our results indicate that dysregulation of AS may have a pivotal role in determining the pathogenesis of UC. In addition, our study uncovers genes with potential therapeutic implications for UC treatment.

The RNA-binding protein CsrA is a global posttranscriptional regulator and controls many physiological processes and virulence traits. Deletion of csrA caused loss of virulence, reduced motility and production of xanthan gum and substantial increase in glycogen accumulation, as well as enhanced bacterial aggregation and cell adhesion in Xanthomonas spp. How CsrA controls these traits is poorly understood. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis was conducted to compare the protein profile of wild-type strain Xanthomonas citri subsp. citri and the isogenic ΔcsrA strain. A total of 2,374 proteins were identified, and 284 were considered to be differentially expressed proteins (DEPS), among which 151 proteins were up-regulated and 133 were down-regulated in the ΔcsrA strain with respect to the wild-type strain. Enrichment analysis and a protein-protein interaction network analysis showed that CsrA regulates bacterial secretion systems, flagella, and xanthan gum biosynthesis. Several proteins encoded by the gumB operon were down-regulated, whereas proteins associated with flagellum assembly and the type IV secretion system were up-regulated in the ΔcsrA strain relative to the Xcc306 strain. These results were confirmed by β-glucuronidase assay or Western blot. RNA secondary structure prediction and a gel-shift assay indicated that CsrA binds to the Shine-Dalgarno sequence of virB5. In addition, the iTRAQ analysis identified 248 DEPs that were not previously identified in transcriptome analyses. Among them, CsrA regulates levels of eight regulatory proteins (ColR, GacA, GlpR, KdgR, MoxR, PilH, RecX, and YgiX), seven TonB-dependent receptors, four outer membrane proteins, and two ferric enterobactin receptors. Taken together, this study greatly expands understanding of the regulatory network of CsrA in X. citri subsp. citri.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

As an intermediate molecule in the Insulin/Insulin‐like growth factor signalling pathway (IIS), the insulin receptor (IR) plays vital roles linking nutritional signals to the downstream regulation of metabolic homeostasis, development, metamorphosis, reproduction and stress responses. In the present study, we describe the molecular characteristics of IR in the cosmopolitan fruit boring pest, Grapholita molesta, and its predicted posttranscription regulator miR‐982490, and elucidate its regulatory roles in glucolipid homeostasis and metamorphosis. Phylogenetic and domain analyses indicate that lepidopteran IRs normally cluster within families, and that four main domains are conserved in GmIR and those of other Lepidoptera. Bio‐informatic prediction, synchronic expression profile evaluation and dual luciferase reporter assays indicated negative regulation of GmIR by miR‐982490. Injection of miR‐982490 agomir into fifth instar larvae yielded effects similar to dsGmIR injection, resulting in enhanced levels of trehalose and triglyceride in haemolymph, and reduced pupation success and pupal weight, both of which could be rescued by co‐injection of dsGmIR and miR‐982490 antagomir. We infer that GmIR regulates glucolipid homeostasis and affects G. molesta metamorphosis via interactions with its posttranscriptional regulator miR‐982490. This study expands our understanding of the regulatory network of IIS in insect nutritional homeostasis and development. [ABSTRACT FROM AUTHOR]

A growing number of variants associated with risk for neurodevelopmental disorders have been identified by genome-wide association and whole genome sequencing studies. As common risk variants often fall within large haplotype blocks covering long stretches of the noncoding genome, the causal variants within an associated locus are often unknown. Similarly, the effect of rare noncoding risk variants identified by whole genome sequencing on molecular traits is seldom known without functional assays. A massively parallel reporter assay (MPRA) is an assay that can functionally validate thousands of regulatory elements simultaneously using high-throughput sequencing and barcode technology. MPRA has been adapted to various experimental designs that measure gene regulatory effects of genetic variants within cis- and trans-regulatory elements as well as posttranscriptional processes. This review discusses different MPRA designs that have been or could be used in the future to experimentally validate genetic variants associated with neurodevelopmental disorders. Though MPRA has limitations such as it does not model genomic context, this assay can help narrow down the underlying genetic causes of neurodevelopmental disorders by screening thousands of sequences in one experiment. We conclude by describing future directions of this technique such as applications of MPRA for gene-by-environment interactions and pharmacogenetics. [ABSTRACT FROM AUTHOR]

In the current study, we investigated dairy cows' circulating microRNA (miRNA) expression signature during several key time points around calving, to get insights into different aspects of metabolic adaptation. In a trial with 32 dairy cows, plasma samples were collected on days-21, 1, 28, and 63 relative to calving. Individually extracted total RNA was subjected to RNA sequencing using NovaSeq 6,000 (Illumina, CA) on the respective platform of IGA Technology Services, Udine, Italy. MiRDeep2 was used to identify known and novel miRNA according to the miRbase collection. Differentially expressed miRNA (DEM) were assessed at a threshold of fold-change > 1.5 and false discovery rate < 0.05 using the edgeR package. The MiRWalk database was used to predict DEM targets and their associated KEGG pathways. Among a total of 1,692 identified miRNA, 445 known miRNA were included for statistical analysis, of which 84, 59, and 61 DEM were found between days-21 to 1, 1 to 28, and 28 to 63, respectively. These miRNA were annotated to KEGG pathways targeting the insulin, MAPK, Ras, Wnt, Hippo, sphingolipid, T cell receptor, and mTOR signaling pathways. MiRNA-mRNA network analysis identified miRNA as master regulators of the biological process including miR-138, miR-149-5p, miR-2466-3p, miR-214, miR-504, and miR-6523a. This study provided new insights into the miRNA signatures of transition to the lactation period. Calving emerged as a critical time point when miRNA were most affected, while the following period appeared to be recovering from massive parturition changes. The primarily affected pathways were key signaling pathways related to establishing metabolic and immune adaptations. [ABSTRACT FROM AUTHOR]

Prader–Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11–q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic–pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder. [ABSTRACT FROM AUTHOR]

Abstract Objective RNA-binding proteins (RBPs) are important regulators of gene expression that influence mRNA splicing, stability, localization, transport, and translational control. In particular, RBPs play an important role in neurons, which have a complex morphology. Previously, we showed that there are many RBPs that play a conserved role in dendrite development in Drosophila dendritic arborization neurons and Caenorhabditis elegans (C. elegans) PVD neurons including the cytoplasmic polyadenylation element binding proteins (CPEBs), Orb in Drosophila and CPB-3 in C. elegans, and the DEAD box RNA helicases, Me31B in Drosophila and CGH-1 in C. elegans. During these studies, we observed that fluorescently-labeled CPB-3 and CGH-1 localize to cytoplasmic particles that are motile, and our research aims to further characterize these RBP-containing particles in live neurons. Results Here we extend on previous work to show that CPB-3 and CGH-1 localize to motile particles within dendrites that move at a speed consistent with microtubule-based transport. This is consistent with a model in which CPB-3 and CGH-1 influence dendrite development through the transport and localization of their mRNA targets. Moreover, CPB-3 and CGH-1 rarely localize to the same particles suggesting that these RBPs function in discrete ribonucleoprotein particles (RNPs) that may regulate distinct mRNAs.

The recent discovery of vast non-coding RNA-based regulatory networks that can be easily modulated by nucleic acid-based drugs has opened numerous new therapeutic possibilities. Long non-coding RNA, and natural antisense transcripts (NATs) in particular, play a significant role in networks that involve a wide variety of disease-relevant biological mechanisms such as transcription, splicing, translation, mRNA degradation and others. Currently, significant efforts are dedicated to harnessing these newly emerging NAT-mediated biological mechanisms for therapeutic purposes. This review will highlight the recent clinical and pre-clinical developments in this field and survey the advances in nucleic acid-based drug technologies that make these developments possible.

ABSTRACT Copper is essential to most living beings but also highly toxic and as such is an important player at the host-pathogen interface. Bacteria have thus developed homeostatic mechanisms to tightly control its intracellular concentration. Known Cu export and import systems are under transcriptional control, whereas posttranscriptional regulatory mechanisms are yet to be characterized. We identified a three-gene operon, bp2923-bfrG-bp2921, downregulated by copper and notably encoding a TonB-dependent transporter in Bordetella pertussis. We show here that the protein encoded by the first gene, which is a member of the DUF2946 protein family, represents a new type of upstream Open Reading Frame (uORF) involved in posttranscriptional regulation of the downstream genes. In the absence of copper, the entire operon is transcribed and translated. Perception of copper by the nascent bp2923-coded protein via its conserved CXXC motif triggers Rho-dependent transcription termination between the first and second genes by relieving translation arrest on a conserved C-terminal RAPP motif. Homologs of bp2923 are widespread in bacterial genomes, where they head operons predicted to participate in copper homeostasis. This work has thus unveiled a new mode of genetic regulation by a transition metal and identified a regulatory function for a member of an uncharacterized family of bacterial proteins that we have named CruR, for copper-responsive upstream regulator. IMPORTANCE Copper is a transition metal necessary for living beings but also extremely toxic. Bacteria thus tightly control its homeostasis with transcriptional regulators. In this work, we have identified in the whooping cough agent Bordetella pertussis a new control mechanism mediated by a small protein called CruR, for copper-responsive upstream regulator. While being translated by the ribosome CruR is able to perceive intracellular copper, which shuts down the transcription of downstream genes of the same operon, coding for a copper uptake system. This mechanism limits the import of copper in conditions where it is abundant for the bacterium. This is the first report of “posttranscriptional regulation” in response to copper. Homologs of CruR genes head many operons harboring copper-related genes in various bacteria, and therefore the regulatory function unveiled here is likely a general property of this new protein family.

In the current study, we investigated dairy cows’ circulating microRNA (miRNA) expression signature during several key time points around calving, to get insights into different aspects of metabolic adaptation. In a trial with 32 dairy cows, plasma samples were collected on days −21, 1, 28, and 63 relative to calving. Individually extracted total RNA was subjected to RNA sequencing using NovaSeq 6,000 (Illumina, CA) on the respective platform of IGA Technology Services, Udine, Italy. MiRDeep2 was used to identify known and novel miRNA according to the miRbase collection. Differentially expressed miRNA (DEM) were assessed at a threshold of fold-change > 1.5 and false discovery rate < 0.05 using the edgeR package. The MiRWalk database was used to predict DEM targets and their associated KEGG pathways. Among a total of 1,692 identified miRNA, 445 known miRNA were included for statistical analysis, of which 84, 59, and 61 DEM were found between days −21 to 1, 1 to 28, and 28 to 63, respectively. These miRNA were annotated to KEGG pathways targeting the insulin, MAPK, Ras, Wnt, Hippo, sphingolipid, T cell receptor, and mTOR signaling pathways. MiRNA-mRNA network analysis identified miRNA as master regulators of the biological process including miR-138, miR-149-5p, miR-2466-3p, miR-214, miR-504, and miR-6523a. This study provided new insights into the miRNA signatures of transition to the lactation period. Calving emerged as a critical time point when miRNA were most affected, while the following period appeared to be recovering from massive parturition changes. The primarily affected pathways were key signaling pathways related to establishing metabolic and immune adaptations.

Abstract N6-methyladenosine (m6A) is the most prevalent internal modification present in the mRNAs of all higher eukaryotes. However, the role of the m6A methylomes in rice is still poorly understood. With the development of the MeRIP-seq technique, the in-depth identification of mRNAs with m6A modification has become feasible. A study suggested that m6A modification is crucial for posttranscriptional regulation related to Cd2+-induced malignant transformation, but the association between m6A modification in plants and Cd tolerance has not been reported. We investigated the m6A methylomes in the roots of a cadmium (Cd)-treated group and compared them with the roots in the control (CK) group by m6A sequencing of cv. 9311 and cv. Nipponbare (NIP) plants. The results indicated that Cd leads to an altered modification profile in 3,406 differential m6A peaks in cv. 9311 and 2,065 differential m6A peaks in cv. NIP. KEGG pathway analysis of the genes with differentially modified m6A peaks indicated that the “phenylalanine”, “tyrosine and tryptophan biosynthesis”, “glycine”, “adherens junctions”, “glycerophospholipid metabolism” and “threonine metabolism” signalling pathways may be associated with the abnormal root development of cv. 9311 rice due to exposure to Cd. The “arginine”, “proline metabolism”, “glycerolipid”, and “protein processing in endoplasmic reticulum” metabolism pathways were significantly enriched in genes with differentially modified m6A peaks in cv. NIP. Unlike that in Arabidopsis, the m6A-modified nucleotide position on mRNAs (m6A peak) distribution in rice exhibited a preference towards both the stop codon and 3′ untranslated regions (3′ UTRs). These findings provide a resource for plant RNA epitranscriptomic studies and further increase our knowledge on the function of m6A modification in RNA in plants.

Abstract UDP-glucuronosyltransferases (UGTs) are the main phase II drug-metabolizing enzymes mediating the most extensive glucuronidation-binding reaction in the human body. The UGT1A family is involved in more than half of glucuronidation reactions. However, significant differences exist in the distribution of UGT1As in vivo and the expression of UGT1As among individuals, and these differences are related to the occurrence of disease and differences in metabolism. In addition to genetic polymorphisms, there is now interest in the contribution of epigenetics and noncoding RNAs (especially miRNAs) to this differential change. Epigenetics regulates UGT1As pretranscriptionally through DNA methylation and histone modification, and miRNAs are considered the key mechanism of posttranscriptional regulation of UGT1As. Both epigenetic inheritance and miRNAs are involved in the differences in sex expression and in vivo distribution of UGT1As. Moreover, epigenetic changes early in life have been shown to affect gene expression throughout life. Here, we review and summarize the current regulatory role of epigenetics in the UGT1A family and discuss the relationship among epigenetics and UGT1A-related diseases and treatment, with references for future research.