Structure and function of Campylobacter jejuni polynucleotide phosphorylase (PNPase): Insights into the role of this RNase in pathogenicity.

Ribonucleases are in charge of the processing, degradation and quality control of all cellular transcripts, which makes them crucial factors in RNA regulation. This post-transcriptional regulation allows bacteria to promptly react to different stress conditions and growth phase transitions, and also to produce the required virulence factors in pathogenic bacteria. Campylobacter jejuni is the main responsible for human gastroenteritis in the world. In this foodborne pathogen, exoribonuclease PNPase (CjPNP) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. Here we report the crystallographic structure of CjPNP, complemented with SAXS, which confirms the characteristic doughnut-shaped trimeric arrangement and evaluates domain arrangement and flexibility. Mutations in highly conserved residues were constructed to access their role in RNA degradation and polymerization. Surprisingly, we found two mutations that altered CjPNP into a protein that is only capable of degrading RNA even in conditions that favour polymerization. These findings will be important to develop new strategies to combat C. jejuni infections.

Methodologies for bacterial ribonuclease characterization using RNA-seq.

Bacteria adjust gene expression at the post-transcriptional level through an intricate network of small regulatory RNAs and RNA-binding proteins, including ribonucleases (RNases). RNases play an essential role in RNA metabolism, regulating RNA stability, decay, and activation. These enzymes exhibit species-specific effects on gene expression, bacterial physiology, and different strategies of target recognition. Recent advances in high-throughput RNA sequencing (RNA-seq) approaches have provided a better understanding of the roles and modes of action of bacterial RNases. Global studies aiming to identify direct targets of RNases have highlighted the diversity of RNase activity and RNA-based mechanisms of gene expression regulation. Here, we review recent RNA-seq approaches used to study bacterial RNases, with a focus on the methods for identifying direct RNase targets.

The Potential Role of the T2 Ribonucleases in TME-Based Cancer Therapy.

In recent years, there has been a growing interest in developing innovative anticancer therapies targeting the tumor microenvironment (TME). The TME is a complex and dynamic milieu surrounding the tumor mass, consisting of various cellular and molecular components, including those from the host organism, endowed with the ability to significantly influence cancer development and progression. Processes such as angiogenesis, immune evasion, and metastasis are crucial targets in the search for novel anticancer drugs. Thus, identifying molecules with "multi-tasking" properties that can counteract cancer cell growth at multiple levels represents a relevant but still unmet clinical need. Extensive research over the past two decades has revealed a consistent anticancer activity for several members of the T2 ribonuclease family, found in evolutionarily distant species. Initially, it was believed that T2 ribonucleases mainly acted as anticancer agents in a cell-autonomous manner. However, further investigation uncovered a complex and independent mechanism of action that operates at a non-cell-autonomous level, affecting crucial processes in TME-induced tumor growth, such as angiogenesis, evasion of immune surveillance, and immune cell polarization. Here, we review and discuss the remarkable properties of ribonucleases from the T2 family in the context of "multilevel" oncosuppression acting on the TME.

Analogs of the Catechol Derivative Dynasore Inhibit HIV-1 Ribonuclease H, SARS-CoV-2 nsp14 Exoribonuclease, and Virus Replication.

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.

Isolation, Characterization, and Biocompatibility of Bisporitin, a Ribotoxin-like Protein from White Button Mushroom (Agaricus bisporus).

White button mushroom (Agaricus bisporus (J.E. Lange) Imbach) is one of the widely consumed edible mushrooms. Indeed, A. bisporus fruiting bodies are a rich source of nutrients and bioactive molecules. In addition, several enzymes with biotechnological applications are found in A. bisporus (e.g., enzymes for lignocellulose degradation). Here, a novel ribotoxin-like protein (RL-P) from the edible mushroom A. bisporus was purified and characterized. This RL-P, named bisporitin, is a monomeric protein (17-kDa) exhibiting specific ribonucleolytic activity by releasing the α-fragment (hallmark of RL-Ps) when incubated with rabbit ribosomes. In addition, bisporitin shows magnesium-dependent endonuclease activity and displays a similar far-UV CD spectrum as ageritin, the prototype of RL-Ps, isolated from Cyclocybe aegerita fruiting bodies. Interestingly, bisporitin is the first member of RL-Ps to have noticeably lower thermal stability (Tm = 48.59 ± 0.98 °C) compared to RL-Ps isolated in other mushrooms (Tm > 70 °C). Finally, this protein is only partially hydrolyzed in an in vitro digestive system and does not produce adverse growing effects on eukaryotic cell lines. This evidence paves the way for future investigations on possible bioactivities of this RL-P in the digestive system.

Virus-Like Cytosolic and Cell-Free Oxidatively Damaged Nucleic Acids Likely Drive Inflammation, Synapse Degeneration, and Neuron Death in Alzheimer's Disease.

Oxidative stress, inflammation, and amyloid-ß are Alzheimer's disease (AD) hallmarks that cause each other and other AD hallmarks. Most amyloid-ß-lowering, antioxidant, anti-inflammatory, and antimicrobial AD clinical trials failed; none stopped or reversed AD. Although signs suggest an infectious etiology, no pathogen accumulated consistently in AD patients. Neuropathology, neuronal cell culture, rodent, genome-wide association, epidemiological, biomarker, and clinical studies, plus analysis using Hill causality criteria and revised Koch's postulates, indicate that the virus-like oxidative damage-associated molecular-pattern (DAMP) cytosolic and cell-free nucleic acids accumulated in AD patients' brains likely drive neuroinflammation, synaptotoxicity, and neurotoxicity. Cytosolic oxidatively-damaged mitochondrial DNA accumulated outside mitochondria dose-dependently in preclinical AD and AD patients' hippocampal neurons, and in AD patients' neocortical neurons but not cerebellar neurons or glia. In oxidatively-stressed neural cells and rodents' brains, cytosolic oxidatively-damaged mitochondrial DNA accumulated and increased antiviral and inflammatory proteins, including cleaved caspase-1, interleukin-1ß, and interferon-ß. Cytosolic double-stranded RNA and DNA are DAMPs that induce antiviral interferons and/or inflammatory proteins by oligomerizing with various innate-immune pattern-recognition receptors, e.g., cyclic GMP-AMP synthase and the nucleotide-binding-oligomerization-domain-like-receptor-pyrin-domain-containing-3 inflammasome. In oxidatively-stressed neural cells, cytosolic oxidatively-damaged mitochondrial DNA caused synaptotoxicity and neurotoxicity. Depleting mitochondrial DNA prevented these effects. Additionally, cell-free nucleic acids accumulated in AD patients' blood, extracellular vesicles, and senile plaques. Injecting cell-free nucleic acids bound to albumin oligomers into wild-type mice's hippocampi triggered antiviral interferon-ß secretion; interferon-ß injection caused synapse degeneration. Deoxyribonuclease-I treatment appeared to improve a severe-AD patient's Mini-Mental Status Exam by 15 points. Preclinical and clinical studies of deoxyribonuclease-I and a ribonuclease for AD should be prioritized.

Conformational exchange divergence along the evolutionary pathway of eosinophil-associated ribonucleases.

The evolutionary role of conformational exchange in the emergence and preservation of function within structural homologs remains elusive. While protein engineering has revealed the importance of flexibility in function, productive modulation of atomic-scale dynamics has only been achieved on a finite number of distinct folds. Allosteric control of unique members within dynamically diverse structural families requires a better appreciation of exchange phenomena. Here, we examined the functional and structural role of conformational exchange within eosinophil-associated ribonucleases. Biological and catalytic activity of various EARs was performed in parallel to mapping their conformational behavior on multiple timescales using NMR and computational analyses. Despite functional conservation and conformational seclusion to a specific domain, we show that EARs can display similar or distinct motional profiles, implying divergence rather than conservation of flexibility. Comparing progressively more distant enzymes should unravel how this subfamily has evolved new functions and/or altered their behavior at the molecular level.

The role of RNA regulators, quorum sensing and c-di-GMP in bacterial biofilm formation.

Biofilms provide an ecological advantage against many environmental stressors, such as pH and temperature, making it the most common life-cycle stage for many bacteria. These protective characteristics make eradication of bacterial biofilms challenging. This is especially true in the health sector where biofilm formation on hospital or patient equipment, such as respirators, or catheters, can quickly become a source of anti-microbial resistant strains. Biofilms are complex structures encased in a self-produced polymeric matrix containing numerous components such as polysaccharides, proteins, signalling molecules, extracellular DNA and extracellular RNA. Biofilm formation is tightly controlled by several regulators, including quorum sensing (QS), cyclic diguanylate (c-di-GMP) and small non-coding RNAs (sRNAs). These three regulators in particular are fundamental in all stages of biofilm formation; in addition, their pathways overlap, and the significance of their role is strain-dependent. Currently, ribonucleases are also of interest for their potential role as biofilm regulators, and their relationships with QS, c-di-GMP and sRNAs have been investigated. This review article will focus on these four biofilm regulators (ribonucleases, QS, c-di-GMP and sRNAs) and the relationships between them.

Developing New Tools to Fight Human Pathogens: A Journey through the Advances in RNA Technologies.

A long scientific journey has led to prominent technological advances in the RNA field, and several new types of molecules have been discovered, from non-coding RNAs (ncRNAs) to riboswitches, small interfering RNAs (siRNAs) and CRISPR systems. Such findings, together with the recognition of the advantages of RNA in terms of its functional performance, have attracted the attention of synthetic biologists to create potent RNA-based tools for biotechnological and medical applications. In this review, we have gathered the knowledge on the connection between RNA metabolism and pathogenesis in Gram-positive and Gram-negative bacteria. We further discuss how RNA techniques have contributed to the building of this knowledge and the development of new tools in synthetic biology for the diagnosis and treatment of diseases caused by pathogenic microorganisms. Infectious diseases are still a world-leading cause of death and morbidity, and RNA-based therapeutics have arisen as an alternative way to achieve success. There are still obstacles to overcome in its application, but much progress has been made in a fast and effective manner, paving the way for the solid establishment of RNA-based therapies in the future.

Structure of angiogenin dimer bound to double-stranded RNA.

Angiogenin is an unusual member of the RNase A family and is of great interest in multiple pathological contexts. Although it has been assigned various regulatory roles, its core catalytic function is that of an RNA endonuclease. However, its catalytic efficiency is comparatively low and this has been linked to a unique C-terminal helix which partially blocks its RNA-binding site. Assuming that binding to its RNA substrate could trigger a conformational rearrangement, much speculation has arisen on the topic of the interaction of angiogenin with RNA. To date, no structural data on angiogenin-RNA interactions have been available. Here, the structure of angiogenin bound to a double-stranded RNA duplex is reported. The RNA does not reach the active site of angiogenin and no structural arrangement of the C-terminal domain is observed. However, angiogenin forms a previously unobserved crystallographic dimer that makes several backbone interactions with the major and minor grooves of the RNA double helix.

RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site.

Knowledge of the cleavage specificity of ribonucleases is critical for their application in RNA modification mapping or RNA-protein binding studies. Here, we detail the cleavage specificity and efficiency of ribonuclease MC1 and cusativin using a customized RNA sequence that contained all dinucleotide combinations and homopolymer sequences. The sequencing of the oligonucleotide digestion products by a semi-quantitative liquid chromatography coupled with mass spectrometry (LC-MS) analysis documented as little as 0.5-1% cleavage levels for a given dinucleotide sequence combination. While RNase MC1 efficiently cleaved the [A/U/C]pU dinucleotide bond, no cleavage was observed for the GpU bond. Similarly, cusativin efficiently cleaved Cp[U/A/G] dinucleotide combinations along with UpA and [A/U]pU, suggesting a broader specificity of dinucleotide preferences. The molecular interactions between the substrate and active site as determined by the dinucleotide docking studies of protein models offered additional evidence and support for the observed substrate specificity. Targeted alteration of the key amino acid residues in the nucleotide-binding site confirms the utility of this in silico approach for the identification of key interactions. Taken together, the use of bioanalytical and computational approaches, involving LC-MS and ligand docking of tertiary structural models, can form a powerful combination to help explain the RNA cleavage behavior of RNases.

The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence.

Secreted RNase proteins have been reported from only a few pathogens, and relatively little is known about their biological functions. Fusarium oxysporum is a soilborne fungal pathogen that causes Fusarium wilt, one of the most important diseases on tomato. During the infection of F. oxysporum, some proteins are secreted that modulate host plant immunity and promote pathogen invasion. In this study, we identify an RNase, FoRnt2, from the F. oxysporum secretome that belongs to the ribonuclease T2 family. FoRnt2 possesses an N-terminal signal peptide and can be secreted from F. oxysporum. FoRnt2 exhibited ribonuclease activity and was able to degrade the host plant total RNA in vitro dependent on the active site residues H80 and H142. Deletion of the FoRnt2 gene reduced fungal virulence but had no obvious effect on mycelial growth and conidial production. The expression of FoRnt2 in tomato significantly enhanced plant susceptibility to pathogens. These data indicate that FoRnt2 is an important contributor to the virulence of F. oxysporum, possibly through the degradation of plant RNA.

Bulge-Forming miRNases Cleave Oncogenic miRNAs at the Central Loop Region in a Sequence-Specific Manner.

The selective degradation of disease-associated microRNA is promising for the development of new therapeutic approaches. In this study, we engineered a series of bulge-loop-forming oligonucleotides conjugated with catalytic peptide [(LeuArg)2Gly]2 (BC-miRNases) capable of recognizing and destroying oncogenic miR-17 and miR-21. The principle behind the design of BC-miRNase is the cleavage of miRNA at a three-nucleotide bulge loop that forms in the central loop region, which is essential for the biological competence of miRNA. A thorough study of mono- and bis-BC-miRNases (containing one or two catalytic peptides, respectively) revealed that: (i) the sequence of miRNA bulge loops and neighbouring motifs are of fundamental importance for efficient miRNA cleavage (i.e., motifs containing repeating pyrimidine-A bonds are more susceptible to cleavage); (ii) the incorporation of the second catalytic peptide in the same molecular scaffold increases the potency of BC-miRNase, providing a complete degradation of miR-17 within 72 h; (iii) the synergetic co-operation of BC-miRNases with RNase H accelerates the rate of miRNA catalytic cleavage by both the conjugate and the enzyme. Such synergy allows the rapid destruction of constantly emerging miRNA to maintain sufficient knockdown and achieve a desired therapeutic effect.

Innovative developments and emerging technologies in RNA therapeutics.

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.

The nsp15 Nuclease as a Good Target to Combat SARS-CoV-2: Mechanism of Action and Its Inactivation with FDA-Approved Drugs.

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

The effect of long-chain noncoding RNA SCAMP1-AS1 on the proliferation and migration of esophageal cancer cells by targeting and regulating miR-483-5p / 中国医师进修杂志

Objective:To observe the expression of long-chain noncoding RNA (lncRNA) SCAMP1-AS1 in esophageal cancer tissues, and explore the effect of SCAMP1-AS1 on the proliferation and migration of esophageal cancer cells and the possible molecular mechanism.Methods:Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of SCAMP1-AS1 in 37 cases of esophageal cancer tissues and adjacent tissues surgically resected in Huangshi Central Hospital of Edong Medical Group from March 2017 to August 2020. RT-qPCR was also used to detect the expression level of SCAMP1-AS1 in 4 types of esophageal cancer cells (EC9706, TE-13, KYSE30, Eca109) and normal esophageal epithelial cells HET-1A. The cells with the lowest expression were selected, the negative control lentivirus (LV-NC) infection was used as the control group, and the recombinant lentivirus carrying SCAMP1-AS1 sequence (LV-SCAMP1-AS1) infection was used as the experimental group. RT-qPCR was used to detect the expression of SCAMP1-AS1 in esophageal cancer cells after infection. Cell counting kit 8 (CCK-8) and Transwell chamber method were used to detect the proliferation and migration ability of esophageal cancer cells. Bioinformatics methods predicted the target genes of SCAMP1-AS1, and dual luciferase reporter experiments verified the interaction of SCAMP1-AS1 with target gene. RT-qPCR detected the expression of target genes. Western blotting detected the expression of cell proliferation and migration phenotype proteins.Results:The relative expression level of SCAMP1-AS1 in esophageal cancer tissue was significantly lower than that in adjacent tissues (1.26 ± 0.48 vs. 8.03 ± 1.17, P<0.01). The relative expression levels of SCAMP1-AS1 in esophageal cancer cells EC9706, TE-13, KYSE30, Eca109 were all lower than that in normal esophageal epithelial cells (0.54 ± 0.05, 0.14 ± 0.02, 0.46 ± 0.07, 0.77 ± 0.05 vs.1.00 ± 0.06, P<0.05), and the expression of SCAMP1-AS1 in TE-13 cells was the lowest ( P<0.01). Compared with the control group, the expression of SCAMP1-AS1 in TE-13 cells in the experimental group was up-regulated ( P<0.01), the proliferation ability of the cells was reduced ( P<0.01), and the migration ability of the cells was reduced ( P<0.01). miR-483-5p was the direct target of SCAMP1-AS1. Compared with the control group, the expression of miR-483-5p was down-regulated in TE-13 cells in the experimental group ( P<0.01), and the expression of cell proliferation and migration phenotype proteins was down-regulated. Conclusions:The expression of lncRNA SCAMP1-AS1 is down-regulated in esophageal cancer. SCAMP1-AS1 can inhibit the proliferation and migration of esophageal cancer TE-13 cells by targeting the expression of miR-483-5p. SCAMP1-AS1 is expected to become a potential molecular therapeutic target for esophageal cancer.

Study on the mechanism of the expression of microRNA-206 in chronic obstructive pulmonary disease and its effect on the proliferation of human airway smooth muscle cells / 中国医师进修杂志

Objective:To investigate the expression of microRNA (miR)-206 in chronic obstructive pulmonary disease (COPD) and its effect on the proliferation of human airway smooth muscle cells (HASMCs) and to explore its mechanism.Methods:Lung tissue samples of 15 patients with COPD (COPD group) who underwent lung volume reduction surgery in the General Hospital of Ningxia Medical University from September 2017 to September 2018 and of 15 patients with benign lung tumors without a history of COPD were collected. Microarray technology was used to analyze the miR and RNA omics in lung tissues of 4 COPD patients and normal controls, and reverse transcriptase polymerase chain reaction(RT-PCR) was used to verify the results. Bioinformatics and double luciferase gene reporting assay were used to detect the target genes of miR-206 in HASMCs. The miR-206 mimic/inhibitor was transfected into HASMCs by liposome transfection technology, and the expression level of miR-206 was detected by RT-PCR. Methyl thiazolyl tetrazolium (MTT), flow cytometry and apoptosis assay were used to detect the effects of miR-206 on the proliferation, cell cycle and apoptosis of HASMCs. The expression of PTEN, cell cycle and apoptotic protein in HASMCs was detected by Western blot.Results:The results of miR and mRNA omics analysis showed that the expressions of miR-206, miR-3187-5p and miR-124 in COPD group were significantly up-regulated (0.09 ± 0.01 vs. 2.17 ± 0.57, 0.60 ± 0.04 vs. 1.32 ± 0.15, 0.22 ± 0.08 vs. 1.09 ± 0.23) ( P<0.05), while the expressions of miR-574 and miR-337-3p decreased significantly (0.79 ± 0.03 vs. 0.15 ± 0.02, 0.95 ± 0.02 vs. 0.17 ± 0.01) ( P<0.05). RT-PCR was used to detect the expression of these five miRNAs in 15 COPD lung tissues, and the results showed that their expression was consistent with that in microarray. The prediction results of miRNA target genes showed that miR-206 could directly inhibit the expression of PTEN. RT-PCR results showed that the expression of miR-206 in miR-206 transfected HASMCs was significantly higher than that in miR-NC transfected group(7.44 ± 0.51 vs. 4.02 ± 0.19), and miR-206 inhibitor could significantly inhibit the expression of miR-206 in cells (1.86 ± 0.32), the difference was statistically significant ( P<0.05); MTT and apoptosis experiments showed that miR-206 mimcs could significantly promote the proliferation rate of cells compared with normal HASMCs or miR-NC transfected cells (0.62 ± 0.14 or 0.57 ± 0.09 vs. 0.83 ± 0.05), inhibit cell apoptosis (9.13 ± 1.71 or 10.02 ± 1.15 vs. 3.06 ± 0.82), the differences were statistically significant ( P<0.05), while miR-206 inhibitor could significantly inhibit cell proliferation and promote cell apoptosis ( P<0.05) The results of cell cycle distribution showed that compared with HASMCs group, the proportion of cells in S phase and G2/M phase in miR-206 mimcs group increased significantly ( P<0.05), while the proportion of cells in S phase and G2/M phase in miR-206 inhibitor group decreased significantly ( P<0.05), and there was no significant difference in miR-NC group ( P>0.05). The results of Western blot showed that compared with normal HASMCs or miR-NC transfected cells, miR-206 mimcs could significantly upregulate the expression of cyclin D1 (0.43 ± 0.07 or 0.41 ± 0.02 vs. 0.63 ± 0.17), and cyclin B1 (0.47 ± 0.13 or 0.50 ± 0.09 vs. 0.79 ± 0.31), and inhibit the expression of PTEN (0.34 ± 0.10 or 0.29 ± 0.05 vs. 0.14 ± 0.02), cyclin p21 (0.34 ± 0.03 or 0.30 ± 0.05 vs. 0.11 ± 0.02), and apoptosis related protein caspase-3 (0.29 ± 0.03 or 0.31 ± 0.05 vs. 0.15 ± 0.03), the differences were statistically significant ( P<0.05). miR-206 inhibitor could significantly inhibit the expression of cyclin D1 and cyclin B1, and promote the expression of PTEN, cyclin p21 and caspase-3 ( P<0.05). Conclusions:In COPD patients, miR-206 could targeted inhibit the expression of PTEN protein in airway smooth muscle cells and regulate the progress of cell cycle, so as to up regulate the proliferation of cells and inhibit their apoptosis.

Effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism / 中国基层医药

Objective:To investigate the effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism.Methods:J82 cells were transfected with random sequence molecules (NC group) and miR-5011-5p sequence molecules (miR-5011-5p group). Flow cytometry and scratch experiment were performed to analyze the effects of miR-5011-5p on apoptosis and migration of J82 cells. The target gene of miR-5011-5p was predicted by bioinformatics. Real-time fluorescent quantitative polymerase chain reaction and western blot assay were performed to investigate the effects of miR-5011-5p on target gene expression.Results:The relative expression of miR-5011-5p in J82 cells in the miR-5011-5p group was significantly higher than that in the NC group (10.73 ± 1.67 vs. 1.04 ± 0.16, t = 5.81, P < 0.01). There was significant difference in the apoptosis rate of J82 cells between NC and miR-5011-5p groups [(8.83 ± 1.67)% vs. (34.96 ± 3.80)%, t = 6.30, P < 0.01]. The migration rate of J82 cells differed significantly between NC and miR-5011-5p groups [(71.31 ± 7.69)% vs. (37.43 ± 5.01)%, t = 3.69, P < 0.05]. The target gene of miR-5011-5p may be Yes-related protein 1 (YAP1). Compared with the NC group, miR-5011-5p exhibited an obvious inhibitory effect on the YAP1 expression in J82 cells ( P < 0.01). Conclusion:miR-5011-5p may promote the apoptosis of J82 cells and inhibit their migration in bladder cancer through targeted inhibition of YAP1 gene expression.

Maturation of UTR-Derived sRNAs Is Modulated during Adaptation to Different Growth Conditions.

Small regulatory RNAs play a major role in bacterial gene regulation by binding their target mRNAs, which mostly influences the stability or translation of the target. Expression levels of sRNAs are often regulated by their own promoters, but recent reports have highlighted the presence and importance of sRNAs that are derived from mRNA 3' untranslated regions (UTRs). In this study, we investigated the maturation of 5' and 3' UTR-derived sRNAs on a global scale in the facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides. Including some already known UTR-derived sRNAs like UpsM or CcsR1-4, 14 sRNAs are predicted to be located in 5 UTRs and 16 in 3' UTRs. The involvement of different ribonucleases during maturation was predicted by a differential RNA 5'/3' end analysis based on RNA next generation sequencing (NGS) data from the respective deletion strains. The results were validated in vivo and underline the importance of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) during processing and maturation. The abundances of some UTR-derived sRNAs changed when cultures were exposed to external stress conditions, such as oxidative stress and also during different growth phases. Promoter fusions revealed that this effect cannot be solely attributed to an altered transcription rate. Moreover, the RNase E dependent cleavage of several UTR-derived sRNAs varied significantly during the early stationary phase and under iron depletion conditions. We conclude that an alteration of ribonucleolytic processing influences the levels of UTR-derived sRNAs, and may thus indirectly affect their mRNA targets.

Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis.

Knowledge of the structural information is essential for understanding the functional details of modified RNA. Cellular non-coding RNA such as rRNA, tRNA and even viral RNAs contain a number of post-transcriptional modifications with varied degree of diversity and density. In this chapter, we discuss the use of a combination of biochemical and analytical tools such as ribonucleases and liquid chromatography coupled with mass spectrometry approaches for characterization of modified RNA. We present the protocols and alternate strategies for obtaining confident modified sequence information to facilitate the understanding of function.