Ribonuclease Degradation Research Articles

Surface engineering of metal-organic framework nanoparticles-based miRNA carrier: Boosting RNA stability, intracellular delivery and synergistic therapy

MicroRNAs (miRNAs) are small noncoding RNAs that are critical for the regulation of multiple physiological and pathological processes, thus holding great clinical potential. However, the therapeutic applications of miRNAs are severely limited by their biological instability and poor intracellular delivery. Herein, we describe a dual-layers surface engineering strategy to design an efficient miRNA delivery nanosystem based on metal–organic frameworks (MOFs) incorporating lipid coating. The resulting nanoparticle system was demonstrated to protect miRNA from ribonuclease degradation, enhance cellular uptake and facilitate lysosomal escape. These ensured effective miRNA mediated gene therapy, which synergized with MOF-specific photodynamic therapy and pre-encapsulated doxorubicin (Dox) chemotherapy to provide a multifunctional with therapeutic effectiveness against cencer cells The mechanisms of miRNA binding and Dox loading were revealed, demonstrating the potential of the present MOFs surface-engineered strategy to overcome their inherent pore-size restriction for macromolecular miRNA carrying, enableefficient co-delivery. In vitro studies revealed the potential of our multifunctional system for miRNA delivery and the demonstrated the therapeutic effectiveness against cancer cells, thereby providing a versatile all-in-one MOFs strategy for delivery of nucleic acids and diverse therapeutic molecules in synergistic therapy.

MetaLnc9-Antisense RNA Contributes to Lung Cancer Metastasis via Modulating RNA-RNA Duplex with MetaLnc9.

Lung cancer is a common life-threatening tumor with high malignancy and high invasiveness. Long non-coding RNAs (lncRNAs) are involved in almost every stage of tumor initiation and progression. Here, we identified an antisense lncRNA, MetaLnc9 antisense (Metalnc9-AS), which arises from the antisense strand of Metalnc9, located on chr9q34.11, while its biological function and mechanism are not clear in lung cancer. In this study, we demonstrated that the expression of Metalnc9-AS was upregulated in non-small cell lung cancer (NSCLC) tissues compared with corresponding non-tumorous tissues. The gain of MetaLnc9-AS was highly associated with the malignant features of NSCLC. Overexpression of MetaLnc9-AS enhanced tumor metastasis in vitro and in vivo. Mechanically, MetaLnc9-AS could form an RNA-RNA hybrid with its cognate sense counterpart, MetaLnc9, to regulate its expression in NSCLC cells, and that such complexes were protected from ribonuclease degradation. Thus, Metalnc9-AS might be a potential and effective treatment for NSCLC.

Exosomes Immunity Strategy: A Novel Approach for Ameliorating Intervertebral Disc Degeneration.

Low back pain (LBP), which is one of the most severe medical and social problems globally, has affected nearly 80% of the population worldwide, and intervertebral disc degeneration (IDD) is a common musculoskeletal disorder that happens to be the primary trigger of LBP. The pathology of IDD is based on the impaired homeostasis of catabolism and anabolism in the extracellular matrix (ECM), uncontrolled activation of immunologic cascades, dysfunction, and loss of nucleus pulposus (NP) cells in addition to dynamic cellular and biochemical alterations in the microenvironment of intervertebral disc (IVD). Currently, the main therapeutic approach regarding IDD is surgical intervention, but it could not considerably cure IDD. Exosomes, extracellular vesicles with a diameter of 30–150 nm, are secreted by various kinds of cell types like stem cells, tumor cells, immune cells, and endothelial cells; the lipid bilayer of the exosomes protects them from ribonuclease degradation and helps improve their biological efficiency in recipient cells. Increasing lines of evidence have reported the promising applications of exosomes in immunological diseases, and regarded exosomes as a potential therapeutic source for IDD. This review focuses on clarifying novel therapies based on exosomes derived from different cell sources and the essential roles of exosomes in regulating IDD, especially the immunologic strategy.

An essential pentatricopeptide repeat protein in the apicomplexan remnant chloroplast.

The malaria parasite Plasmodium and other apicomplexans such as Toxoplasma evolved from photosynthetic organisms and contain an essential, remnant plastid termed the apicoplast. Transcription of the apicoplast genome is polycistronic with extensive RNA processing. Yet little is known about the mechanism of apicoplast RNA processing. In plants, chloroplast RNA processing is controlled by multiple pentatricopeptide repeat (PPR) proteins. Here, we identify the single apicoplast PPR protein, PPR1. We show that the protein is essential and that it binds to RNA motifs corresponding with previously characterized processing sites. Additionally, PPR1 shields RNA transcripts from ribonuclease degradation. This is the first characterization of a PPR protein from a nonphotosynthetic plastid.

The Alleviation of Heat Damage to Photosystem II and Enzymatic Antioxidants by Exogenous Spermidine in Tall Fescue.

Tall fescue (Festuca arundinacea Schreb) is a typical cool-season grass that is widely used in turf and pasture. However, high temperature as an abiotic stress seriously affects its utilization. The objective of this study was to explore the effect of spermidine (Spd) on heat stress response of tall fescue. The samples were exposed to 22°C (normal condition) or 44°C (heat stress) for 4 h. The results showed that exogenous Spd partially improved the quality of tall fescue leaves under normal temperature conditions. Nevertheless, after heat stress treatment, exogenous Spd significantly decreased the electrolyte leakage of tall fescue leaves. Spd also profoundly reduced the H2O2 and O2⋅- content and increased antioxidant enzymes activities. In addition, PAs can also regulate antioxidant enzymes activities including SOD, POD, and APX which could help to scavenge ROS. Moreover, application of Spd could also remarkably increase the chlorophyll content and had a positive effect on the chlorophyll α fluorescence transients under high temperature. The Spd reagent enhanced the performance of photosystem II (PSII) as observed by the JIP-test. Under heat stress, the Spd profoundly improved the partial potentials at the steps of energy bifurcations (PIABS and PItotal) and the quantum yields and efficiencies (φP0, δR0, φR0, and γRC). Exogenous Spd could also reduce the specific energy fluxes per QA- reducing PSII reaction center (RC) (TP0/RC and ET0/RC). Additionally, exogenous Spd improved the expression level of psbA and psbB, which encoded the proteins of PSII core reaction center complex. We infer that PAs can stabilize the structure of nucleic acids and protect RNA from the degradation of ribonuclease. In brief, our study indicates that exogenous Spd enhances the heat tolerance of tall fescue by maintaining cell membrane stability, increasing antioxidant enzymes activities, improving PSII, and relevant gene expression.

SiRNA delivery system based on magnetic nanovectors: Characterization and stability evaluation

Gene therapy and particularly small interfering RNA (siRNA) is a promising therapeutic method for treatment of various human diseases, especially cancer. However the lack of an ideal delivery system limits its clinical applications. Effective anticancer drug development represents the key for translation of research advances into medicines. Previously we reported, the optimization of magnetic siRNA nanovectors (MSN) formulation based on superparamagnetic iron oxide nanoparticles (SPION) and chitosan for systemic administration.This work aimed at using rational design to further optimize and develop MSN. Therefore, formulated MSN were first purified, then their physical and chemical properties were studied mainly through capillary electrophoresis. 95% of siRNA was found enclosed within the purified MSN (pMSN). pMSN showed colloidal stability at pH 7.4, effective protection of siRNA against ribonuclease degradation up to 24 hours and few siRNA release (less than 10%) at pH 7.4. These findings push toward further evaluation studies in vitro and/or in vivo, indicating the appropriateness of pMSN for cancer theranostics.

A direct quantification method for measuring plasma MicroRNAs identified potential biomarkers for detecting metastatic breast cancer.

Circulating miRNAs are protected from ribonuclease degradation by assembly into microvesicles and exosomes. Releasing miRNAs completely from these particles is the key step to quantify the circulating miRNAs. Currently purified RNA-based quantitative analysis is widely used while it is time and cost consuming with high risk for those circulating miRNAs with low abundance due to partial loss of RNA during the steps of total RNA extraction and small RNA enrichment. Herein, we optimized a simple, effective and time-saving method to directly measure plasma miRNAs without RNA isolation. It is based on complete miRNA release from the protein complexes, followed by miRNA-specific reverse transcription and quantitative real-time PCR amplification. By comparison to the RNA-based approach, the direct quantification method showed more efficiency for circulating miRNA analysis, higher accuracy and specificity. By application of the direct quantification method to clinical samples combined with the RNA-based miRNA screening analysis, upregulation of miR-106a in blood was validated in metastatic breast cancer patients, indicating miR-106a are a potential biomarker for metastatic breast cancer.

Hydrogen exchange and proteolytic degradation of ribonuclease A. The local splitting of the native structure and the conformation of loop segments

The limited proteolytic sites or nicksites are present only in one of the five loops of the RNase A molecule. The splitted loop 15-23 connects two structural domains in the hinge region of the interdomain contacts of the V-shaped molecule. The other four loops are inside two domains, 64-71 and 112-115 in the domain I (1-19, 47-81, 102-106) and 36-42 and 88-95 in the domain II (20-46, 82-101). Because of enhanced chain flexibility of the splitted loop in the pH-dependent conformational isomerization, deformation of its structure is slighter under the influence of the intermolecular contacts in the crystal lattice and more significant changes occur in loop conformation at the formation of the 3D swapped dimer of the RNase A molecule. The proteolytic splitting of the 15-23 loop proceeds due to the local fluctuation of the native protein structure.

SiRNA-optimized Modifications for Enhanced In Vivo Activity

Current modifications used in small interfering RNAs (siRNAs), such as 2'-methoxy (2'-OMe) and 2'-fluoro (2'-F), improve stability, specificity or immunogenic properties but do not improve potency. These modifications were previously designed for use in antisense and not siRNA. We show, for the first time, that the siRNA-optimized novel 2'-O modifications, 2'-O-benzyl, and 2'-O-methyl-4-pyridine (2'-O-CH2Py(4)), are tolerated at multiple positions on the guide strand of siRNA sequences in vivo. 2'-O-benzyl and 2'-O-CH2Py(4) modifications were tested at each position individually along the guide strand in five sequences to determine positions that tolerated the modifications. The positions were combined together and found to increase potency and duration of siRNAs in vivo compared to their unmodified counterparts when delivered using lipid nanoparticles. For 2'-O-benzyl, four incorporations were tolerated with similar activity to the unmodified siRNA in vivo, while for 2'-O-CH2Py(4) six incorporations were tolerated. Increased in vivo activity was observed when the modifications were combined at positions 8 and 15 on the guide strand. Understanding the optimal placement of siRNA-optimized modifications needed for maximal in vivo activity is necessary for development of RNA-based therapeutics.

The design and characterization of an RNA nanoring for use in RNA interference drug delivery

While RNA interference (RNAi) offers tremendous promise as a selective means to silence gene expression, novel packaging and delivery methods are needed to improve its overall therapeutic effectiveness. The following RNA nanoring was previously designed as a simple means to package and deliver RNAi. Characterization of the RNA nanoring and the contribution of the RNAI/RNAII inverse interaction responsible for its specific assembly were carried out through the rational design of monomers possessing helical stems with altered degrees of stability, twist, and rigidity. Experimental analysis of the different monomer assemblies confirms that the geometry of the loop‐loop interaction is the dominant factor in the formation of RNA nanorings. Subsequent studies on one specific RNA nanoring design have demonstrated a promising level of resistance toward ribonuclease degradation and confirm its potential as a platform for RNAi drug delivery. This work was funded by the National Institutes of Health (RO1 GM079604‐01) to Luc Jaeger.

Recycling of a regulatory protein by degradation of the RNA to which it binds.

When Bacillus subtilis is grown in the presence of excess tryptophan, transcription of the trp operon is regulated by binding of tryptophan-activated TRAP to trp leader RNA, which promotes transcription termination in the trp leader region. Transcriptome analysis of a B. subtilis strain lacking polynucleotide phosphorylase (PNPase; a 3'-to-5' exoribonuclease) revealed a striking overexpression of trp operon structural genes when the strain was grown in the presence of abundant tryptophan. Analysis of trp leader RNA in the PNPase(-) strain showed accumulation of a stable, TRAP-protected fragment of trp leader RNA. Loss of trp operon transcriptional regulation in the PNPase(-) strain was due to the inability of ribonucleases other than PNPase to degrade TRAP-bound leader RNA, resulting in the sequestration of limiting TRAP. Thus, in the case of the B. subtilis trp operon, specific ribonuclease degradation of RNA in an RNA-protein complex is required for recycling of an RNA-binding protein. Such a mechanism may be relevant to other systems in which limiting concentrations of an RNA-binding protein must keep pace with ongoing transcription.

Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding.

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Import of a Cytosolic Protein into Lysosomes by Chaperone-mediated Autophagy Depends on Its Folding State

Intracellular ribozyme-catalyzed trans-cleavage of RNA monitored by fluorescence resonance energy transfer.

Small catalytic RNAs like the hairpin ribozyme are proving to be useful intracellular tools; however, most attempts to demonstrate trans-cleavage of RNA by ribozymes in cells have been frustrated by rapid cellular degradation of the cleavage products. Here, we describe a fluorescence resonance energy transfer (FRET) assay that directly monitors cleavage of target RNA in tissue-culture cells. An oligoribonucleotide substrate was modified to inhibit cellular ribonuclease degradation without interfering with ribozyme cleavage, and donor (fluorescein) and acceptor (tetramethylrhodamine) fluorophores were introduced at positions flanking the cleavage site. In simple buffers, the intact substrate produces a strong FRET signal that is lost upon cleavage, resulting in a red-to-green shift in dominant fluorescence emission. Hairpin ribozyme and fluorescent substrate were microinjected into murine fibroblasts under conditions in which substrate cleavage can occur only inside the cell. A strong FRET signal was observed by fluorescence microscopy when substrate was injected, but rapid decay of the FRET signal occurred when an active, cognate ribozyme was introduced with the substrate. No acceleration in cleavage rates was observed in control experiments utilizing a noncleavable substrate, inactive ribozyme, or an active ribozyme with altered substrate specificity. Subsequently, the fluorescent substrates were injected into clonal cell lines that expressed cognate or noncognate ribozymes. A decrease in FRET signal was observed only when substrate was microinjected into cells expressing its cognate ribozyme. These results demonstrate trans-cleavage of RNA within mammalian cells, and provide an experimental basis for quantitative analysis of ribozyme activity and specificity within the cell.

DNA technology in the clinical laboratory.

Abstract Objectives.—To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Eighth Annual William Beaumont Hospital Symposium. Data Sources.—The 10 manuscripts submitted were reviewed, and their major findings were compared with literature on the same topic. Study Selection.—Two manuscripts addressed specimen (nucleic acid) stability, 2 described novel analytic approaches, 3 discussed detection of B- or T-cell clonality in lymphoproliferative disorders, and 3 reported the frequency of a variety of genetic polymorphisms found in cardiac disorders. Data Synthesis.—DNA from dried blood spots is stable and may be purified rapidly for amplification and mutation analysis. RNA is much less stable, and a variety of methods may be used to reduce ribonuclease degradation of enteroviral RNA. False-negative reactions may be reduced by genomic amplification of ligated padlock probes by cascade rolling circle or polymerase...

DNA technology in the clinical laboratory.

To review the advances in clinically useful molecular biological techniques and to identify their applications in clinical practice, as presented at the Eighth Annual William Beaumont Hospital Symposium. The 10 manuscripts submitted were reviewed, and their major findings were compared with literature on the same topic. Two manuscripts addressed specimen (nucleic acid) stability, 2 described novel analytic approaches, 3 discussed detection of B- or T-cell clonality in lymphoproliferative disorders, and 3 reported the frequency of a variety of genetic polymorphisms found in cardiac disorders. DNA from dried blood spots is stable and may be purified rapidly for amplification and mutation analysis. RNA is much less stable, and a variety of methods may be used to reduce ribonuclease degradation of enteroviral RNA. False-negative reactions may be reduced by genomic amplification of ligated padlock probes by cascade rolling circle or polymerase chain reaction. A multiplex polymerase chain method using fluorescence-labeled products that separate both the wild-type and mutant hemochromatosis gene alleles by capillary gel electrophoresis represents another approach for detecting the 2 major missense mutations (C282Y and H63D) in hemochromatosis. Southern blotting and polymerase chain reaction have been used to detect B- and T-cell clonality in lymphoproliferative diseases, including mantle cell lymphoma and lymphoma of the breast. Genetic polymorphisms in a variety of coagulation factors and platelet glycoprotein IIIa are associated with ischemic heart disease. As the Human Genome Project continues to define disease-associated mutations, the number of clinically useful molecular pathologic techniques and assays will expand. Clinical outcome analysis is still required to document a decrease in the patient's length of stay to offset the cost of introducing molecular biological assays in the routine clinical pathology laboratory.

A GM-colony-stimulating factor (CSF) activated ribonuclease system transregulates M-CSF receptor expression in the murine FDC-P1/MAC myeloid cell line.

The murine myeloid precursor cell line FDC-P1/MAC simultaneously expresses receptors for multi-colony-stimulating factor (CSF), granulocyte-macrophage (GM)-CSF, and macrophage (M)-CSF. Growth of FDC-P1/MAC cells in either multi-CSF or GM-CSF results in the posttranscriptional suppression of M-CSF receptor (c-fms proto-oncogene) expression. We use the term transregulation to describe this control of receptor expression and have further characterized this regulatory process. The removal of FDC-P1/MAC cells from GM-CSF stimulation resulted in the re-expression of c-fms mRNA independent of M-CSF stimulation and new protein synthesis. Switching FDC-P1/MAC cells from growth in M-CSF to GM-CSF caused the selective degradation of c-fms mRNA within 6 h after factor switching. Blocking protein synthesis or gene transcription with metabolic inhibitors effectively prevented GM-CSF stimulated degradation of c-fms mRNA. These results suggest that the transregulation of c-fms transcripts by GM-CSF requires the transcriptional activation of a selective mRNA degradation factor. In vitro analysis, the use of cytoplasmic cell extracts, provided evidence that a ribonuclease is preferentially active in GM-CSF stimulated cells, although the specificity for mRNA degradation in vitro is broader than seen in vivo. Together, these data suggest that GM-CSF can dominantly transregulate the level of c-fms transcript through the transcriptional activation of a ribonuclease degradation system.

Kinetic Characterization of Ribonuclease-Resistant 2′-Modified Hammerhead Ribozymes

The incorporation of 2'-fluoro- and 2'-aminonucleotides into a hammerhead ribozyme was accomplished by automated chemical synthesis. The presence of 2'-fluorouridines, 2'-fluorocytidines, or 2'-aminouridines did not appreciably decrease catalytic efficiency. Incorporation of 2'-aminocytidines decreased ribozyme activity approximately by a factor of 20. The replacement of all adenosines with 2'-fluoroadenosines abolished catalysis in the presence of MgCl2 within the limits of detection, but some activity was retained in the presence of MnCl2. This effect on catalysis was localized to a specific group of adenines within the conserved single-stranded region of the ribozyme. The decrease in catalytic efficiency was caused by a decrease in the rate constant; the Michaelis constant was unaltered. The 2'-fluoro and 2'-amino modifications conferred resistance toward ribonuclease degradation. Ribozymes containing 2'-fluoro- or 2'-aminonucleotides at all uridine and cytidine positions were stabilized against degradation in rabbit serum by a factor of at least 10(3) compared to unmodified ribozyme.

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts.

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.

Erythrocyte-mediated microinjection, a technique to study protein degradation in muscle cells.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.