Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

Human RNASET2 acts as a powerful oncosuppressor protein in in vivo xenograft-based murine models of human cancer. Secretion of RNASET2 in the tumor microenvironment seems involved in tumor suppression, following recruitment of M1-polarized macrophages. Here, we report a murine Rnaset2-based syngeneic in vivo assay. BALB/c mice were injected with parental, empty vector-transfected or murine Rnaset2-overexpressing mouse C51 or TS/A syngeneic cells and tumor growth pattern and immune cells distribution in tumor mass were investigated. Compared to control cells, mouse Rnaset2-expressing C51 cells showed strong delayed tumor growth. CD86⁺ M1 macrophages were massively recruited in Rnaset2-expressing C51-derived tumors, with concomitant inhibition of MDSCs and CD206⁺ M2 macrophages recruitment. At later times, a relevant expansion of intra-tumor CD8⁺ T cells was also observed. After re-challenge with C51 parental cells, most mice previously injected with Rnaset2-expressing C51 cells still rejected C51 tumor cells, suggesting a Rnaset2-mediated T cell adaptive immune memory response. These results point at T2 RNases as evolutionary conserved oncosuppressors endowed with the ability to inhibit cancer growth in vivo through rebalance of intra-tumor M1/M2 macrophage ratio and concomitant recruitment of adaptive anti-tumor CD8⁺ T cells.

The diverse roles of RNAs depend on their ability to fold so as to form biologically functional structures. Thus, understanding the function of a given RNA molecule often requires experimental analysis of its secondary structure by in vitro RNA probing, which is more accurate than using prediction programs only. This chapter presents in vitro RNA probing protocols that we routinely use, from RNA transcript production and purification to RNA structure determination using enzymatic (RNases T1, T2, and V1) and chemical (DMS, CMCT, kethoxal, and Pb(2+)) probing performed on both unlabeled and end-labeled RNAs.

UNASSIGNED

Enzymes belonging to the RNase T2 family are essential for normal rRNA turnover in eukaryotes. In Arabidopsis thaliana, this function is performed by RNS2. The null mutant rns2-2 has increased rRNA half-life and constitutive autophagy. The aim of this work was to determine the molecular changes that take place in the rns2-2 mutant that may lead to altered cellular homeostasis, manifested by the observed cellular phenotype.

UNASSIGNED

To determine the effect of defective rRNA turnover on cellular homeostasis, comparative transcriptome and metabolome analyses of 10-day-old wild-type and rns2-2 seedlings were used to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy.

UNASSIGNED

Few genes were differentially expressed in the rns2-2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the post-transcriptional level. Among differentially expressed genes, transcripts related to carbon flux processes, particularly the pentose phosphate pathway (PPP), were identified. Metabolite analyses confirmed changes in the levels of PPP intermediates. Genes related to cell wall loosening were also differentially expressed in the mutant, and a decrease in monosaccharide components of cell wall hemicellulose were found. As a potential effect of weaker cell walls, rns2-2 plants are larger than wild-type controls, due to larger cells and increased water content. Elevated levels of reactive oxygen species (ROS) were also measured in rns2-2, and the constitutive autophagy phenotype was blocked by preventing ROS production via NADPH oxidase.

UNASSIGNED

Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

UNASSIGNED

Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis. RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.

Human ribosomal protein S5 is a homologue of prokaryotic ribosomal protein S7 that binds to the 16S rRNA region formed by helices H28-30 and H41-43 and contacts this site within the 30S ribosomal subunit. To date, one of the most available approaches to study the structure of the eukaryotic ribosome is based on studying binding of rRNA transcripts with recombinant ribosomal proteins. In the present work, we report studying interaction of human recombinant protein S5 with an RNA transcript "corresponding to the region 1203-1236/1521-1698 (helices H28-30 and H41-43) of human 18S rRNA. The homologous region of 16S rRNA is known to bind protein S7. The apparent association constant of S5 with the transcript was determined. Nucleotide residues of the transcript changing their accessibility for cleavage with RNases T1 and T2 in the presence of S5 were determined by the enzymatic footprinting. It was shown that these nucleotides are located preferentially in the internal loop of the fragment formed by basal parts of the helices H29, H30 and H41 that agrees with the data on the S7 binding to 16S rRNA and on its location within the 30S ribosomal subunit. Besides, we also found nucleotides in the transcript regions corresponding to 16S rRNA regions that do not contact S7 in the 30S subunit.

Ribonucleases (RNases) are hydrolytic enzymes endowed with the ability to either process or degrade ribonucleic acids. Among the many biological functions assigned to RNases, a growing attention has been recently devoted to the control of cancer growth, in the attempt to bring novel therapeutic approaches to clinical oncology. Indeed, several enzymes belonging to different ribonuclease families have been reported in the last decade to display a marked oncosuppressive activity in a wide range of experimental models. The human RNASET2 gene, the only member of the highly conserved T2/Rh/S family of endoribonucleolytic enzymes described in our species, has been shown to display oncosuppressive roles in both in vitro and in vivo models representing several human malignancies. In the present study, we extend previous findings obtained in ovarian cancer models to shed further light on the cell-autonomous roles played by this gene in the context of its oncosuppresive role and to show that RNASET2 silencing can significantly affect the transcriptional output in one of the most thoroughly investigated human ovarian cancer cell lines. Moreover, we report for the first time that RNASET2-mediated changes in the cell transcriptome are in part mediated by its apparent ability to affect the cell's microRNA expression pattern.

Pestiviruses express the unique essential envelope protein E^rns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of E^rns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host's innate immune response to pestivirus infection. The unusual membrane anchor of E^rns raises questions with regard to proteolytic processing of the viral polyprotein at the E^rns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of E^rns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the E^rns membrane anchor.

Keywords: ER retention; RNA virus polyprotein processing; amphipathic helix; charge zipper; extracellular vesicles; membrane anchor; pestivirus; secreted T2 RNase; signal peptidase; transport of virus particles; virus assembly; virus budding.

The pestivirus envelope protein E^rns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the E^rns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the E^rns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an E^rns-only expression construct, these mutations enhanced the naturally occurring E^rns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.

Keywords: RNA virus polyprotein processing; amphipathic helix; charge zipper; membrane anchor; pestivirus; secreted T2 RNase; signal peptidase.

Poly[2'-O-(2,4-dinitrophenyl)]poly(A)[DNP-poly(A)] has been found to be a potent inhibitor in solution for RNases A, B, S, T1, T2 and H as well as phosphodiesterases I and II. Kinetic measurements with RNase B and RNase T1 showed DNP-poly(A) to be a reversible competitive inhibitor with K1 equal to 1.03 and 1.05 microM, respectively. Data on the quenching of fluorescence of RNase T1 by DNP-poly(A) indicate the existence of more than one RNase-binding site in each DNP-poly(A) molecule. By attaching each DNP-poly(A) molecule at one end covalently to oxirane acrylic beads, an affinity column was prepared for selective removal of RNases from aqueous solutions by simple filtration. It was found that a 1000-fold reduction in RNase concentration can be obtained by passing either 7.0 microM or 7.0 nM RNase A solution through a 5-cm-long column. The column can be saturated by passing through a concentrated RNase solution and subsequently regenerated by washing with salt solution. The regenerated column can be used repeatedly with no significant decrease in RNase-binding affinity and capacity. By titration of the derivatized beads with RNase, the first dissociation constant (Kd) and binding capacity for the bound enzyme can be determined. The (Kd) was found to be 0.66 microM for RNase B and 0.48 microM for RNase T1; the corresponding binding capacities were found to be 21.0 x (10)-8 and 9.6 x (10)-8 mol/g, respectively.

Hen oviduct N alpha-acetyltransferase was clarified to have a nucleic acid as an existing constituent by the following three results: (i) an ultraviolet absorption spectrum of the purified N alpha-acetyltransferase free of S-acetyl coenzyme A (Ac-CoA) had an absorption maximum at 260 nm. (ii) A nucleic acid band stained with ethidium bromide was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (iii) An ethidium bromide band co-migrated with a fluorescent band of the protein treated with N-(7-dimethylamino-4-methylcoumarinyl)maleimide, a reagent specific for thiol groups, on polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate. N alpha-Acetyltransferase lost its activity partially or completely by digestion with bovine pancreatic RNase A, Staphylococcus aureus nuclease, or proteinase K, showing that both the nucleic acid and the protein subunit were necessary for the enzyme activity. The nucleic acid component was identified as an RNA but not a DNA because the RNase T2 digest of the nucleic acid was composed of four 3'-ribomononucleotides and completely separated from 3'- and 5'-deoxyribomononucleotides on TLC. The chain length of the nucleic acid of 260 nucleotides estimated by formamide-polyacrylamide gel electrophoresis was calculated to be about 83,000 of the molecular weight. The contents of RNA (35.0%) and protein (65.0%) in N alpha-acetyltransferase determined on weight basis corresponded reasonably well to the contents of RNA (34.4%) and protein (65.6%) calculated based on the assumption that N alpha-acetyltransferase consisted of one molecule of 7 S RNA (Mr 83,000) and two identical Mr 79,000 protein subunits. The total molecular weight (241,000) of the holoenzyme calculated based on the above result was identical to the molecular weight (240,000) of N alpha-acetyltransferase estimated by Sepharose 6B gel filtration.

The present work deals with the processes involved in the abiogenic polycondensation of nucleotides adsorbed on the clay mineral kaolinite under the action of ultraviolet (UV) radiation. The dependence of the yield of synthesis products on irradiation dose was studied. The maximum yield corresponds to a 6-h exposure. The newly synthesized substances were analyzed by ion-exchange chromatography. Some fractions were studied for the type of bonds they contained by venom phosphodiesterase and RNase T2 enzymatic hydrolysis. It was determined that some of the products synthesized by exposure of AMP adsorbed on the surface of clay particles to UV radiation may be looked upon as oligonucleotides in which some fragments have 2'-5'-bonded and others 3'-5'-bonded nucleotides.

The helix content of rRNA species (Escherichia coli, Caldariella acidophila, rat liver) and the G . C content of their bihelical domains have been investigated by chemical modification of uracil and cytosine residues with probes specific for sterically exposed bases. By using radioactively labelled rRNA, G . C base pairs and the sum of A . U plus G . U base pairs have been quantified assuming that they are numerically identical with the unreactive cytosine and uracil rings, respectively. Exposed uracil bases were probed by their conversion to alkali-labile, nonultraviolet-absorbing sulphonated adducts, with 1.33 M bisulfite pH 7, at 20 degrees C; the adducts can be separated from unreacted uracil, and quantified, by cation-exchange chromatography of RNase T2 plus pancreatic RNase digests of bisulfite-modified rRNA. Exposed cytosines were probed by their conversion to methoxyaminated, alkali-stable, derivatives with 1 M methoxyamine, pH 5.5, at 37 degrees C, and quantified by monitoring the CMP/AMP radioactivity ratio after alkaline hydrolysis of modified rRNA. Exposed uracil rings can also be estimated spectrophotometrically by the alkali-catalyzed reversal of the non-ultraviolet-absorbing sulphonated adducts after separation of the latter from unreacted uracil. The cytosine deamination reaction, catalyzed by bisulfite at pH 6, has also been investigated and found to exhibit little specificity for sterically exposed bases of rRNA, the (G + C)-richer rRNA species of C. acidophila being considerably less susceptible to non-specific deamination than the (G + C)-poorer rRNA of E. coli. A high degree of congruence is shown to exist between results obtained by chemical modification and melting hyperchromicity experiments.

RNase LE from cultured tomato cells is a member of the RNase T2 family. It is, however, distinguishable from RNase Rh from Rhizopus niveus, a typical RNase of the RNase T2 family, by its CD spectrum in the 200-250 nm region. In order to reinvestigate the base specificity of RNase LE and to study the role of Asn44 in RNase LE, which is considered to correspond to the base recognition site Asp51 of RNase Rh, RNase LE, and its Asp mutant at the 44th position were expressed from yeast cells with the same expression system as RNase Rh [K. Ohgi, et al., J. Biochem., 109, 776-785 (1991)]. RNase LE with four extra amino acid residues at the 2nd amino acid residue of mature RNase LE and its Asp44 mutant were secreted from yeast cells to give a yield of 10 mg/liter and 0.5 mg/liter culture broth, respectively. The expressed RNase LE (RNase RNAP LE) had the same characteristics as native RNase LE in the CD spectrum and specific activity. This is the first example of the expression of plant RNase from microbes and in sufficient amount to perform further enzymological research. The base specificity of RNase LE was guanylic acid preferential and that of N44D was changed to a more adenylic acid preference as compared to that of RNase LE. These experiments showed that Asn44 of RNase LE is crucial for base recognition as the case of Asp51 in RNASE Rh, and also suggested that the base recognition mechanism of RNase LE is very similar to that of RNase Rh.

The secondary structure of [32P] end-labeled 5S rRNA from Tetrahymena thermophilia (strain B) has been investigated using the enzymes S1 nuclease, cobra venom ribonuclease and T2 ribonuclease. The results, analyzed by scanning microdensitometry and illustrated by three-dimensional computer graphics, support the secondary structure model of Curtiss and Vournakis for 5S rRNA. Aberrent mobility of certain RNA fragments on sequencing gels was observed as regions of band compression. These regions are postulated to be caused by stable internal base-pairing. The molecule was probed with T2 RNase in neutral (pH 7.5) and acidic (pH 4.5) buffers and only minor structural differences were revealed. One of the helices was found to be susceptible to enzymatic attack by both the single-strand and double-strand specific enzymes. These observations are evidence for the existence of dynamic structural equilibria in 5S rRNA.

An ultrasensitive electrochemical genosensor has been fabricated for the double determination of two different specific sequences deduced from the maternally expressed gene3 (MEG3) lncRNA (long noncoding RNA), which was demonstrated by coupling RNase A-aided target recycling with DNA supersandwich-induced signal enhancement, based on a composite interface of graphene-like tungsten disulfide/dendritic gold nanostructures (WS2/DGN). Firstly, duple target sequences of T1 and T2 were captured by the primer probes of P1/P2 functionalized Fe3O4@C magnetic nanoparticles, via the DNA/RNA hybridization between T1/T2 and P1/P2. In the presence of RNase A, T1 and T2 were released to trigger the target recycling, accompanied by the generation of numerous intermediate DNAs designated as IT1 and IT2, respectively. After the magnetic separation, the IT1 and IT2 were liberated and hybridized with the capture probes of CP1/CP2 loaded DGN/WS2 modified electrode. Subsequently, the stepwise DNA hybridization chain reactions (HCR) labeled with ferrocene (Fc) and methyleneblue (MB) were processed, respectively. The DPV current values of Fc and MB were recorded, which were proportional with the concentration of T1 and T2, respectively. Using the multiplexed amplification strategy, this newly designed genosensor provided a wide linear range from 1 fM to 100 pM with a low detection limit of 0.25 fM for T1 and 0.3 fM for T2. The application of the genosensor in real serum sample has also been studied, confirming the excellent selectivity and sensitivity for the application in bioanalysis and clinical diagnostics.

The antisense RNA ArrS is complementary to a sequence in the 5' untranslated region of the gadE T3 mRNA, the largest transcript of gadE, which encodes a transcriptional activator of the glutamate-dependent acid resistance system in Escherichia coli. Expression of arrS is strongly induced during the stationary growth phase, particularly under acidic conditions, and transcription is dependent on σ(S) and GadE. The aim of the present study was to clarify the role of ArrS in controlling gadE expression by overexpressing arrS in E. coli. The results showed a marked increase in the survival of arrS-overexpressing cells at 2 h after a shift to pH 2.5. This was accompanied by increased expression of gadA, gadBC and gadE. The level of gadE T3 mRNA decreased markedly in response to arrS overexpression, and was accompanied by a marked increase in gadE mRNA T2. T2 mRNA had a monophosphorylated 5' terminus, which is usually found in cleaved mRNAs, and no T2 mRNA was observed in an RNase III-deficient cell strain. In addition, T2 mRNA was not generated by a P3-deleted gadE-luc translational fusion. These results suggest strongly that T2 mRNA is generated via the processing of T3 mRNA. Moreover, the T2 mRNA, which was abundant in arrS-overexpressing cells, was more stable than T3 mRNA in non-overexpressing cells. These results suggest that overexpression of ArrS positively regulates gadE expression in a post-transcriptional manner.

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.

Lentinus edodes (shiitake) produces three base non- specific and acid ribonucleases, RNase Le2, RNase Le37 and RNase Le45. The primary structures of the former two RNases, having molecular masses about 24 and 37 kDa, respectively, have been elucidated to be members of the RNase T2 family. The latter two are excreted from mycelia into the medium. In this report, we estimated the primary structure of RNase Le45 using the following experimental evidence. (i) The partial amino acid sequence of RNase Le45 determined that up to about 60% of total protein was identical with that of RNase Le37. (ii) The amino acid composition of RNase Le45 was identical to that of RNase Le37. (iii) The elution profiles on HPLC of lysylendopeptidase and Staphylococcus aureus V8 protease digests of RCM-RNase Le45 (reduced and S-carboxymethylated RNase Le45) were very similar to those of RNase Le37, except for the absence of C-terminus peptide which contained O-glycosylated peptides. However, RNase Le45 contained about 70 residues of mannose and 4 residues of hexosamine. These values were more than twice those of RNase Le37. (iv) RNase Le45 was immunologically indistinguishable from RNase Le37. (v) After treatment with both glycosidase EndoH and alpha-mannosidase, RNases Le37 and Le45 gave complex bands by slab-gel electrophoresis. However, one of the major bands with the highest mobility from RNase Le45 and Le37 showed the molecular mass of 29 kDa in common, which is slightly larger than that of RNase Le2 containing no carbohydrate. These results indicated that RNase Le45 is an enzyme which is a heavily glycosylated species of RNase Le37.

The action of ribonucleases on poly and oligoribonucleotides containing cytosine bases modified by methoxyamine and bisulphite was examined. Resistance of phosphodiester bonds in (Cp)nXp (where n greater than or equal to 1 and X stands for A, G or U) to T2 RNase hydrolysis was observed if substrates were modified chemically. The phenomenon formed the basis for isolation of (Cp)nXp blocks as an additional tool in sequence investigations. After modification of cytosine pancreatic RNase was unable to hydrolyse (Cp)nUp blocks. Therefore the specificity of pyrimidyl RNase may be narrowed to 'uridyl RNase'.

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a phosphoprotein highly enriched in concentration in the neurons of the limbic striatum. It is likely a third messenger in the intracellular cascade of events following neuronal stimulation by first-messenger activators of the adenylate cyclase system, including dopamine via the D1 receptor. ARPP-21 expression is restricted to telencephalic post-mitotic, post-migrational neurons, and its precise pattern of temporal and spatial expression makes it an attractive candidate for the study of transcriptional regulation of neuronal maturation. To define genomic regions likely to contain functional promoter elements, we isolated the murine ARPP-21 gene. Primer extension and T2 RNase protection analyses identified multiple transcription start sites, but 1.3 kb of 5'-flanking DNA revealed few consensus transcription factor binding sequences. A series of transient transfection assays in clonal cell lines which do not express ARPP-21 identified a basal promoter active in both neuronal and non-neuronal lines. Expression in all lines was decreased by the inclusion of regions further upstream, and extinguished by the inclusion of the first intron. Further analyses are likely to reveal cell specific regulatory sequences.

The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosine-labeled 35S viral RNA.

A kinetic study has been made of the RNase-T2-catalyzed transphosphorylation of two adenine nucleotides, adenylyl(3'-5')uridine and adenosine 3'-(1-naphthyl)phosphate. Rates were measured at pH values ranging from 2.6 to 8.2. The observed shape of the plot of log kcat against pH for both the natural and the synthetic substrate suggests that there exist two parallel rate-determining pathways. Two pH-independent rate constants and three ionization constants of the enzyme-substrate complexes were obtained by nonlinear iterative least-squares analysis. Detailed interpretation of the pH profiles was carried out and it is proposed that carboxylate anion is likely to deprotonate O-2' at 4 less than pH less than 6, but at pH greater than 6 an alternative general base would play this role more effectively than the carboxylate group. Another base in its protonated cationic form is responsible for the general acid catalysis.

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)