Subcellular localization of a bacterial regulatory RNA

Eukaryotes and bacteria regulate the activity of some proteins by localizing them to discrete subcellular structures, and eukaryotes localize some RNAs for the same purpose. To explore whether bacteria also spatially regulate RNAs, the localization of tmRNA was determined using fluorescence in situ hybridization. tmRNA is a small regulatory RNA that is ubiquitous in bacteria and that interacts with translating ribosomes in a reaction known as trans-translation. In Caulobacter crescentus, tmRNA was localized in a cell-cycle–dependent manner. In G₁-phase cells, tmRNA was found in regularly spaced foci indicative of a helix-like structure. After initiation of DNA replication, most of the tmRNA was degraded, and the remaining molecules were spread throughout the cytoplasm. Immunofluorescence assays showed that SmpB, a protein that binds tightly to tmRNA, was colocalized with tmRNA in the helix-like pattern. RNase R, the nuclease that degrades tmRNA, was localized in a helix-like pattern that was separate from the SmpB-tmRNA complex. These results suggest a model in which tmRNA-SmpB is localized to sequester tmRNA from RNase R, and localization might also regulate tmRNA-SmpB interactions with ribosomes.     

Long noncoding RNAs in hepatitis B virus-related hepatocellular carcinoma

AIM: To study the expression of long noncoding RNAs (lncRNAs) in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).METHODS: The lncRNA profiles between HBV-related HCC tissues and corresponding normal liver tissues were generated using microarray analysis. Datasets were analyzed using multiple algorithms to depict alterations in gene expression on the basis of gene ontology (GO), pathway analysis, and lncRNA levels.RESULTS: The microarray revealed that 1772 lncRNAs and 2508 mRNAs were differently expressed. The pathway analysis demonstrated that the cell cycle, cytokine-cytokine receptor interaction, chemokine signaling pathway, and phosphoinositide 3-kinase-protein kinase B signaling pathway may play important roles in HCC. Several GO terms, such as cell cycle, DNA replication, immune response, and signal transduction, were enriched in gene lists, suggesting a potential correlation with HBV-related HCC. The upregulated large intergenic noncoding RNA ULK4P2 was physically combined with enhancer of zeste homolog 2. Therefore, the lncRNAs may participate in regulating HBV-related HCC.CONCLUSION: lncRNAs play important roles in HCC, future studies should verify whether large intergenic noncoding ULK4P2 functions by combining with enhancer of zeste homolog 2 in HCC.     

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.     

The genomic binding sites of a noncoding RNA

Long noncoding RNAs (lncRNAs) have important regulatory roles and can function at the level of chromatin. To determine where lncRNAs bind to chromatin, we developed capture hybridization analysis of RNA targets (CHART), a hybridization-based technique that specifically enriches endogenous RNAs along with their targets from reversibly cross-linked chromatin extracts. CHART was used to enrich the DNA and protein targets of endogenous lncRNAs from flies and humans. This analysis was extended to genome-wide mapping of roX2, a well-studied ncRNA involved in dosage compensation in Drosophila. CHART revealed that roX2 binds at specific genomic sites that coincide with the binding sites of proteins from the male-specific lethal complex that affects dosage compensation. These results reveal the genomic targets of roX2 and demonstrate how CHART can be used to study RNAs in a manner analogous to chromatin immunoprecipitation for proteins.     

Role of noncoding RNAs in liver fibrosis

Liver fibrosis is a wound-healing response following chronic liver injury caused by hepatitis virus infection, obesity, or excessive alcohol. It is a dynamic and reversible process characterized by the activation of hepatic stellate cells and excess accumulation of extracellular matrix. Advanced fibrosis could lead to cirrhosis and even liver cancer, which has become a significant health burden worldwide. Many studies have revealed that noncoding RNAs (ncRNAs), including microRNAs, long noncoding RNAs and circular RNAs, are involved in the pathogenesis and development of liver fibrosis by regulating signaling pathways including transforming growth factor-β pathway, phosphatidylinositol 3-kinase/protein kinase B pathway, and Wnt/β-catenin pathway. NcRNAs in serum or exosomes have been reported to tentatively applied in the diagnosis and staging of liver fibrosis and combined with elastography to improve the accuracy of diagnosis. NcRNAs mimics, ncRNAs in mesenchymal stem cell-derived exosomes, and lipid nanoparticles-encapsulated ncRNAs have become promising therapeutic approaches for the treatment of liver fibrosis. In this review, we update the latest knowledge on ncRNAs in the pathogenesis and progression of liver fibrosis, and discuss the potentials and challenges to use these ncRNAs for diagnosis, staging and treatment of liver fibrosis. All these will help us to develop a comprehensive understanding of the role of ncRNAs in liver fibrosis.     

非编码RNAs在慢性阻塞性肺疾病所致肺动脉高压致病机制中的作用

慢性阻塞性肺疾病所致肺动脉高压(COPD-PH)的发病率和病死率高,患者3年生存率仅为41%。目前COPD-PH的常规对症治疗以及针对PH的靶向治疗,效果均不理想。非编码RNAs作为重要的内源基因调节因子,严格调控生理和病理过程。COPD-PH患者中存在非编码RNAs表达的改变,且发现其在COPD-PH发病机制中发挥重...     

Inhibition of P-glycoprotein facilitated glycosaminoglycan accumulation during chondrogenesis of human bone marrow mesenchymal stem cells

Aim: P‐glycoprotein (P‐gp) is an adenosine‐5‐triphosphate Binding Cassettes B 1 (ABCB1) transporter that exports various substrates on cellular membrane. Surface expression of P‐gp was decreased during chondrogenesis of human bone marrow mesenchymal stem cells (BM‐MSCs). We examined the role of P‐gp in extracellular matrix deposition during chondrogenesis of human BM‐MSCs. Method: BM‐MSCs were isolated from 16 volunteers after informed consent and incubated for 28 days using three‐dimensional culture methods in chondrogenic medium with and without P‐gp inhibitor (verapamil, 10 μmol/L). Results: Hematoxylin and eosin staining revealed a cartilaginous structure with chondrogenic cells in the lacunae after 2 weeks of culture. Total glycosaminoglycan (GAG) content was increased and rose during pellet culture. Hyaluronan (HA) content of the culture medium decreased with P‐gp inhibitor. Type II collagen deposition decreased with P‐gp inhibitor. Conclusion: Inhibition of P‐gp facilitated GAG accumulation via HA export inhibition during chondrogenic differentiation of human BM‐MSCs. Modulation of P‐gp expression during chondrogenesis would be a possible therapeutic approach for articular cartilage regeneration.     

RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.     

PolyU tail of rho-independent terminator of bacterial small RNAs is essential for Hfq action

Major bacterial small RNAs (sRNAs) regulate the translation and stability of target mRNAs through base pairing with the help of the RNA chaperone Hfq. The Hfq-dependent sRNAs consist of three basic elements, mRNA base-pairing region, Hfq-binding site, and rho-independent terminator. Although the base-pairing region and the terminator are well documented in many sRNAs, the Hfq-binding site is less well-defined except that Hfq binds RNA with a preference for AU-rich sequences. Here, we performed mutational and biochemical studies to define the sRNA site required for Hfq action using SgrS as a model sRNA. We found that shortening terminator polyU tail eliminates the ability of SgrS to bind to Hfq and to silence ptsG mRNA. We also demonstrate that the SgrS terminator can be replaced with any foreign rho-independent terminators possessing a polyU tail longer than 8 without losing the ability to silence ptsG mRNA in an Hfq-dependent manner. Moreover, we found that shortening the terminator polyU tail of several other sRNAs also eliminates the ability to bind to Hfq and to regulate target mRNAs. We conclude that the polyU tail of sRNAs is essential for Hfq action in general. The data also indicate that the terminator polyU tail plays a role in Hfq-dependent stabilization of sRNAs.