Association Of RNase Research Articles

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress.

Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher’s exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

Regulation of angiogenin expression and epithelial-mesenchymal transition by HIF-1α signaling in hypoxic retinal pigment epithelial cells

Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. Epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1α signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1α. Blockage of HIF-1α signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1α and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1α signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1α signaling. It provides molecular evidence for potential therapy strategies of treating CNV.

Association of RNase L with a Ras GTPase‐activating‐like protein IQGAP1 in mediating the apoptosis of a human cancer cell‐line

Mammalian intracellular ribonuclease L (RNase L) is a latent endoribonuclease that functions against viral infections as an apoptosis-inducing protein, and its activity requires intracellular 5'-end-triphosphorylated-2',5' oligoadenylates (2-5A) as an activator. Previously, we showed that RNase L can be activated in human cancer cell line HT1080 by an RNA polymerase I inhibitor, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (3'-ethynylcytidine; ECyd). In ECyd-treated cells, knockdown of the RNase L resulted in a marked decrease in c-jun N-terminal kinase (JNK) phosphorylation, thereby inhibiting apoptosis. We investigate RNase L binding partners by focused proteomic approach using immunoprecipitation with anti-RNase L IgG and mass spectrometry. We found that the IQ motif-containing Ras GTPase-activating-like protein 1 (IQGAP1) can associate with RNase L, and that phosphorylation occurs on the IQGAP1. ECyd-induced JNK phosphorylation and apoptosis were inhibited when IQGAP1 was knocked down with a small interfering RNA. These results raise the interesting possibility that the RNase L-IQGAP1 association may regulate JNK phosphorylation in RNase L-madiated apoptosis. It is likely IQGAP1 works as a regulator in apoptosis.

Role of RNase L in apoptosis induced by 1-(3-C-ethynyl-β-d-ribo-pentofuranosyl)cytosine

1-(3-C-Ethynyl-beta-D: -ribo-pentofuranosyl)cytosine (ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. The present studies have been performed to elucidate the overall mechanisms of ECyd-induced apoptotic cell death. Cultured cells of mouse mammary carcinoma FM3A and human fibrosarcoma HT 1080 lines were used. The efficacy of RNA synthesis inhibition by ECyd was assessed by kinetic analysis using nuclei isolated from FM3A cells. RNA status in ECyd-treated cells was investigated by Northern blots, and the cleavage sites of RNA were identified by rapid amplification of 5' cDNA ends (5'-RACE). The effect of protein functions on the ECyd-induced apoptotic pathway was analyzed by siRNA and immunohistochemical techniques. Apoptotic cells were detected by TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assay. ECyd induces inhibition of RNA synthesis in vitro and in vivo, which appears to be a major cause for the apoptosis. It is known that ECyd is converted inside the cell into its 5'-triphosphate (ECTP). We have now found in test-tube experiments that ECTP strongly inhibits the activity of RNA polymerase I by competing with CTP. In the absence of robust RNA synthesis, the cellular RNAs would be destined to break down. RNase L was found to be playing a role in the breakdown: thus, the 28S rRNA-fragmentation pattern observed for the ECyd-treated cells was very similar to that observable in an in vitro treatment of the 28S ribosomes with RNase L. Association of RNase L with the cytotoxic action of ECyd was confirmed by use of the siRNA-mediated suppression of the cellular RNase L. Thus, the cells in which the RNase L was knocked-down were highly resistant to the cytotoxic action of ECyd. Further events, downstream of the RNase L action that can lead to the eventual apoptosis, would conceivably involve the phosphorylation of c-jun N-terminal kinase and subsequent decrease in mitochondrial membrane-potential. Evidence to support this flow of events was obtained by siRNA-experiments. The results from this study demonstrated that RNase L is activated after the inhibition of RNA polymerase, and induces mitochondria-dependent apoptotic pathway. We propose this new role for RNase L in the apoptotic mechanism. These findings may open up the possibility of finding new targets for anticancer agents.

Characterization of RNABP, an RNA binding protein that associates with RNase L.

The 2',5'-oligoadenylate (2-5A)/RNase L pathway is one of several enzymatic pathways induced by interferons (IFN). RNase L is a latent endoribonuclease that is activated on its binding by 2-5A and inhibited by the ribonuclease L inhibitor (RLI). We have shown previously by coimmunoprecipitation that RNase L may be associated with a 90-kDa RNA binding protein (RNABP), identified with a monoclonal antibody (mAb) raised against an RNase L complex purified under native conditions on 2-5A-sepharose. Here we confirm, by gel-filtration and pull-down analysis, the association of RNase L and RNABP, and we demonstrate that this association is significantly increased in the presence of 2-5A. Moreover, we found that RNABP protein levels decrease during terminal differentiation in various cell lines but do not vary during vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) infection or following IFN-alpha/beta treatment. In this latter case, although total cellular RNABP levels do not vary, the amount of RNABP found in the cytoplasm increases in comparison to that found in the nucleus, indicating a cytoplasmic localization of RNABP after IFN-alpha/beta treatment. Finally, we demonstrate the interaction between RNase L and RNABP in intact cells. Microinjection of an mAb against RNABP into HeLa cells inhibits RNase L antiviral activity and partially inhibits the IFN-alpha/beta-induced antiviral activity.

Human Immunodeficiency Virus Reverse Transcriptase-associated RNase H Activity

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.

Association of RNase H activity with yeast RNA polymerase A.

EUKARYOTIC RNA polymerases were isolated as large multimeric protein complexes containing two high molecular weight subunits and a collection of smaller polypeptide chains1–3. This structural complexity suggests a multifunctional system, organised around a core enzyme combined with specificity determinants and possibly other proteins involved in regulation of chromatin activity. The purpose of this report is to describe the association of a ribonuclease H activity with yeast RNA polymerase A. This nuclease activity, which specifically degrades RNA–DNA hybrids, is inseparable from the polymerase by various fractionation procedures.

Association of Ribonuclease I with Ribosomes and Their Subunits

Abstract RNase I of Salmonella typhimurium has been purified to a homogeneous state. It has a molecular weight of 2 x 104. Association of RNase I with the 30 S subunits of ribosomes has been investigated by using an inhibition assay. 70 S ribosomes and 30 S subunits, but not the 50 S subunits, inhibit the hydrolysis of polyribonucleotides (only poly adenylic acid was used in the present investigation) by RNase I. This inhibition is Mg++-dependent. Each 30 S subunit appears to bind approximately 3 molecules of enzyme. The three-dimensional configuration of the ribosomes is essential for the inhibition since removal of even a small amount of protein by salt extraction leads to considerable loss of inhibitory activity; the detached proteins have no inhibitory activity. Although ribosomal RNA has some inhibitory activity, it is much less than that of the intact ribosomes. Reconstitution of the ribosomes from the salttreated particles and detached proteins leads to complete recovery of the inhibitory activity. This inhibition assay can be very conveniently used in measuring the purity of subunit preparations from 70 S ribosomes as well as in the study of the reconstitution of the ribosomes.