We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.

DNA content and sensitivity of DNA in situ to denaturation by acid were analyzed by flow cytometry of cell nuclei freshly isolated from the bladder tumors of 32 patients and were compared with normal urothelium of 8 subjects. DNA sensitivity to denaturation was assessed in RNase treated cells by acridine orange metachromasia following partial denaturation with hydrochloric acid; the extent of denatured DNA is given as an index (alpha t), representing the ratio of single stranded to total DNA per nucleus. Of the low stage tumors (papillomas, Ta, Tis, T1) 11 of 18 (61%) were aneuploid. Of the high stage tumors (T2 and T3a) 11 of 14 (79%) were aneuploid. DNA in nuclei of normal transitional epithelium was very sensitive to denaturation, as was papilloma, characterized by nuclear alpha t indices of 0.73 +/- 0.01 (SD) and 0.73 +/- 0.04, respectively. Nuclear DNA of noninvasive carcinomas (Ta, Tis) was significantly more resistant to denaturation (alpha t = 0.69), and DNA of invasive carcinomas was most resistant, ranging from alpha t = 0.61 (T1 tumors) to alpha t = 0.59 (T2 tumors) to alpha t = 0.57 (T3 tumors). High stage tumors as a group (T2, T3) had significantly different (lower) alpha t values than low stage tumors (Ta, Tis, T1). In model cell culture systems it is known that a decrease in alpha t index, i.e., greater resistance to denaturability, occurs as cells transit from resting phase into the cell cycle. Whether the alpha t index can be used to estimate resting vesus cycling cells of human tumors is still speculative; changes in DNA denaturability also are known to occur with changes in chromatin structure during cell differentiation and in transformation. However, the empirical relationship between alpha t index and tumor stage, of itself, may prove clinically useful in identifying more advanced and perhaps more aggressive tumors.

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N3-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3'-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The 3'-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T2.

A base-nonspecific and acid ribonuclease (RNase Ok2) was purified from the liver of a salmon (Oncorhnchus keta) to a homogeneous state by SDS-PAGE. The primary structure of RNase Ok2 was determined by protein chemistry and molecular cloning. The RNase Ok2 was a glycoprotein and consisted of 216 amino acid residues. Its molecular mass of protein moiety was 25,198, and its amino acid sequence showed that it belongs to the RNase T2 family of enzymes. The optimal pH of RNase Ok2 was around 5.5. The base preferences at the B1 and B2 sites were estimated from the rates of hydrolysis of 16 dinucleoside phosphates to be G>)A>U, C, and G>A>U>C respectively. In this enzyme, one of the three histidine residues which have been thought to be important for catalysis of RNase Rh, a typical RNase of this family of enzymes, His104 was replaced by tyrosine residue. Based on the results, the role of H104, which has been proposed to be a phosphate binding site with a substrate, was reconsidered, and we proposed a revised role of this His residue in the hydrolysis mechanism of RNase T2 family enzymes.

The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.

A 125-kilodalton (kDa) phosphoprotein was isolated from nucleoli of Novikoff hepatoma cells in the presence of various inhibitors of proteases, alkaline phosphatase, and RNase. This protein was the most highly phosphorylated protein found thus far in the nucleolus. The half-life of [32P]phosphate in the 125-kDa phosphoprotein was approximately 60 min. Amino acid analysis of the protein showed it had a high serine content (15.5 mol %), a high glutamine plus glutamic acid content (15.5 mol %), and a high lysine content (10.3 mol %). Phosphoserine was the only phosphorylated amino acid identified. After alkaline hydrolysis of the 32P-labeled protein, ribonucleotides were found which accounted for approximately 8.5% of the [32P]phosphate. After cytidine 3',5'-[32P]diphosphate ([32P]pCp) labeling by RNA ligase, several oligoribonucleotide sequences were purified including GGGCOH and GGGGCOH. The binding of oligonucleotides to peptides was stable under denaturing fractionation conditions including 6 M urea treatment and incubation at 100 degrees C for 10 min in sodium dodecyl sulfate and beta-mercaptoethanol. Furthermore, when nucleotide-peptide complex was treated with ribonuclease T2 followed by snake venom phosphodiesterase, the junctional nucleotide pCp was released. These results suggest that one or more ribonucleotides are covalently bound to the 125-kDa phosphoprotein.

The isoleucine acceptance of tRNA from Escherichia coli C6 was previously shown to be influenced by the synthetase level (Marashi, F. and Harris, C.L. 1977. Biochim. Biophys. Acta 477, 84-88). We show here that the increased acceptance observed at higher enzyme levels is accompanied by an increase in the aminoacylation of one tRNAile species. Hence, tRNAile, a minor species at low enzyme levels, is a major isoacceptor after full aminoacylation. The two major isoleucine species have been purified using a combination of BD-cellulose, DEAE-Sephadex A-50 and methylated albumin kieselguhr chromatography. tRNAile (1511 pmoles ile/A260 of tRNA) was found to be slowly acylated, with 2a Vmax one-seventh that observed with tRNAil3le (1475 pmoles ile/A260). Two-dimensional TLC analysis of RNase T2 digests revealed differences in nucleotide content between the purified tRNAs. These results are discussed in terms of the presence of slow and fast tRNAile species in E. coli.

The 5'-terminal structures of murine alpha- and beta-globin mRNA were determined after incubating cells of the erythropoietic spleens of mice with [methyl-3H]methionine. Globin mRNA was obtained from total cellular RNA by oligo(dT)-cellulose chromatography followed by elution of mRNA from formamide gels after electrophoresis. The globin mRNA was then hydrolyzed with KOH or digested with a combination of RNase T2 and bacterial alkaline phosphatase, and 5'-terminal structures were isolated by DEAE-cellulose chromatography. The methylated nucleotides of these 5'-structures were determined following digestion with specific ribonucleases and bacterial alkaline phosphatase. Analyses of mRNA fractions enriched for either alpha- or beta-mRNA gave similar results. Our data indicate that murine alpha- and beta-globin mRNAs are identical through the first three nucleotides and that partial dimethylation exists at the second position: m7G(5')ppp(5') [m6Am/Am]pCmpNp.

Partially purified RNase T2 (EC 2.7.7.17) from Aspergillus oryzae was bound through its carbohydrate moiety to Concanavalin A-Sepharose. The retention of activity was high, ranging from 70% at low enzyme load to approximately 9% at high enzyme load. Though there was no change in the pH and temperature optima, the pH stability and the Km decreased after immobilization. Compared to the soluble enzyme, the immobilized RNase T2 showed enhanced temperature stability and more resistance to metal ions. Both soluble and immobilized enzymes were stable to 8 M urea. On repeated use, the bound enzyme retained more than 60% of its initial activity after six cycles.

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.

Two ribonucleases (RNase Phya and RNase Phyb) were purified to homogeneity on SDS-PAGE from the culture filtrate of the fungus Physarum polycephalum. The apparent molecular weights of RNases Phya and Phyb were about 20,000. The pH optima of these two RNases were around 4.5-4.75. The RNases released mononucleotides from RNA in the order of 3'-GMP, 3'-AMP, and 3'-pyrimidine nucleotides. RNase Phya and RNase Phyb have the N-terminal amino acid sequences STSFD--- and KSTSF--, respectively. This finding and the similar amino acid compositions of both RNases indicated that they might share the same protein moiety except for the N-terminus Lys. The complete primary structure of RNase Phyb was determined, mostly by analysis of the peptides generated by trypsin, V8 protease, and lysylendopeptidase digestions. The molecular weight of the protein moiety was 19,704. The locations of four half cystine residues were almost superimposable on those in five known fungal RNase T2 family RNases, but two others were not. The sequence homology between RNase Phyb and five known fungal RNases amounted to 53-59 residues, which are concentrated around the three histidine residues, supposed to form the active site in enzymes of the RNase T2 family. However, the amino acid sequence of RNase Phyb more closely resembles those of plant RNases such as RNases from Nicotiana alata [McClure, B.A. et al. (1989) Nature 342, 955-957], tomato [RNase Le, Yost et al. (1991) Eur. J. Biochem. 198, 1-6], and Momoridica charantia [RNase MC1, Ide et al. (1991) FEBS Lett. 284, 161-164].

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase LE37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.

The RNase T2 family consists of evolutionarily conserved endonucleases that express in many different species, including animals, plants, protozoans, bacteria, and viruses. The main biological roles of these ribonucleases are cleaving or degrading RNA substrates. They preferentially cleave single-stranded RNA molecules between purine and uridine residues to generate two nucleotide fragments with 2'3'-cyclic phosphate adenosine/guanosine terminus and uridine residue, respectively. Accumulating studies have revealed that RNase T2 is critical for the pathophysiology of inflammation and cancer. In this review, we introduce the distribution, structure, and functions of RNase T2, its differential roles in inflammation and cancer, and the perspective for its research and related applications in medicine.

Keywords: RNase T2; cancer; immunity; inflammation; toll-like receptors.

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and belongs to the RNase T2 family, including fungal RNases typified by RNase Rh from Rhizopus niveus. We expressed RNase MC1 in Escherichia coli cells and made use of site-directed mutagenesis to identify essential amino acid residues for catalytic activity. Mutations of His34 and His88 to Ala completely abolished the enzymatic activity, and considerable decreases in the enzymatic activity were observed in cases of mutations of His83, Glu84, and Lys87, when yeast RNA was used as a substrate. Kinetic parameters for the enzymatic activity of the mutants of His83, Glu84, and Lys87 were analyzed using a dinucleoside monophosphate CpU. Km values for the mutants were approximately like that for wild-type, while k(cat) values were decreased by about 6 to 25-fold. These results suggest that His34, His83, Glu84, Lys87, and His88 in RNase MC1 may be involved in the catalytic function. These observation suggests that RNase MC1 from a plant catalyzes RNA degradation in a similar manner to that of fungal RNases.

A base-nonspecific and acid ribonuclease (RNase Os) belonging to the RNase T2 family was purified from rice bran to a homogeneous state by SDS-PAGE. The primary structure of RNase Os was determined by protein chemistry and molecular cloning. The RNase Os was a simple protein and consisted of 205 amino acid residues. Its molecular weight was 22578 and its amino acid sequence showed that it was most similar to barley RNase among the known RNase T2 family enzymes having 157 amino acid residues identical with barley RNase. However, its N-terminus was blocked by a gamma-pyroglutamyl residue. The optimal pH of RNase Os was around 5.5. The base preference at the B1 and B2 site of RNase Os was estimated from the rates of hydrolysis of 16 dinucleoside phosphates, to be guanine as the case of RNase LE from tomato. RNase Os was successfully expressed from yeast cells using the E. coli yeast expression vector pYE-RNAP.

Tupanviruses are giant viruses recently discovered in Brazil from extreme environments: Tupanvirus soda lake (TPV-SL) and Tupanvirus deep ocean (TPV-DO). Unexpected features in Tupanviruses is the cytotoxic effect observed during infection, where the virus degrades the ribosomal RNA (rRNA) of its amoebal host. Interestingly, only TPV-SL causes this rRNA shutdown. We performed a genomic comparison of the two strains to determine potential modifications explaining the absence of rRNA degradation by TPV-DO. Whole genome comparisons were performed as well as more in-depth analysis at the gene level. We also calculated selective pressure on the orthologous genes between the two viruses. Our computational and evolutionary investigations revealed a potential target: a ribonuclease T2. These enzymes are known to be involved in cellular RNA catabolism such as in lysosomal degradation of rRNA. Our results suggest a functional ribonuclease localized in acid compartment closely related to ribonuclease T2 from eukaryotes. Silencing of the RNAse T2 gene of TPV-SL abolished its rRNA shutdown ability thereby correlating in silico assumption to the experimental evidence. In conclusion, all our results pointed to RNAse T2 as a target for explaining the difference for rRNA degradation ability between both strains.

Keywords: amoeba; giant virus; rRNA shutdown; ribonuclease T2; tupanvirus.

We have developed an assay that uses phenyl boronate agarose column chromatography to measure the capping efficiencies of RNA polymerases used for in vitro transcription of cloned cDNAs. Capped 32P-labeled ovalbumin mRNAs were synthesized by in vitro run-off transcription with SP6 or T7 RNA polymerase in the presence of cap analogs and digested to completion with T1 and T2 RNase. The resulting 3'-nucleoside monophosphates (NMPs) and cap structures were separated by chromatography on phenyl boronate agarose, and the ratio of radioactivity between the two was used to estimate the extent of transcript capping.

A nontoxic high molecular substance associated with some paralytic shellfish poisons was separated by Sephadex G-50 gel filtration from the toxic digestive glands of the scallop Patinopecten yessoensis fed the causative plankton Protogonyaulax tamarensis. Unlike the corresponding fraction from the nontoxic digestive glands, the substance released gonyautoxins II and III on digestion with RNase T2, suggesting that it is associated with an RNA of P. tamarensis. It is possible that the toxification of scallop is partly due to the toxins already accumulated in Protogonyaulax cells, and partly due to incorporation of this precursor, which releases the toxins as a result of enzymic processes in the shellfish.

Recombinant human ribosomal protein S16 (rpS16) is shown to bind specifically to a fragment of its own pre-mRNA that includes exons 1 and 2, intron 1, and part of intron 2, and to inhibit the splicing of the fragment in vitro. The weaker binding of other recombinant human ribosomal proteins, S10 and S13, to this pre-mRNA fragment indicated that the binding of rpS16 was specific. Besides, poly(AU) and rpS16 mRNA fragment affected poorly the binding of rpS16 to its pre-mRNA, providing another evidence that the interaction was specific. RpS16 specifically inhibited the pre-mRNA fragment splicing whereas recombinant rpS10 and rpS16 did not affect excision of intron from this pre-mRNA fragment in contrast to rpS16. Those positions in rpS16 pre-mRNA fragment that were protected by rpS16 against cleavage by RNases T1, T2 and V1 were found to be located closely to the branch point and 3' splice site in the pre-mRNA. Results obtained support the possibility of the autoregulation of rpS13 pre-mRNA splicing through feedback mechanism.

The 3' flanking region of the Escherichia coli rnpB gene-encoding M1 RNA, the RNA component of RNase P, contains a 113 bp repeated sequence. This sequence, successively reiterating 3.5 times, includes the region for intrinsic termination. In vivo termination of transcription occurs mostly at the first terminator (T1). Analysis of deletions at the 3' flanking region revealed that the second terminator (T2) and third (T3) are functional in vivo and that the sequences preceding the region coding for an RNA-terminator hairpin and U-rich 3' tail are essential for efficient termination. Transcripts terminating at T2 and T3 were also processed at the 3' end in a manner similar to those terminating at T1.

Imino proton resonances in the downfield region (10-14 ppm) of the 500-MHz 1H NMR spectrum of Torulopsis utilis 5S RNA are identified (A X U, G X C, or G X U) and assigned to base pairs in helices I, IV, and V via analysis of homonuclear Overhauser enhancements (NOE) from intact T. utilis 5S RNA, its RNase T1 and RNase T2 digested fragments, and a second yeast (Saccharomyces cerevisiae) 5S RNA whose nucleotide sequence differs at only six residues from that of T. utilis 5S RNA. The near-identical chemical shifts and NOE behavior of most of the common peaks from these four RNAs strongly suggest that helices I, IV, and V retain the same conformation after RNase digestion and that both T. utilis and S. cerevisiae 5S RNAs share a common secondary and tertiary structure. Of the four G X U base pairs identified in the intact 5S RNA, two are assigned to the terminal stem (helix I) and the other two to helices IV and V. Seven of the nine base pairs of the terminal stem have been assigned. Our experimental demonstration of a G X U base pair in helix V supports the 5S RNA secondary structural model of Luehrsen and Fox [Luehrsen, K. R., & Fox, G.E. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2150-2154]. Finally, the base-pair proton peak assigned to the terminal G X U in helix V of the RNase T2 cleaved fragment is shifted downfield from that in the intact 5S RNA, suggesting that helices I and V may be coaxial in intact T. utilis 5S RNA.

The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.

Ribonuclease T2, nuclease S1, and snake venom phosphodiesterase were used as a structural probe for investigation of the interaction between Escherichia coli tRNAfMet and methionyl-tRNA synthetase, and the cleavage sites were analyzed by a rapid sequencing gel electrophoresis of 5'-32P-labeled tRNA. Both endonucleases cleaved the D-loop of synthetase-bound tRNA much more extensively than that of the free tRNA. Positions of A14, G15, A22, and G23 in the D-loop and C35 in the anticodon of the synthetase-bound tRNA were more susceptible to RNase T2. The synthetase-bound tRNA was predominantly cleaved by nuclease S1 at position of G15, G19, G20, and G23 in the D-loop and G2 in the acceptor stem. In contrast, the synthetase-bound tRNA was more resistant to the 3'-exonuclease, snake venom phosphodiesterase, than was the free tRNA molecule. These results suggest conformational change of the tRNA by the synthetase binding which weakened tertiary interaction between the D-loop and T psi C-loop/extra-loop. Production of acid-soluble radioactivity was also examined in the limited digestion of 5'-32P-labeled tRNA or 3'-14C-labeled methionyl-tRNA. The synthetase enhanced the release of acid-soluble oligonucleotides from the 5'-end of the tRNA but suppressed that from the 3'-end of the molecule. These results are consistent with that obtained by gel electrophoresis.