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PubMed1998

Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types.

Human extracellular ribonucleases (RNase), together with other members of the mammalian RNase A superfamily, can be classified into four different RNase families on the basis of their structural, catalytic and/or biological properties. Their occurrence and main distinctive features have been described, and the information available on their catalytic properties has been analysed and discussed in comparison with those of other animal RNases. On the basis of some results obtained with various single- and double-stranded polyribonucleotides, it has been proposed that while pancreatic-type (pt) RNases could be defined as single-strand/pyrimidine 'preferring' ribonucleases, mammalian nonpancreatic-type (npt) RNases may be referred to as single-strand/pyrimidine 'specific' ribonucleases. In addition, some data concerning human nptRNases may support the suggestion [Cuchillo et al. (1993) FEBS Lett. 333: 207-210] that the enzyme 'ribonuclease' should be reclassified as 'transferase'.

Amino Acid Sequence↗
PubMed2000

Analysis and origins of the human and mouse RNase L genes: mediators of interferon action.

The 2',5'-oligoadenylate-activated enzyme, RNase L, is an endoribonuclease implicated in the antiviral and apoptotic activities of interferons. To probe the genetics of the 2-5A system, the human and mouse genes were cloned, characterized, and compared. The first coding exon of both genes encodes the regulatory regions of RNase L, 67-70% of the proteins including nine ankyrin repeats, the 2-5A binding domain, and several protein kinase homology motifs. In contrast, the coding sequence for the ribonuclease domain in the mouse and human gene is divided among three exons. The transcriptional start site of the human RNase L gene was located in noncoding exon I by primer extension analysis. A complete coding sequence of mouse RNase L was obtained revealing a 735-amino acid protein with 64% identity to human RNase L. A hypothesis is presented concerning the evolutionary relationship of RNase L to both an ankyrin repeat protein kinase and the kinase-endoribonuclease. IRE1, that mediates the unfolded protein response.

5' Untranslated Regions↗
PubMed2006

Detection and transcript expression of S-RNase gene associated with self-incompatibility in apricot (Prunus armeniaca L.).

The identity and expression of S-RNase genotypes in the self-compatible (SC) apricot cultivar 'Katy' and the self-incompatible (SI) cultivar 'Xinshiji' were examined. We used allele specific polymerase chain reaction (AS-PCR) and designated the alleles in 'Katy' and 'Xinshiji' as S(8)Sc and S(9)S(10), respectively. The S-RNase gene was expressed in style at the balloon stage in both genotypes. Using real-time fluorescence quantification RT-PCR technology (FQRT-PCR), spatio-temporal expression patterns of S-RNase gene between 'Katy' and 'Xinshiji' were compared. The results revealed that the expression of the S-RNase gene in 'Katy' and 'Xinshiji' were different. The transcript abundance was distinctly diverse at the key stage (i.e., at 24 h after self-pollination) in both genotypes, and was greater in 'Xinshiji' (SI) than 'Katy' (SC). In addition, the abundance of the S-RNase transcript was higher in upper-half of style than in the lower-half of style or in the ovary. In the SI cultivar 'Xinshiji', the expression of S-RNase reminded a relatively high level after cross-pollination, but it dropped continuously after self-pollination and un-pollination.

Alleles↗
PubMed2004

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes.

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S -locusan S-RNase and a recently described pollen expressed S -haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21 kb in total of the S -locus region in 3 different apricot S -haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunus species, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-box and the S-RNase genes was determined exactly in the S2-haplotype (2.9 kb) and inferred approximately in the S 1-haplotype (< 49 kb) confirming that these genes are linked. Sequence analysis of the 5' flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S1, S2 and S4) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB1, SFB2 and SFB4) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S1- and S2-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB1 and SFB2 are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.

5' Flanking Region↗
PubMed1996

RNase E can inhibit the decay of some degradation intermediates: degradation of Desulfovibrio vulgaris cytochrome c3 mRNA in E coli.

In Escherichia coli, ribonuclease E (RNase E) is a key endonuclease in mRNA decay. We have analysed the role of E coli RNase E on the degradation of a heterologous cytochrome c3 (cyc) mRNA from Desulfovibrio vulgaris Hildenborough. The decay of the cyc transcript in wild-type and mutant E coli cells was followed and the degradation intermediates analysed by Northern blotting and S1 protection analysis. The half-life of total cyc mRNA intermediates was increased in the RNase E mutant. A number of degradation intermediates were stabilised, and new species arose. However, some species decayed faster in the met5 mutant at the non-permissive temperature, suggesting that RNase E might inhibit their degradation. The results indicate that RNase E is involved in cyc mRNA degradation, and, interestingly, decay of certain intermediates could be reduced by this enzyme activity. This may suggest a functional interaction between RNase E and exonucleases, like polynucleotide phosphorylase.

Blotting, Northern↗
PubMed2003

Cooperative regulation for Okazaki fragment processing by RNase HII and FEN-1 purified from a hyperthermophilic archaeon, Pyrococcus furiosus.

A reconstitution system that recapitulates the processing of Okazaki-primer RNA was established by the heat-stable recombinant enzymes RNase HII and FEN-1 (termed Pf-RNase HII and Pf-FEN-1, respectively) prepared from a hyperthermophilic archaeon, Pyrococcus furiosus. A 35-mer RNA-DNA/DNA hybrid substrate mimicking an Okazaki fragment was used to investigate the properties of the processing reaction in vitro at 50 degrees C. Pf-RNase HII endonucleolytically cleaves the RNA primer region, but does not cut the junction between RNA and DNA. Removal of the RNA of the RNA-DNA junction was brought about by Pf-FEN-1 after Pf-RNase HII digestion. In the presence of 0.25-5mM MnCl(2), Pf-FEN-1 alone weakly cleaved the junction. The addition of Pf-RNase HII to the reaction mixture increased removal efficiency and optimal Pf-FEN-1 activity was achieved at an equal amount of the two enzymes. These results indicate that there are at least two steps in the degradation of primer RNA requiring a step-specific enzyme. It is likely that Pf-RNase HII and Pf-FEN-1 cooperatively process Okazaki fragment during lagging-strand DNA replication.

Base Sequence↗
PubMed2004

Molecular requirements for duplex recognition and cleavage by eukaryotic RNase III: discovery of an RNA-dependent DNA cleavage activity of yeast Rnt1p.

Members of the double-stranded RNA (dsRNA) specific RNase III family are known to use a conserved dsRNA-binding domain (dsRBD) to distinguish RNA A-form helices from DNA B-form ones, however, the basis of this selectivity and its effect on cleavage specificity remain unknown. Here, we directly examine the molecular requirements for dsRNA recognition and cleavage by the budding yeast RNase III (Rnt1p), and compare it to both bacterial RNase III and fission yeast RNase III (Pac1). We synthesized substrates with either chemically modified nucleotides near the cleavage sites, or with different DNA/RNA combinations, and investigated their binding and cleavage by Rnt1p. Substitution for the ribonucleotide vicinal to the scissile phosphodiester linkage with 2'-deoxy-2'-fluoro-beta-d-ribose (2' F-RNA), a deoxyribonucleotide, or a 2'-O-methylribonucleotide permitted cleavage by Rnt1p, while the introduction of a 2', 5'-phosphodiester linkage permitted binding, but not cleavage. This indicates that the position of the phosphodiester link with respect to the nuclease domain, and not the 2'-OH group, is critical for cleavage by Rnt1p. Surprisingly, Rnt1p bound to a DNA helix capped with an NGNN tetraribonucleotide loop indicating that the binding of at least one member of the RNase III family is not restricted to RNA. The results also suggest that the dsRBD may accommodate B-form DNA duplexes. Interestingly, Rnt1p, but not Pac1 nor bacterial RNase III, cleaved the DNA strand of a DNA/RNA hybrid, indicating that A-form RNA helix is not essential for cleavage by Rnt1p. In contrast, RNA/DNA hybrids bound to, but were not cleaved by Rnt1p, underscoring the critical role for the nucleotide located at 3' end of the tetraloop and suggesting an asymmetrical mode of substrate recognition. In cell extracts, the native enzyme effectively cleaved the DNA/RNA hybrid, indicating much broader Rnt1p substrate specificity than previously thought. The discovery of this novel RNA-dependent deoxyribonuclease activity has potential implications in devising new antiviral strategies that target actively transcribed DNA.

Bacterial Proteins↗
PubMed2005

An important role for RNase R in mRNA decay.

mRNA decay is a major determinant of gene expression. In Escherichia coli, message degradation initiates with an endoribonucleolytic cleavage followed by exoribonuclease digestion to generate 5'-mononucleotides. Although the 3' to 5' processive exoribonucleases, PNPase and RNase II, have long been considered to be mediators of this digestion, we show here that another enzyme, RNase R, also participates in the process. RNase R is particularly important for removing mRNA fragments with extensive secondary structure, such as those derived from the many mRNAs that contain REP elements. In the absence of RNase R and PNPase, REP-containing fragments accumulate to high levels. RNase R is unusual among exoribonucleases in that, by itself, it can digest through extensive secondary structure provided that a single-stranded binding region, such as a poly(A) tail, is present. These data demonstrate that RNase R, which is widespread in prokaryotes and eukaryotes, is an important participant in mRNA decay.

Base Sequence↗
PubMed2005

Crystal structure of Escherichia coli RNase D, an exoribonuclease involved in structured RNA processing.

RNase D (RND) is one of seven exoribonucleases identified in Escherichia coli. RNase D has homologs in many eubacteria and eukaryotes, and has been shown to contribute to the 3' maturation of several stable RNAs. Here, we report the 1.6 A resolution crystal structure of E. coli RNase D. The conserved DEDD residues of RNase D fold into an arrangement very similar to the Klenow fragment exonuclease domain. Besides the catalytic domain, RNase D also contains two structurally similar alpha-helical domains with no discernible sequence homology between them. These closely resemble the HRDC domain previously seen in RecQ-family helicases and several other proteins acting on nucleic acids. More interestingly, the DEDD catalytic domain and the two helical domains come together to form a ring-shaped structure. The ring-shaped architecture of E. coli RNase D and the HRDC domains likely play a major role in determining the substrate specificity of this exoribonuclease.

Amino Acid Motifs↗
PubMed2006

Herpes simplex virus type 2-mediated disease is reduced in mice lacking RNase L.

RNase L helps mediate the antiviral state induced by type I interferons (IFNalphabeta). Although herpes simplex virus (HSV) encodes inhibitors of the IFNalphabeta-induced antiviral response, the IFNalphabeta system serves the body as a first line of defense against HSV. We investigated whether RNase L limits HSV-2 replication and virulence. RNaseL(-/-) and wild-type C57BL/6 mice were infected intravaginally with HSV-2 strain 333. Although initial replication in the genital epithelium was similar, mice lacking RNase L developed less severe genital and neurologic disease than wild-type mice, survived longer, and contained lower viral titers in the nervous system. CD4(+) T cell infiltration into the genital tract and spinal cord of RNase L(-/-) mice was reduced, suggesting that a restricted inflammatory response may account for reduction in disease. Thus, RNase L does not play a significant role in control of HSV-2 infection in vivo; instead, RNase L may regulate aspects of the inflammatory response that contribute to disease.

Animals↗
PubMed2003

Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme.

Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor. Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P. The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined. However, an understanding of the structure of the RNase P holoenzyme (i.e. the ribonucleoprotein complex) is lacking. We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P. The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate. These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.

Amino Acid Sequence↗
PubMed2001

A critical survey of the structure-function of the antisense oligo/RNA heteroduplex as substrate for RNase H.

The aim of this review is to draw a correlation between the structure of the DNA/RNA hybrid and its properties as a substrate for the RNase H, as well as to point the crucial structural requirements for the modified AONs to preserve their RNase H potency. The review is divided into the following parts: (1) mechanistic considerations, (2) target RNA folding-AON folding-RNase H assistance in AON/RNA hybrid formation, (3) carbohydrate modifications, (4) backbone modifications, (5) base modifications, (6) conjugated AONs, (7) importance of the tethered chromophore in AON for the AON/RNA hybrid interactions with the RNase H. The structural changes in the AON/RNA hybrid duplexes brought by different modifications of the sugar, backbone or base in the antisense strand, and the effect of these changes on the RNase H recognition of the modified substrates have been addressed. Only those AON modifications and the corresponding AON/RNA hybrids, which have been structurally characterized by spectroscopic means and functionally analyzed by their ability to elicit RNase H potency in comparison with the native counterpart have been presented here.

Carbohydrates↗
PubMed2000

Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes.

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.

Base Sequence↗
PubMed2003

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis.

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.

Animals↗
PubMed1997

Cloning and cytotoxicity of a human pancreatic RNase immunofusion.

BACKGROUND: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. OBJECTIVES: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. STUDY DESIGN: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. RESULTS: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. CONCLUSION: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Animals↗
PubMed1998

Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P.

Human RNase P recognizes a small model substrate consisting of only the 5' leader sequence, aminoacyl acceptor stem, and T stem and loop of a tRNA precursor. It was demonstrated here that a bimolecular construct in which the T loop is opened between G57 and A58 (tRNA numbering system) is still processed by RNase P. The strand that is cleaved can be considered the target RNA, whereas the other strand serves as an external guide sequence (EGS). The nucleotides corresponding to nt 58-60 in the T loop could be deleted without affecting cleavage of the substrate. Thus, the complete T loop can be replaced by the single-stranded sequence UUCG or UUCA (nt 55-57 in the T loop). The four nucleotides UUCR possibly form a structure that resembles the uridine turn in the T loop of tRNA. Because recognition by RNase P is independent of the helical sequence, this motif can be used for targeting RNA molecules for EGS-directed cleavage by human RNase P. Chemically modified EGSs with 2'-O-methyl groups also showed activity in inducing RNase P cleavage. Several 13-mer EGSs targeted to the 2.1-kb surface antigen mRNA of hepatitis B virus (HBV) were designed and tested using a co-transcriptional cleavage assay with a 2.1-kb HBV transcript. Some of the new EGSs were capable of inducing cleavage of the HBV RNA by RNase P.

Base Sequence↗
PubMed1991

Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase.

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.

Blotting, Western↗
PubMed1991

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Antiviral Agents↗