The role of mammalian ribonucleases (RNases) in cancer

Ribonucleases (RNases) are a group of enzymes that cleave RNAs at phosphodiester bonds resulting in remarkably diverse biological consequences. This review focuses on mammalian RNases that are capable of, or potentially capable of, cleaving messenger RNA (mRNA) as well as other RNAs in cells and play roles in the development of human cancers. The aims of this review are to provide an overview of the roles of currently known mammalian RNases, and the evidence that associate them as regulators of tumor development. The roles of these RNases as oncoproteins and/or tumor suppressors in influencing cell growth, apoptosis, angiogenesis, and other cellular hallmarks of cancer will be presented and discussed. The RNases under discussion include RNases from the conventional mRNA decay pathways, RNases that are activated under cellular stress, RNases from the miRNA pathway, and RNases with multifunctional activity.     

The eight human “canonical” ribonucleases: Molecular diversity, catalytic properties, and special biological actions of the enzyme proteins

Human ribonucleases (RNases) are members of a large superfamily of rapidly evolving homologous proteins. Upon completion of the human genome, eight catalytically active RNases (numbered 1-8) were identified. These structurally distinct RNases, characterized by their various catalytic differences on different RNA substrates, constitute a gene family that appears to be the sole vertebrate-specific enzyme family. Apart from digestion of dietary RNA, a wide variety of biological actions, including neurotoxicity, angiogenesis, immunosuppressivity, and anti-pathogen activity, have been recently reported for almost all members of the family. Recent evolutionary studies suggest that RNases started off in vertebrates as host defence or angiogenic proteins.     

RNase T affects Escherichia coli growth and recovery from metabolic stress.

To determine the essentiality and role of RNase T in RNA metabolism, we constructed an Escherichia coli chromosomal rnt::kan mutation by using gene replacement with a disrupted, plasmid-borne copy of the rnt gene. Cell extracts of a strain with mutations in RNases BN, D, II, and I and an interuppted rnt gene were devoid of RNase T activity, although they retained a low level (less than 10%) of exonucleolytic activity on tRNA-C-C-[14C]A due to two other unidentified RNases. A mutant lacking tRNA nucleotidyltransferase in addition to the aforementioned RNases accumulated only about 5% as much defective tRNA as did RNase T-positive cells, indicating that this RNase is responsible for essentially all tRNA end turnover in E. coli. tRNA from rnt::kan strains displayed a slightly reduced capacity to be aminoacylated, raising the possibility that RNase T may also participate in tRNA processing. Strains devoid of RNase T displayed slower growth rates than did the wild type, and this phenotype was accentuated by the absence of the other exoribonucleases. A strain lacking RNase T and other RNases displayed a normal response to UV irradiation and to the growth of bacteriophages but was severely affected in its ability to recover from a starvation regimen. The data demonstrate that the absence of RNase T affects the normal functioning of E. coli, but it can be compensated for to some degree by the presence of other RNases. Possible roles of RNase T in RNA metabolism are discussed.     

RNase T2 genes from rice and the evolution of secretory ribonucleases in plants

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.     

The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor

Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here we show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which we have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3'-phosphate and 5'-hydroxyl groups, typical of EDTA-resistant degradative RNases. This is the first example of an antitoxin-toxin system in B. subtilis.     

Ribonucleases of different origins with a wide spectrum of medicinal applications

Ribonucleases (RNases) are a type of nucleases that catalyze the degradation of RNA into smaller components. They exist in a wide range of life forms from prokaryotes to eukaryotes. RNase-controlled RNA degradation is a determining factor in the control of gene expression, maturation and turnover, which are further associated with the progression of cancers and infectious diseases. Over the years, RNases purified from multiple origins have drawn increasing attention from medical scientists due to their remarkable antitumor properties. In this review, we present a brief summary of the representative RNases of fungal, bacterial, plant, and animal origins and outline their potential medicinal value in the treatment of tumor and AIDS. Among them, the most clinically promising RNases are mushroom RNases, Binase and Barnase from bacteria, ginseng RNases, and Onconase from frog (Rana pipiens). Fast developing protein engineering of RNases, which display more potent cytotoxic activity on and greater selectivity for malignant cells, has also aroused the interest of researchers. The multiple anti-cancer mechanisms of RNases are also included. To sum up, these inspiring studies unveil a new perspective for RNases as potential therapeutic agents.     

Ribonucleases and angiogenins from fish

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities.     

Inhibitors of ribonucleases

RNases are the most important enzymes of cellular metabolism. They influence gene expression, cell growth and differentiation, participate in cell protection against pathogens and induction of apoptosis. Since intracellular RNases exist mainly in complexes with their inhibitors, the latter are also involved in all above-mentioned processes. The review describes natural protein inhibitors of animal, plant, and bacterial RNases along with synthetic low molecular-weight inhibitors. Special attention is paid to the perspectives of application of RNase inhibitors to therapy of oncological and allergic diseases. Despite wide distribution of RNases and their numerous studies, the number of available natural and synthetic inhibitors of these enzymes remains limited. Creation of highly efficient low-molecular inhibitors of RNase activity of angiogenin and eosinophil-associated RNases, aimed at the therapy of oncological and allergic diseases, still remains quite actual.     

Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes

Human non-secretory neutral ribonucleases (RNases) from kidney,liver and spleen have been purified and characterized. SDS—PAGEindicates that all three RNases are highly purified and haveapparent mol. wts of 17–18 kDa. Kinetic analysis indicatesthat all three RNases have a broad pH optimum centred around6.5, and all three have similar substrate specificities withsignificant preference for RNA and poly(U) when compared topoly(C), poly (A) and poly(G). All of the above data, as wellas immunoblotting data using three polyclonal antibodies (anti-humanliver RNase, anti-human pancreatic RNase, anti-human eosino-phil-derivedneurotoxin), indicate that the three proteins are highly purifiedand are non-secretory RNases (IIN). Further characterizationby cyanogen bromide peptide mapping and extensive lectin blottingindicated no significant differences between the three humanRNases. All three RNases appear to have very similar, if notidentical, protein backbones and all three are glycoproteinswhich are recognized by lectins with specificity for GlcNAc,Fuc and, to a lesser extent, with specificity for Galß(1–4)GlcNAc.No significant tissuespecific differences were found among thethree human non-secretory RNases. lectin blotting non-secretory RNases peptide mapping     

Cytosolic RNase inhibitor only affects RNases with intrinsic cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.     

Ribonucleases with angiogenic and bactericidal activities from the Atlantic salmon

The importance of fish in vertebrate evolution has been better recognized in recent years after the intense work carried out on fish genomics. The recent discovery that fish genomes comprise homologs of ribonucleases, studied before only in tetrapods, and the isolation of ribonucleases from zebrafish have suggested an experimental model for studying fish and vertebrate evolution. Thus, the cDNAs encoding the RNases from the Atlantic salmon were expressed, and the recombinant RNases (Ss-RNase-1 and Ss-RNase-2) were isolated and characterized as both proteins and for their biological activities. Salmon RNases are less active than RNase A in degrading RNA, but are both sensitive to the action of the human cytosolic RNase inhibitor. The two enzymes possess both angiogenic and bactericidal activities. However, catalytically inactivated Ss-RNases do not exert any angiogenic activity, but preserve their full bactericidal activity, which is surprisingly preserved even when the enzyme proteins are fully denatured. Analyses of the conformational stability of the two RNases has revealed that they are as stable as typical RNases of the superfamily, and Ss-RNase-2, the most active as an enzyme, is also the most resistant to thermal and chemical denaturation. The implications of these findings in terms of the evolution of early RNases, in particular of the physiological significance of the angiogenic and bactericidal activities of fish RNases, are analyzed and discussed.     

Discrimination between mammalian RNases H-1 and H-2.

The two principal RNases H in mammalian cells, H-1 and H-2, differ in their responses to sale, divalent metal, and sulfhydryl inhibition. Specific reaction conditions that provide unambiguous discrimination between RNases H-1 and H-2 with only two assays are described. The assays were used for identification in a new purification procedure for RNases H-1 and H-2.     

Self-incompatibility RNases from three plant families: homology or convergence?

In the Rosaceae, Scrophulariaceae, and Solanaceae, the stylar product of the self-incompatibility (S-) locus is an RNase. Using protein sequence data from 34 RNase genes (three fungal RNases, seven angiosperm non-S RNases, 11 Rosaceae S-alleles, three Scrophulariaceae S-alleles, and ten Solanaceae S-alleles) we reconstructed the genealogy of angiosperm RNases using the neighbor joining method and two distance metrics in order to assess whether use of S-RNases in these families is the result of homology or convergence. Four monophyletic groups of angiosperm RNases were found: the S-RNases of each of the three families and a group comprising most of the angiosperm non-S RNases. The S-RNases of the Scrophulariaceae and Solanaceae were found to be homologous but strong inference concerning the homology or convergence of S-RNases from the Rosaceae with those of the other families was not possible because of uncertain placement of both the root and two of the angiosperm non-S RNases. The most recent common ancestor of the Rosaceae and both the Scrophulariaceae and Solanaceae is shared by ~80% of dicot families. If the -RNases of the Rosaceae are homologous to those of the Scrophulariaceae and Solanaceae, then many other dicot families might be expected to share RNases as the mechanism of gametophytic self-incompatibility.     

Zebrafish ribonucleases are bactericidal: implications for the origin of the vertebrate RNase A superfamily

Understanding the evolutionary origin of the ribonuclease (RNase) A superfamily is of great interest because the superfamily is the sole vertebrate-specific enzyme family known to date. Although mammalian RNases have a diverse array of biochemical and physiological functions, the original function of the superfamily at its birth is enigmatic. Such information may be obtained by studying basal lineages of the vertebrate phylogeny and is necessary for discerning how and why this superfamily originated. Here, we clone and characterize 3 RNase genes from the zebrafish, the most basal vertebrate examined for RNases. We report 1) that all the 3 zebrafish RNases are ribonucleolytically active, with one of them having an RNase activity comparable to that of bovine RNase A, the prototype of the superfamily; 2) that 2 zebrafish RNases have prominent expressions in adult liver and gut, whereas the 3rd is expressed in adult eye and heart; and 3) that all 3 RNases have antibacterial activities in vitro. These results, together with the presence of antibacterial and/or antiviral activities in multiple distantly related mammalian RNases, strongly suggest that the superfamily started as a host-defense mechanism in vertebrate evolution.     

Ribonuclease inhibitors

RNases are important enzymes of cell metabolism, influencing gene expression, affecting cell growth and differentiation, and participating in cell defense against pathogens and induction of apoptosis. Since RNases mostly occur in complex with their inhibitors in the cell, the inhibitors also play a role in the above processes. The review considers natural protein RNase inhibitors of animals, plants, and bacteria, as well as synthetic low-molecular-weight inhibitors. Special emphasis is placed on the prospective use of RNase inhibitors in the therapy of cancer and allergy. While RNases are widespread, the number of the available natural and synthetic inhibitors is limited. A pressing problem is to design highly effective low-molecular-weight inhibitors of the RNase activity of angiogenin and eosinophil-associated RNases for anticancer and antiallergy therapy.     

小麦及其近缘物种中小分子量核糖核酸酶的初步分析

应用ＲＮａｓｅ功能胶体系,在小麦及其相关物物中发现１组小分子量核糖核酸酶（ＲＮａｓｅ）。ＲＮａｓｅ的分子量变化范围在６．５～１４ｋＤａ间,低于已报道的大多数植物ＲＮａｓｅ的分子量。小分子量ＲＮａｓｅ在幼苗中大量表达,并且在降解ＲＮＡ底物时,随着缓冲液ＰＨ值及离子浓度的不同而有所变化。在休眠及发芽泪科种 现几种可能不同的小分子量ＲＮａｓｅ。在发芽过程中,其中２种ＲＮａｓｅ的少变化不明显,而另外２种Ｒ     

Ribonucleases as antiviral agents

Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. The review discusses the well-known RNases that possess established antiviral effects, including both intracellular RNases (RNase L, MCPIP1 protein, and eosinophil-associated RNases) and exogenous RNases (RNase A, BS-RNase, onconase, binase, and synthetic RNases). Attention is paid to two important, but not always obligatory, aspects of molecules of RNases that have antiviral properties, i.e., catalytic activity and ability to dimerize. The hypothetic scheme of virus elimination by exogenous RNases that reflects possible types of interaction of viruses and RNases with a cell is proposed. The evidence for RNases as classical components of immune defense and thus perspective agents for the development of new antiviral therapeutics is proposed.