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PubMed1996

Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family.

The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our ongoing efforts to trace the evolutionary history of the ribonuclease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino acid polypeptide with eight cysteines and histidine and lysine residues corresponding to those found in the active site of the prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous sequences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human tissues tested, with lung representing the most abundant source. At the cellular level, transcripts encoding RNase k6 were detected in normal human monocytes and neutrophils (but not in eosinophils) suggesting a role for this ribonuclease in host defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence identity; slight cross-reactivity between RNase k6 and EDN was observed on Western blots probed with polyclonal anti-EDN antiserum. The catalytic constants determined, Km = 5.0 microM and Kcat = 0.13 s-1, indicate that recombinant RNase k6 has approximately 40-fold less ribonuclease activity than recombinant EDN. The identification and characterization of RNase k6 has extended the ribonuclease gene family and suggests the possibility that there are others awaiting discovery.

Amino Acid Sequence↗

PubMed1998

RNase L dimerization in a mammalian two-hybrid system in response to 2',5'-oligoadenylates.

RNase L, a key enzyme in the anti-viral activity of interferons, requires activation by 2',5'-linked oligoadenylates (2-5A) to cleave viral and cellular single-stranded RNA. Here we demonstrate that 2-5A causes formation of stable dimers of RNase L in intact human cells as measured with a mammalian two-hybrid system. Hybrid proteins consisting of the GAL4 DNA binding domain fused to RNase L and the VP16 transactivation domain fused to RNase L were able to associate and drive transcription of a reporter gene, but only after cells were transfected with 2-5A. Several functional forms of 2-5A, such as p3A2'p5'A2'p5'A, were capable of activating transcription in human HeLa cells. In contrast, p3A2'p5'A, which can neither activate nor dimerize RNase L, did not induce gene expression. Evidence for the involvement of the C-terminal region of RNase L in dimerization was obtained by expressing truncated forms of RNase L. These findings describe a convenient, high-throughput screening method for RNase L activators which could lead to the discovery of novel anti-viral and anti-cancer agents.

Adenine Nucleotides↗

PubMed1998

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.

Amino Acid Sequence↗

PubMed1998

Oxidative refolding of bovine pancreatic RNases A and B promoted by Asn-glycans.

It was previously revealed [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477] that both intra- and extramolecular high-mannose type Asn-glycans promote the renaturation of reductively denatured bovine pancreatic RNases A and B under oxidation conditions. To characterize the conformational changes of the polypeptides during the renaturation promoted by the intramolecular Asn-glycans, RNase B was compared with its nonglycosylated form, RNase A, as to the features of the regeneration from their reductively denatured species under Cu2+-catalyzed oxidation conditions. The refolding intermediates of RNase B, as compared with those of RNase A, seemed to contain much less impaired disulfide linkages. In agreement with this finding, the proper refolding of RNase B was much faster than that of RNase A, as revealed by the intrinsic fluorescence and 1-anilino-8-naphthalenesulfonate binding of the refolding intermediates. Such a promoting effect was also observed for extramolecular Asn-glycans of the complex as well as of the high-mannose type. In contrast, common mono-, oligo-, and polysaccharides, but not yeast mannan, exhibited much lower stimulatory effects on the oxidative refolding of RNase A.

Anilino Naphthalenesulfonates↗

PubMed1995

Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus.

In order to elucidate on the mechanism of action of RNase Rh from Rhizopus niveus, we investigated the role of Lys108, which is conserved among the RNase T2 family RNases except for two cases. The RNase activities of Lys108 mutant RNases, RNase RNAP K108R and K108L, are about 33.5 and 3.1% of that of the wild type enzyme, respectively. The relative rates of cleavage of dinucleoside phosphates by these two mutant enzymes were comparable to those with RNA as a substrate. The kinetic parameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, G, U, and C) were measured. The data indicated that the Km values of the two mutant enzymes are similar to those of the wild-type enzyme. The rates of release of the four nucleotides from RNA by digestion with the mutant enzymes were in the order A > G > U > C, which is qualitatively the same as that of the wild-type enzyme. From these data, we concluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispensable for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition state as proposed in the case of Lys41 in RNase A, or may polarize the phosphate moiety of the substrate.

Amino Acid Sequence↗

PubMed1981

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2.

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.

Adenosine Monophosphate↗

PubMed2000

Comparative sequence analyses of the ribonuclease P (RNase P) RNA genes from LL-2,6-diaminopimelic acid-containing actinomycetes.

Ribonuclease P (RNase P) RNA genes of LL-2,6-diaminopimelic acid (LL-A2pm)-containing actinomycetes, except Streptomyces species, were sequenced after PCR-amplification and cloning. By using the sequence data, the relationships between species within genera and the relationships between taxa above genus level were investigated and the usefulness of the RNase P RNA gene as another phylogenetic marker was evaluated. RNase P RNA gene sequences of all strains used in this study contained relatively conserved regions along with highly variable regions. The mean RNase P RNA gene similarity value was approximately 82 +/- 18% and the mean RNase P RNA gene similarity value when gaps were included was approximately 76 +/- 24%. The nucleotide similarities between the RNase P RNA genes of different strains were mostly fewer than the 16S rDNA similarities. The RNase P RNA gene was more useful than 16S rDNA for clearly differentiating the relationships between species belonging to a genus and the relationships between some genera. However, nucleotide sequences of RNase P RNA genes were not necessarily appropriate for comparisons at all taxonomic levels (such as those between species, between genera and between families).

Actinomycetales↗

PubMed1990

RNase III-dependent hydrolysis of lambda cII-O gene mRNA mediated by lambda OOP antisense RNA.

The 77-nucleotide OOP antisense RNA of bacteriophage lambda complements lambda cII-O mRNA in a region that includes 55 nucleotides at the 3' end of the cII gene and 22 nucleotides in the intercistronic region between the cII and O genes. OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression by approximately 100-fold through an RNase III-dependent mechanism. Using primer extension analysis of cellular RNA isolated from an induced lambda lysogen that contains an OOP DNA plasmid, we have identified a cleavage site in cII-O mRNA within the region of complementarity with OOP RNA, at 13 nucleotides from the 3' end of that region. Ribonuclease protection experiments demonstrate that almost all cII-O mRNA in this overlap region is cleaved when OOP RNA is overproduced in RNase III+ cells but not in RNase III- cells. RNA fragments are detected that extend into the O gene from the cleavage sites, while the sister fragments that extend into the cII gene cannot be detected and must be eliminated by additional hydrolytic events. Differences in levels of uncleaved mRNA between RNase III+ and RNase III- cells are much less at several hundred nucleotides to either side of the target region. An alternate OOP RNA-dependent hydrolytic process occurs in RNase III- cells that results in cleavages in one of two regions, one close to the cleavage site observed in RNase III+ cells, and the second several nucleotides beyond the end of the complementary region between OOP RNA and cII-O mRNA. In this latter case, the fragments that extend into the cII gene are stable, while the sister O gene fragments are destroyed, in direct contrast to the RNase III-dependent process.

Bacteriophage lambda↗

PubMed1999

Production of an S RNase with dual specificity suggests a novel hypothesis for the generation of new S alleles.

Gametophytic self-incompatibility in plants involves rejection of pollen when pistil and pollen share the same allele at the S locus. This locus is highly multiallelic, but the mechanism by which new functional S alleles are generated in nature has not been determined and remains one of the most intriguing conceptual barriers to a full understanding of self-incompatibility. The S(11) and S(13) RNases of Solanum chacoense differ by only 10 amino acids, but they are phenotypically distinct (i.e., they reject either S(11) or S(13) pollen, respectively). These RNases are thus ideally suited for a dissection of the elements involved in recognition specificity. We have previously found that the modification of four amino acid residues in the S(11) RNase to match those in the S(13) RNase was sufficient to completely replace the S(11) phenotype with the S(13) phenotype. We now show that an S(11) RNase in which only three amino acid residues were modified to match those in the S(13) RNase displays the unprecedented property of dual specificity (i.e., the simultaneous rejection of both S(11) and S(13) pollen). Thus, S(12)S(14) plants expressing this hybrid S RNase rejected S(11), S(12), S(13), and S(14) pollen yet allowed S(15) pollen to pass freely. Surprisingly, only a single base pair differs between the dual-specific S allele and a monospecific S(13) allele. Dual-specific S RNases represent a previously unsuspected category of S alleles. We propose that dual-specific alleles play a critical role in establishing novel S alleles, because the plants harboring them could maintain their old recognition phenotype while acquiring a new one.

Alleles↗

PubMed1993

S-RNase gene of Nicotiana alata is expressed in developing pollen.

In the solanaceous plant Nicotiana alata, self-incompatibility is controlled by a single, multiallelic locus (S locus) expressed in both pollen and pistil. Previously, we have shown cosegregation between alleles of the S locus and alleles of a gene that encodes a glycoprotein with ribonuclease activity (S-RNase). Furthermore, expression of the S-RNase gene is apparently confined to the pistil and is correlated with the onset of self-incompatibility. In this paper, we report that the S-RNase gene is also expressed at low levels in developing pollen. A transcript in developing pollen hybridized to a cDNA encoding the S2-RNase allele of the parent plant and did not hybridize to cDNAs encoding other S-RNase alleles. Two cDNAs for the S2-RNase were cloned from a library derived from anthers of a plant homozygous for the S2 allele and both corresponded to the coding sequence of the S2-RNase. The product of the S-RNase gene was detected by immunocytochemistry in the intine of mature, hydrated pollen grains. These results are interpreted in the light of current knowledge of the structure of the S locus.

Base Sequence↗

PubMed2001

Crystallization and preliminary X-ray crystallographic analysis of RNase HIII from Bacillus subtilis.

The genome of Bacillus subtilis contains three different genes encoding RNase H homologs: RNases HI, HII and HIII. RNase HIII from B. subtilis degrades RNA in RNA-DNA hybrids in an Mg(2+)-dependent manner like Escherichia coli RNase HI. However, they belong to different classes; the former belongs to the 'class II' or 'large' RNase H family, while the latter belongs to the 'class I' or 'small' RNase H family. RNase HIII of B. subtilis has been overexpressed in E. coli and crystallized at 296 K using sodium formate as a precipitant. The native X-ray diffraction data have been collected to 2.8 A resolution using synchrotron radiation. The crystals are hexagonal, belonging to the space group P6(1), with unit-cell parameters a = b = 86.89, c = 214.49 A, alpha = beta = 90.0, gamma = 120.0 degrees. A self-rotation function calculation indicated the presence of two monomers of the recombinant RNase HIII in the crystallographic asymmetric unit, giving a V(M) of 3.43 A(3) Da(-1) and a solvent content of 64.2%.

Bacillus subtilis↗

PubMed2001

Three-dimensional structure of human RNase 1 delta N7 at 1.9 A resolution.

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

Amino Acid Sequence↗

PubMed2006

RNase Z in Escherichia coli plays a significant role in mRNA decay.

Genetic and biochemical analysis of RNase Z in eukaryotes, such as Arabadopsis thaliana, and prokaryotes like Bacillus subtilis have demonstrated that this endoribonuclease is essential for the maturation of tRNA precursors that do not contain a chromosomally encoded CCA determinant. As all Escherichia coli tRNA transcripts have chromosomally encoded CCA determinants, the function of its putative RNase Z homologue, the product of the elaC gene, is not clear. Here we demonstrate that the E. coli ElaC protein (RNase Z) endonucleolytically processes B. subtilis tRNA precursors lacking a CCA determinant both in vivo and in vitro. More importantly, E. coli RNase Z plays a significant role in mRNA decay, a previously unidentified activity for the enzyme. The purified RNase Z protein cleaves the rpsT mRNA at locations distinct from those obtained with RNase E. As expected, under physiological conditions E. coli and B. subtilis tRNA precursors containing a CCA determinant are not substrates. These results suggest a potentially important new role for the RNase Z family of proteins in RNA metabolism, particularly in organisms lacking RNase E.

Bacillus subtilis↗

PubMed1985

Regulation of ppp(A2'p)nA-dependent RNase levels during interferon treatment and cell differentiation.

The intracellular effector oligonucleotides ppp(A2'p)nA (n = 2- greater than or equal to 4) regulate the breakdown of RNA by activating ppp(A2'p)nA-dependent RNase. Cellular levels of this RNase were demonstrated to be regulated during differentiation of murine embryonal carcinoma cells. An induction of this RNase by interferon was demonstrated in each of three differentiated cell types (F9 clone 9, PYS, and PSA 5E) by analyzing rRNA breakdown following the introduction of ppp(A2'p)nA into the intact cells. In contrast, in three undifferentiated embryonal carcinoma cell lines (F9, PC13 clone 5, and Nulli 2A) there was little if any ppp(A2'p)nA-dependent RNase either with or without interferon pretreatment. These results were confirmed by affinity labeling of the RNase in cell-free systems. Addition of the proteinase inhibitor, leupeptin, to the cell lysis buffer was necessary to stabilize the RNase against cleavage to discrete breakdown products. Moreover, during differentiation of PC13 clone 5 cells by retinoic acid and N6,O2'-dibutyryl-adenosine 3',5'-monophosphate there was a gradual induction of ppp(A2'p)nA-dependent RNase. The expression of this RNase is, therefore, greatly enhanced during cell differentiation. In addition, the double-stranded-RNA-dependent protein kinase was investigated and was found to be interferon-inducible in all of the cell lines regardless of the state of cell differentiation.

Affinity Labels↗

PubMed1987

On the interaction of bovine seminal RNase with actin in vitro.

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.

Actins↗

PubMed1992

Evidence that axonal tRNAs are resistant to RNase and ATPase and can be aminoacylated in the absence of exogenous ATP.

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.

Adenosine Triphosphatases↗

PubMed2006

The role of electrostatic interactions in the antitumor activity of dimeric RNases.

The cytotoxic action of some ribonucleases homologous to bovine pancreatic RNase A, the superfamily prototype, has interested and intrigued investigators. Their ribonucleolytic activity is essential for their cytotoxic action, and their target RNA is in the cytosol. It has been proposed that the cytosolic RNase inhibitor (cRI) plays a major role in determining the ability of an RNase to be cytotoxic. However, to interact with cRI RNases must reach the cytosol, and cross intracellular membranes. To investigate the interactions of cytotoxic RNases with membranes, cytotoxic dimeric RNases resistant, or considered to be resistant to cRI, were assayed for their effects on negatively charged membranes. Furthermore, we analyzed the electrostatic interaction energy of the RNases complexed in silico with a model membrane. The results of this study suggest that close correlations can be recognized between the cytotoxic action of a dimeric RNase and its ability to complex and destabilize negatively charged membranes.

Animals↗

PubMed2006

RNase E maintenance of proper FtsZ/FtsA ratio required for nonfilamentous growth of Escherichia coli cells but not for colony-forming ability.

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Culture Media↗