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PubMed2002

PEG chains increase aspermatogenic and antitumor activity of RNase A and BS-RNase enzymes.

RNase A (bovine pancreatic ribonuclease) and BS-RNase (bovine seminal ribonuclease) are monomeric and dimeric enzymes, respectively, with aspermatogenic and antitumor activities. While the aspermatogenic and, in some experimental situations, the antitumor effects of the RNase A are only minor, the activity of BS-RNase in these phenomena is very significant. These differences can be annulled by means of conjugation of the enzymes with PEG (polyethylene glycol) chains. Aspermatogenic activity was studied histologically following subcutaneous injections of RNase A and BS-RNase conjugates in ICR mice, and the antitumor activity in athymic nude mice with growing human melanoma with i.p. injection of these conjugated ribonucleases. The experiments proved that RNase A, when conjugated to PEG, produced identical aspermatogenic and antitumour effects as BS-RNase conjugated to this polymer. Immunogenicity of RNase A and BS-RNase did not change substantially after the conjugation with PEG polymers. Binding of produced antibodies to both ribonucleases attached to PEG, however, was substantially reduced.

Animals↗
PubMed2002

Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins.

Although archaeal RNase P RNAs are similar in both sequence and structure to those of Bacteria rather than eukaryotes, and heterologous reconstitution between the Bacillus subtilis RNase P protein and some archaeal RNase P RNAs has been demonstrated, no archaeal protein sequences with similarity to any known bacterial RNase P protein subunit have been identified, and the density of Methanothermobacter thermoautotrophicus RNase P in Cs2SO4 (1.42 g/mL) is inconsistent with a single small bacterial-like protein subunit. Four hypothetical open reading frames (MTH11, MTH687, MTH688, and MTH1618) were identified in the genome of M. thermoautotrophicus that have sequence similarity to four of the nine Saccharomyces cerevisiae RNase P protein subunits: Pop4p, Pop5p, Rpp1p, and Rpr2p, respectively. Polyclonal antisera generated to recombinant Mth11p, Mth687p, Mth688p, and Mth1618p each recognized a protein of the predicted molecular weight in western blots of partially purified M. thermoautotrophicus RNase P, and immunoprecipitated RNase P activity from the same partially purified preparation. RNase P in Archaea is therefore composed of an RNA subunit similar to bacterial RNase P RNA and multiple protein subunits similar to those in the eukaryotic nucleus.

Amino Acid Sequence↗
PubMed1981

Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I).

The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.

Avian Myeloblastosis Virus↗
PubMed2004

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.

Animals↗
PubMed1999

hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes.

RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.

Amino Acid Sequence↗
PubMed2003

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily.

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine-lysine- histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3's cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M.

The endoribonuclease, RNase I, was purified from the periplasm of Escherichia coli. Based on PAGE, it has molecular mass of approximately 27 kDa with a migration rate indistinguishable from that of the recently reported RNase M from E. coli. The amino acid sequence of the two enzymes must be very similar based on two-dimensional mapping of their tryptic peptides and suggests either a post-transcriptional modification to yield different proteins from the same gene or evolution of two genes by gene duplication. However, while RNase I could degrade each of the four ribonucleotide homopolymers, only poly(U) or poly(C) were good substrates for RNase M with possibly some hydrolysis of poly(A). The reaction rate for poly(C) hydrolysis with RNase M was about ten times faster than for poly(U), while for RNase I the rates were about equal. Besides differences in specificity, RNase M was only located in the spheroplasts while RNase I found in the periplasm of growing cells. In terms of function, RNase I is known to cause degradation of rRNA during periods of stress or non-growth, whereas it has been proposed that RNase M is the endonuclease for mRNA degradation in growing cells.

Amino Acid Sequence↗
PubMed2001

Escherichia coli RNase M is a multiply altered form of RNase I.

RNase M, an enzyme previously purified to homogeneity from Escherichia coli, was suggested to be the RNase responsible for mRNA degradation in this bacterium. Although related to the endoribonuclease, RNase I, its distinct properties led to the conclusion that RNase M was a second, low molecular mass, broad specificity endoribonuclease present in E. coli. However, based on sequence analysis, southern hybridization, and enzyme activity, we show that RNase M is, in fact, a multiply altered form of RNase I. In addition to three amino acid substitutions that confer the properties of RNase M on the mutated RNase I, the protein is synthesized from an rna gene that contains a UGA nonsense codon at position 5, apparently as a result of a low level of readthrough. We also suggest that RNase M is just one of several previously described endoribonuclease activities that are actually manifestations of RNase I.

Endoribonucleases↗
PubMed1992

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.

Escherichia coli↗
PubMed1998

Identification of regions in which positive selection may operate in S-RNase of Rosaceae: implication for S-allele-specific recognition sites in S-RNase.

A stylar S-RNase is associated with gametophytic self-incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S-RNase is responsible for S-allele-specific recognition in the self-incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (dS) and non-synonymous (dN) substitutions in rosaceous S-RNases detected four regions with an excess of dN over dS in which positive selection may operate (PS regions). The topology of the secondary structure of the S-RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S-RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S-RNases the PS regions also form two sites and are candidates for the recognition sites for S-allele-specific discrimination.

Alleles↗
PubMed2000

Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae.

RNase MRP and RNase P are both ribonucleoprotein enzymes performing endonucleolytic cleavage of RNA. RNase MRP cleaves at a specific site in the precursor-rRNA transcript to initiate processing of the 5.8S rRNA. RNase P cleaves precursor tRNAs to create the 5' end of the mature tRNAs. In spite of their different specificities, the two RNases have significant structural similarities. For example, the two enzymes in Saccharomyces cerevisiae share eight protein subunits; only one protein is unique to each enzyme. The RNA components of the two nucleases also show striking secondary-structure similarity. To begin to characterize the role of the RNA subunits in enzyme function and substrate specificity, we swapped two hairpin structures (MRP3 and P3) between RNase MRP RNA and RNase P RNA of S. cerevisiae. The hairpins in the two enzymes could be exchanged without loss of function or specificity. On the other hand, when the MRP3 hairpin in RNase MRP of S. cerevisiae was replaced with the corresponding hairpin from the RNA of Schizosaccharomyces pombe or human RNase MRP, no functional enzyme was assembled. We propose that the MRP3 and P3 hairpins in S. cerevisiae perform similar functions and have coevolved to maintain common features that are different from those of MRP3 and P3 hairpins in other species.

Base Sequence↗
PubMed1984

7S RNA, containing 5S ribosomal RNA and the termination stem, is a specific substrate for the two RNA processing enzymes RNase III and RNase E.

The 7S RNA, a precursor of 5S rRNA that contains 5S rRNA and the termination stem and loop, is a substrate for RNase E and is also a substrate for RNase III. The cleavage by RNase III is in the stem, 11 nucleotides downstream from the 3' end of the mature 5S rRNA and 8 nucleotides downstream from the RNase E cleavage site. Near the cleaved nucleotides there are three base pairs that appear in the same relative positions in most known RNase III cleavage sites. The large product of the RNase III cleavage reaction, which is a 5S rRNA that contains 11 extra nucleotides at the 3' end, is a substrate for RNase E. This suggests that the information for the 3'-end cleavage by RNase E resides mainly in the 5S rRNA itself. Using rnc rne strains, carrying the plasmid that leads to the accumulation of 7S RNA, we showed that the 7S RNA does not result from an RNase III cleavage but is apparently a proper transcription termination product.

Base Sequence↗
PubMed2001

RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II.

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.

Amino Acid Sequence↗
PubMed2004

Catalytic activation of multimeric RNase E and RNase G by 5'-monophosphorylated RNA.

RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5' end. To investigate the mechanism by which 5'-terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5' monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5' monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5' phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5'-end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.

Amino Acid Sequence↗
PubMed1995

Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) is a multifunctional protein, containing both DNA polymerase and RNase H activity. The RNase H activity of HIV RT catalyzes the hydrolysis of the RNA strand of RNA.DNA hybrids. While the domain that carries out the RNase H activity in HIV RT can be expressed as an independent, folded polypeptide, it is inactive as an RNase H. Here, we report the overexpression and purification of an active, recombinant HIV RNase H domain in which the sequence corresponding to the Escherichia coli RNase H1 basic helix/loop has been substituted for the corresponding sequence of HIV RNase H. The resulting polypeptide (RNH102) has Mn(2+)-dependent RNase H activity and is more stable than the independently expressed wild-type HIV RNase H domain.

Amino Acid Sequence↗
PubMed1997

Pop3p is essential for the activity of the RNase MRP and RNase P ribonucleoproteins in vivo.

RNase MRP is a ribonucleoprotein (RNP) particle which is involved in the processing of pre-rRNA at site A3 in internal transcribed spacer 1. Although RNase MRP has been analysed functionally, the structure and composition of the particle are not well characterized. A genetic screen for mutants which are synthetically lethal (sl) with a temperature-sensitive (ts) mutation in the RNA component of RNase MRP (rrp2-1) identified an essential gene, POP3, which encodes a basic protein of 22.6 kDa predicted molecular weight. Over-expression of Pop3p fully suppresses the ts growth phenotype of the rrp2-1 allele at 34 degrees C and gives partial suppression at 37 degrees C. Depletion of Pop3p in vivo results in a phenotype characteristic of the loss of RNase MRP activity; A3 cleavage is inhibited, leading to under-accumulation of the short form of the 5.8S rRNA (5.8S(S)) and formation of an aberrant 5.8S rRNA precursor which is 5'-extended to site A2. Pop3p depletion also inhibits pre-tRNA processing; tRNA primary transcripts accumulate, as well as spliced but 5'- and 3'-unprocessed pre-tRNAs. The Pop3p depletion phenotype resembles those previously described for mutations in components of RNase MRP and RNase P (rrp2-1, rpr1-1 and pop1-1). Immunoprecipitation of epitope-tagged Pop3p co-precipitates the RNA components of both RNase MRP and RNase P. Pop3p is, therefore, a common component of both RNPs and is required for their enzymatic functions in vivo. The ubiquitous RNase P RNP, which has a single protein component in Bacteria and Archaea, requires at least two protein subunits for its function in eukaryotic cells.

Alleles↗
PubMed2005

The mouse RNase 4 and RNase 5/ang 1 locus utilizes dual promoters for tissue-specific expression.

The ribonuclease A (RNase A) superfamily has been the subject of extensive studies in the areas of protein evolution, structure and biochemistry and are exciting molecules in that they appear to be responding to unique selection pressures, generating proteins capable of multiple and diverse activities. The RNase 4 and RNase 5/ang 1 shared locus breaks a pattern that is otherwise canonical among the members of the RNase A gene superfamily. Conserved among humans, mice and rats, the locus includes two non-coding exons followed by two distinct exons encoding RNase 4 and RNase 5/ang 1. Transcription from this locus is controlled by differential splicing and tissue-specific expression from promoters located 5' to each of the non-coding exons. Promoter 1, 5' to exon I, is universally active, while Promoter 2, 5' to exon II, is active only in hepatic cells in promoter assays in vitro. Transcription from Promoter 2 is dependent on an intact HNF-1 consensus binding site which binds the transcription factor HNF-1alpha. In summary, RNase 4 and RNase 5/ang 1 are unique among the RNase A ribonuclease genes in that they maintain a complex gene locus that is conserved across species with transcription initiated from tissue-specific dual promoters followed by differential exon splicing.

Amino Acid Sequence↗
PubMed1990

Escherichia coli rna gene encoding RNase I: cloning, overexpression, subcellular distribution of the enzyme, and use of an rna deletion to identify additional RNases.

The cloning and overexpression of the Escherichia coli rna gene encoding RNase I are described. Only a single copy of the rna gene is present on the E. coli chromosome. Although cells with as much as a 100-fold increase in RNase I activity were constructed, little effect on cell growth was observed. Overexpressed RNase I was found in the periplasmic space to the same degree (approximately 85%) as wild-type enzyme, suggesting no limitation in RNase I transport. The rna clone was used to identify a deletion strain totally lacking the rna gene. The normal growth of this strain showed that RNase I is not essential for cell viability. Extracts from the RNase I deletion strain still retained a low level of RNase activity in the presence of EDTA, conclusively demonstrating the existence of additional EDTA-active RNases in E. coli. The possibility of a RNase I inhibitor is also discussed.

Chromosome Deletion↗