Use Of RNase Research Articles

Human RNase P: overview of a ribonuclease of interrelated molecular networks and gene-targeting systems.

The seminal discovery of ribonuclease P (RNase P) and its catalytic RNA by Sidney Altman has not only revolutionized our understanding of life, but also opened new fields for scientific exploration and investigation. This review focuses on human RNase P and its use as a gene-targeting tool, two topics initiated in Altman's laboratory. We outline early works on human RNase P as a tRNA processing enzyme and comment on its expanding nonconventional functions in molecular networks of transcription, chromatin remodeling, homology-directed repair, and innate immunity. The important implications and insights from these discoveries on the potential use of RNase P as a gene-targeting tool are presented. This multifunctionality calls to a modified structure-function partitioning of domains in human RNase P, as well as its relative ribonucleoprotein, RNase MRP. The role of these two catalysts in innate immunity is of particular interest in molecular evolution, as this dynamic molecular network could have originated and evolved from primordial enzymes and sensors of RNA, including predecessors of these two ribonucleoproteins.

The human TRAM1 locus expresses circular RNAs

Numerous indirect and in silico produced evidences suggest circular RNAs (circRNA) in mammals while thorough experimental proofs of their existence have rarely been reported. Biological studies of circRNA, however, should be based on experimentally verified circRNAs. Here, we describe the identification of two circRNAs originating from the gene locus of the translocation associated membrane protein 1 (TRAM1). Linear and potentially circular TRAM1-specific transcripts were identified in a transcriptome analysis of urine RNA of bladder cancer (BCa) patients versus healthy donors. Thus, we first focused on the topology of TRAM1-specific transcripts. We describe conclusive experimental evidence for the existence of TRAM1-specific circRNAs in the human BCa cell lines ECV-304 and RT-4. PCR-based methodology followed by cloning and sequencing strongly indicated the circular topology of two TRAM1 RNAs. Further, studies with exon fusion sequence-specific antisense oligonucleotides (asON) and RNase H as well as studies in the use of RNase R contribute to conclusive set of experiments supporting the circular topology of TRAM1 transcripts. On the biological side, TRAM1-specific circRNAs showed low expression levels and minor differences in BCa cell lines while linear TRAM1 transcripts displayed down-regulated expression in the higher cancer stage model ECV-304 versus more differentiated RT-4 cells.

Biological Activities of Secretory RNases: Focus on Their Oligomerization to Design Antitumor Drugs.

Ribonucleases (RNases) are a large number of enzymes gathered into different bacterial or eukaryotic superfamilies. Bovine pancreatic RNase A, bovine seminal BS-RNase, human pancreatic RNase 1, angiogenin (RNase 5), and amphibian onconase belong to the pancreatic type superfamily, while binase and barnase are in the bacterial RNase N1/T1 family. In physiological conditions, most RNases secreted in the extracellular space counteract the undesired effects of extracellular RNAs and become protective against infections. Instead, if they enter the cell, RNases can digest intracellular RNAs, becoming cytotoxic and having advantageous effects against malignant cells. Their biological activities have been investigated either in vitro, toward a number of different cancer cell lines, or in some cases in vivo to test their potential therapeutic use. However, immunogenicity or other undesired effects have sometimes been associated with their action. Nevertheless, the use of RNases in therapy remains an appealing strategy against some still incurable tumors, such as mesothelioma, melanoma, or pancreatic cancer. The RNase inhibitor (RI) present inside almost all cells is the most efficacious sentry to counteract the ribonucleolytic action against intracellular RNAs because it forms a tight, irreversible and enzymatically inactive complex with many monomeric RNases. Therefore, dimerization or multimerization could represent a useful strategy for RNases to exert a remarkable cytotoxic activity by evading the interaction with RI by steric hindrance. Indeed, the majority of the mentioned RNases can hetero-dimerize with antibody derivatives, or even homo-dimerize or multimerize, spontaneously or artificially. This can occur through weak interactions or upon introducing covalent bonds. Immuno-RNases, in particular, are fusion proteins representing promising drugs by combining high target specificity with easy delivery in tumors. The results concerning the biological features of many RNases reported in the literature are described and discussed in this review. Furthermore, the activities displayed by some RNases forming oligomeric complexes, the mechanisms driving toward these supramolecular structures, and the biological rebounds connected are analyzed. These aspects are offered with the perspective to suggest possible efficacious therapeutic applications for RNases oligomeric derivatives that could contemporarily lack, or strongly reduce, immunogenicity and other undesired side-effects.

Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development.

BackgroundRecently, thousands of circular RNAs (circRNAs) have been discovered in various tissues and cell types from human, mouse, fruit fly and nematodes. However, expression of circRNAs across mammalian brain development has never been examined.ResultsHere we profile the expression of circRNA in five brain tissues at up to six time-points during fetal porcine development, constituting the first report of circRNA in the brain development of a large animal. An unbiased analysis reveals a highly complex regulation pattern of thousands of circular RNAs, with a distinct spatio-temporal expression profile. The amount and complexity of circRNA expression was most pronounced in cortex at day 60 of gestation. At this time-point we find 4634 unique circRNAs expressed from 2195 genes out of a total of 13,854 expressed genes. Approximately 20 % of the porcine splice sites involved in circRNA production are functionally conserved between mouse and human. Furthermore, we observe that “hot-spot” genes produce multiple circRNA isoforms, which are often differentially expressed across porcine brain development. A global comparison of porcine circRNAs reveals that introns flanking circularized exons are longer than average and more frequently contain proximal complementary SINEs, which potentially can facilitate base pairing between the flanking introns. Finally, we report the first use of RNase R treatment in combination with in situ hybridization to show dynamic subcellular localization of circRNA during development.ConclusionsThese data demonstrate that circRNAs are highly abundant and dynamically expressed in a spatio-temporal manner in porcine fetal brain, suggesting important functions during mammalian brain development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0801-3) contains supplementary material, which is available to authorized users.

Flow cytometry reliability analysis and variations in sugarcane DNA content.

The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 μg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 μg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties.

RNA-Friendly Plasmid Preparation

With the advent of miniprep kits for the preparation of plasmids, we have noticed that many investigators use plasmids prepared by these methods as transcription templates or for cell transfection experiments. This can be quite problematic because miniprep (or maxiprep) DNA can often be contaminated with RNases. Indeed, most DNA preparation kits include pancreatic RNase. This RNase is extremely difficult to inactivate and can contaminate benchtops and pipettors. We highly recommend that plasmid DNAs, especially those that will be used repeatedly, be purified using traditional cesium chloride (CsCl) density gradient methods that do not involve the use of RNase. We have found that the protocol described here yields highly pure, noncontaminated plasmid DNA suitable for in vitro transcription and/or transfection analyses. An added benefit to this procedure is that it will yield enough plasmid DNA for many, many experiments.

Diversity and distribution of the bioactive actinobacterial genus Salinispora from sponges along the Great Barrier Reef

Isolates from the marine actinobacterial genus Salinispora were cultured from marine sponges collected from along the length of the Great Barrier Reef (GBR), Queensland, Australia. Strains of two species of Salinispora, Salinispora arenicola and "Salinispora pacifica", were isolated from GBR sponges Dercitus xanthus, Cinachyrella australiensis and Hyattella intestinalis. Phylogenetic analysis of the 16S rRNA gene sequences of representative strains, selected via BOX-PCR screening, identified previously unreported phylotypes of the species "S.pacifica". The classification of these microdiverse 16S rRNA groups was further confirmed by analysis of the ribonuclease P RNA (RNase P RNA) gene through both phylogenetic and secondary structure analysis. The use of RNase P RNA sequences combined with 16S rRNA sequences allowed distinction of six new intraspecies phylotypes of "S. pacifica" within the geographical area of the GBR alone. One of these new phylotypes possessed a localised regional distribution within the GBR.

Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I

Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.5–50 μg/kg) and DNase I (0.02–2.3 mg/kg) inhibits the development of metastasis to 60–90% and RNase A exerts 30% retardation of tumor growth. Remarkably, the increase in RNase A dose from 50 μg/kg to 10 mg/kg leads to a disappearance of antitumor and antimetastatic effects. Simultaneous treatment of tumor-bearing animals with RNase A and DNase I leads to an additive effect and results in almost total absence of metastases. The use of RNase A as an adjuvant in conjunction with conventional cytostatic cyclophosphamide results in a reliable enhancement of antitumor and antimetastatic effect of the therapy compared with the use of these agents individually. The search for possible molecular mechanism of antimetastatic effect of nucleases showed that daily administration of the enzymes reduced the pathologically increased level of extracellular nucleic acids and increased nuclease activity of the blood plasma of tumor-bearing mice back to the level of healthy animals. Thus, we unequivocally show that the proposed protocol of treatment of tumor-bearing animals with RNase A and DNase I has a general systemic and immunomodulatory effect, leads to a drastic suppression of metastasis development, and in perspective may become an effective component of intensive complex therapy of cancer.

Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G(1) cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

BackgroundThe ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H- RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.ResultsOur findings on cellular RNA and in vitro synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H+ RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection.ConclusionWhile RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.

Use of RNase P for Efficient Preparation of Yeast tRNATyr Transcript and Its Mutants

Because T7 RNA polymerase has a strong preference for particular sequences to initiate transcription, some RNAs having pyrimidine-rich sequences at their 5'-end (yeast tRNA(Tyr), for example) are hardly transcribed by this enzyme. To circumvent this inconvenience, we have developed an efficient method for in vitro preparation of such tRNAs. The RNA of interest is first transcribed as a precursor form that has purine-rich extra sequences at its 5'-end, then processed with RNase P to generate the objective tRNAs. By using this protocol, we were able to prepare easily and efficiently yeast tRNA(Tyr) transcript and its mutants harboring base substitutions within the anticodon loop and/or acceptor stem regions. Aminoacylation analyses of these tRNA transcripts with yeast tyrosyl-tRNA synthetase revealed that the replacement of G34 by C34 (mutation to amber suppressor) severely impaired the aminoacylation, whereas the replacement of the U4:G69 wobble base-pair in the acceptor stem region by C4:G69 normal Watson-Crick type base-pair improved it.

138. Purification of Plasmid DNA on an Anion Exchange Column without the Use of RNase To Process Scale

We report here the use of a simple, straightforward RNase free method for the purification of plasmid DNA on ANX Sepharose 4 FF high sub exploiting the use of compacting agents and salts to improve selectivity, capacity and recovery. We have used generic procedures with standard protocols for alkaline lysis with commonly used buffers. The method was applicable to high and low copy number plasmids generated in different bacterial strains. A combination of isopropanol for compaction of supercoiled plasmid during adsorption and removal of residual RNA with ammonium acetate during a wash step allowed omission of RNA pretreatment. Material generated by this one column procedure was sufficiently pure to allow transfection of cell lines.

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe.

Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.

Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations

A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.

Flexible and frozen sugar-modified nucleic acids--modulation of biological activity through furanose ring dynamics in the antisense strand.

A comparison of carbohydrate modified nucleic acids has identified key structural characteristics in antisense oligonucleotides (AON) that are necessary for sufficient clinical utility, including increased duplex stability towards RNA complements and improved hydrolytic resistance towards general serum and cellular nucleases. As such, the exogenous addition of short, synthetic oligonucleotides can influence cellular RNA metabolism at any or all levels of replication, transcription or translation by tight and specific hybridization with a chosen target and subsequently stop further function at that site. Furthermore, appropriate modification of the sugar residue may prove to be a vital design element in future AONs that operate by promoting enzyme assisted catalytic destruction of the mRNA target. Unfortunately, many of the current AON designs have provided little insight on the particular structural role of the AON towards enzymatic discrimination of the resultant hybrid. The use of RNase H as a cellular vehicle to assist the inhibitory potency of an AON as well as possible ways of enhancing activity in pre-existing antisense candidates are presented. Of the emerging criteria in this aspect, a balance between flexibility and rigidity within the AON appears to be a critical mediator of the RNase H assisted antisense effect. Accordingly, this review describes the conformational features and selected biological attributes of some of the more prominent AON contenders with a focus on the conformational criteria by which ribonuclease H activity is recruited to a particular hybrid target.

Evolutionary relationships among self-incompatibility RNases.

T2-type RNases are responsible for self-pollen recognition and rejection in three distantly related families of flowering plants-the Solanaceae, Scrophulariaceae, and Rosaceae. We used phylogenetic analyses of 67 T2-type RNases together with information on intron number and position to determine whether the use of RNases for self-incompatibility in these families is homologous or convergent. All methods of phylogenetic reconstruction as well as patterns of variation in intron structure find that all self-incompatibility RNases along with non-S genes from only two taxa form a monophyletic clade. Several lines of evidence suggest that the best interpretation of this pattern is homology of self-incompatibility RNases from the Scrophulariaceae, Solanaceae, and Rosaceae. Because the most recent common ancestor of these three families is the ancestor of approximately 75% of dicot families, our results indicate that RNase-based self-incompatibility was the ancestral state in the majority of dicots.

Fungal Extracellular RNases (Review)

Studies of certain fungal extracellular RNases mainly isolated from representatives of the genera Aspergillus and Penicillium are reviewed. The isolation of these enzymes in highly purified states in the 1970s and 1980s strongly stimulated further studies of their structure, functions, and mechanisms of action. This also promoted the use of RNases as catalysts in oligoribonucleotide syntheses and as objects of comparative and evolutionary biochemical and other studies. Results of determinations of the primary, secondary, and spatial structures of guanyl-specific fungal RNases are reviewed. These studies revealed considerable homology within the subfamilies of fungal, bacterial, and actinomycete RNases. Characteristics of the nonspecific RNase Pb2 are considered.

Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications.

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.

The Subsites Structure of Bovine Pancreatic Ribonuclease A Accounts for the Abnormal Kinetic Behavior with Cytidine 2′,3′-Cyclic Phosphate

The kinetics of the hydrolysis of cytidine 2',3'-cyclic phosphate (C>p) to 3'-CMP by ribonuclease A are multiphasic at high substrate concentrations. We have investigated these kinetics by determining 3'-CMP formation both spectrophotometrically and by a highly specific and quantitative chemical sampling method. With the use of RNase A derivatives that lack a functional p2 binding subsite, evidence is presented that the abnormal kinetics with the native enzyme are caused by the sequential binding of the substrate to the several subsites that make up the active site of ribonuclease. The evidence is based on the following points. 1) Some of the unusual features found with native RNase A and C>p as substrate disappear when the derivatives lacking a functional p2 binding subsite are used. 2) The kcat/Km values with oligocytidylic acids of increasing lengths (ending in C>p) show a behavior that parallels the specific velocity with C>p at high concentrations: increase in going from the monomer to the trimer, a decrease from tetramer to hexamer, and then an increase in going to poly(C). 3) Adenosine increases the kcat obtained with a fixed concentration of C>p as substrate. 4) High concentrations of C>p protect the enzyme from digestion with subtilisin, which results in a more compact molecule, implying large substrate concentration-induced conformational changes. The data for the hydrolysis of C>p by RNase A can be fitted to a fifth order polynomial that has been derived from a kinetic scheme based on the sequential binding of several monomeric substrate molecules.

Template-dependent biosynthesis of poly(G)·poly(C) and its antiviral activity in vitro and in vivo

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G)·poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G)·poly(C) and poly(I)·poly(C). Template-dependent poly(G)·poly(C) was similar in its in vitro activity to poly(I)·poly(C) or even surpassed it, whereas the `traditional' poly(G)·poly(C) was only slightly active in vitro. However, `traditional' poly(G)·poly(C) and poly(I)·poly(C) had similar activity in vivo, whereas template-dependent poly(G)·poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.