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At least 217 records · Page 12Linked to original sources
PubMed2006

RNase L plays a role in the antiviral response to West Nile virus.

Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce approximately 100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-beta and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, approximately 5- to 10-times-higher yields of WNV were produced. Similarly, about approximately 5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L-/- cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) . poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

RNA processing enzymes RNase III, E and P in Escherichia coli are not ribosomal enzymes.

We recently showed that RNase III can process a small stable RNA, precursor 10Sa RNA, that accumulates in an rne (RNase E) strain at non-permissive temperatures. Precursor 10Sa (p10Sa) RNA is processed to 10Sa RNA in two steps, the first step is catalyzed by RNase III in the presence of Mn2+ but not Mg2+. It was shown that RNase III cosediments with membrane preparation from wild type as well as RNase III overexpressing cells. However, the possibility of membrane preparation contamination with ribosomes could not be ruled out. Here we show that RNase III, E and P are not associated with ribosomes. E. coli cells were opened either by alumina grinding or by sonication and fractionated into cytosolic and pellet fractions. The characterization of membrane preparations was done by assaying NADH oxidase, a bona fide membrane enzyme. Ribosomes prepared by alumina grinding were found to be contaminated with small fragments of membrane which contained RNase III activity. RNase III and NADH oxidase activities were present in the ribosomal preparations which could be solubilized by reagents that dissolve the inner membrane. Isopycnic sucrose gradient centrifugation of the membrane and ribosomal preparations also confirmed that RNase III fractionated with the inner membrane. Similarly RNase P activity was found in the corresponding fractions when isopycnic centrifugation of membrane and ribosome preparations was carried out. RNase E activity was also found to be present mostly in the post-ribosomal supernatant. These findings show that RNase III, E and P are not ribosomal enzymes.

Cell Membrane↗
PubMed1995

Double-stranded RNA: the variables controlling its degradation by RNases.

The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.

Animals↗
PubMed1999

Sequence and comparative structural analysis of the murine leukaemia virus amphotropic strain 4070A RNase H domain.

The sequence of a 900-nucleotide segment (encoding part of the reverse transcriptase, including the entire RNase H domain) of the pol gene of the murine leukaemia virus (MLV) amphotropic strain 4070A is presented. Alignment of the inferred 4070A RNase H amino acid sequence (157 residues) with other MLV RNase H sequences revealed only minor differences compared with the divergence between other retroviral and prokaryotic or eukaryotic RNase H sequences. Only 10 residues were invariant across the entire sample set, but secondary structure predictions for the enzymes from E. coli, yeast, human liver and diverse retroviruses (HIV, Rous sarcoma virus, foamy viruses) supported, in every case, the five beta-strands (1 to 5) and four or five alpha-helices (A, B/C, D, E) that have been identified by crystallography in the RNase H domain of HIV-1 reverse transcriptase and in E. coli RNase H. In the case of MLV, analysis of the RNase H domain sequences inferred from 10 different strains (including the amphotropic 4070A) predicted all five alpha-helices (A-E), as well as beta-strands 4 and 5. However, the N-terminal segment (residues 1-40) was predicted, without exception and with high probability, to fold uniquely into one (or two adjacent) alpha-helix(es) encompassing residues 13-37, instead of the three beta-strands known to exist in the HIV-1 and E. coli enzymes. The unerring consistency between the known and predicted structures of the HIV-1 and E. coli enzymes, and the prediction of the same structural elements (including beta-strands 1-3 within the N-terminal segment) for all other (non-MLV) RNase H proteins examined in this study, suggests that the N-terminal segment of the MLV RNase H domain assumes a conformation distinct from that of other retroviral and cellular RNase H molecules. An additional (sixth) beta-strand was also predicted uniquely within the C-terminal region of foamy virus RNase H domains.

Amino Acid Sequence↗
PubMed1992

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates.

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.

Chromatography, Liquid↗
PubMed2002

AhSL28, a senescence- and phosphate starvation-induced S-like RNase gene in Antirrhinum.

Several species of higher plants have been found to contain S-like ribonucleases (RNases), which are homologous to S-RNases controlling self-incompatibility. No S-like RNase genes have been isolated from self-incompatible Antirrhinum. To investigate the relationship between S- and S-like RNases, we cloned a gene named AhSL28 encoding an S-like RNase in Antirrhinum. Amino acid sequence, genomic structure and phylogenetic analyses indicated that AhSL28 is most similar to RNS2, an S-like RNase from Arabidopsis thaliana and formed a distinct subclass together with several other S-like RNases within the S-RNase superfamily. Unlike S-RNase genes in Antirrhinum, AhSL28 is not only expressed in pistils but also in leaves, petals, sepals and anthers, in particular, showing a strong expression in vascular tissues and transmitting track. Moreover, its RNA transcripts were induced during leaf senescence and phosphate (Pi) starvation but not by wounding, indicating that AhSL28 plays a role in remobilizing Pi and other nutrients, particularly when cells senesce and are under limited Pi conditions in Antirrhinum. Possible evolutionary relations of S- and S-like RNases as well as signal transduction pathways related to S-like RNase action are discussed.

Amino Acid Sequence↗
PubMed2001

An essential protein-binding domain of nuclear RNase P RNA.

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.

Base Sequence↗
PubMed2002

RNase E plays an essential role in the maturation of Escherichia coli tRNA precursors.

Conversion of tRNA precursors to their mature forms requires the action of both endo- and exoribonucleases. Although studies over many years identified the endoribonuclease, RNase P, and several exoribonucleases as the enzymes responsible for generating the mature 5' and 3' termini, respectively, of Escherichia coli tRNAs, relatively little is known about how tRNAs are separated from long multimeric or multifunction transcripts, or from long leader and trailer sequences. To examine this question, the tRNA products that accumulate in mutant strains devoid of multiple exoribonucleases plus one or several endoribonucleases were analyzed by northern analysis. We find that the multifunction tyrT transcript, which contains two tRNA(Tyr)1 sequences separated by a 209-nt spacer region plus a downstream mRNA, is cleaved at three sites in the spacer region by the endoribonuclease, RNase E. When both RNase E and RNase P are absent, a product containing both tRNAs accumulates. Two multimeric tRNA transcripts, those for tRNA Arg-His-Leu-Pro and tRNA Gly-Cys-Leu also require RNase E for maturation. For the former transcript, products with long 3' extensions on tRNA(Arg), tRNA(His), and tRNA(Pro), as well as the primary transcript, accumulate in the absence of RNase E. For the latter transcript, RNase E cleaves downstream of each tRNA. Little processing of either multimeric transcript occurs in the absence of both RNase E and RNase P. These data indicate that RNase E is a major contributor to the initial processing of E. coli tRNA transcripts, providing substrates for final maturation by RNase P and the 3' exoribonucleases. Based on this new information, a detailed model for tRNA maturation is proposed.

Blotting, Northern↗
PubMed2002

The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit.

The RNA subunit of bacterial ribonuclease P (RNase P) requires high concentrations of magnesium ions for efficient catalysis of tRNA 5'-maturation in vitro. The protein component of RNase P, required for cleavage of precursor tRNA in vivo, enhances pre-tRNA binding by directly contacting the 5'-leader sequence. Using a combination of transient kinetics and equilibrium binding measurements, we now demonstrate that the protein component of RNase P also facilitates catalysis by specifically increasing the affinities of magnesium ions bound to the RNase P x pre-tRNA(Asp) complex. The protein component does not alter the number or apparent affinity of magnesium ions that are either diffusely associated with the RNase P RNA polyanion or required for binding mature tRNA(Asp). Nor does the protein component alter the pH dependence of pre-tRNA(Asp) cleavage catalyzed by RNase P, providing further evidence that the protein component does not directly stabilize the catalytic transition state. However, the protein subunit does increase the affinities of at least four magnesium sites that stabilize pre-tRNA binding and, possibly, catalysis. Furthermore, this stabilizing effect is coupled to the P protein/5'-leader contact in the RNase P holoenzyme x pre-tRNA complex. These results suggest that the protein component enhances the magnesium affinity of the RNase P x pre-tRNA complex indirectly by binding and positioning pre-tRNA. Furthermore, RNase P is inhibited by cobalt hexammine (K(I) = 0.11 +/- 0.01 mM) while magnesium, manganese, cobalt, and zinc compete with cobalt hexammine to activate RNase P. These data are consistent with the hypothesis that catalysis by RNase P requires at least one metal-water ligand or one inner-sphere metal contact.

Bacillus subtilis↗
PubMed2006

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Aging↗
PubMed1997

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition.

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extra-cellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata SA2- and SC10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing SA2- and SC10-RNases. However, none of the chimeric S-RNases caused rejection of either SA2- or SC10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.

Alleles↗
PubMed1992

Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase.

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.

Base Sequence↗
PubMed1996

Escherichia coli RNase T functions in vivo as a dimer dependent on cysteine 168.

It was shown that Cys-168 is required for RNase T function and thermostability and that its hydrophobic properties are important for this role (Li, Z., Zhan, L., and Deutscher, M. P. (1996) J. Biol Chem. 271, 1127-1132). To understand the molecular basis for these findings, further studies of Cys-168 and RNase T structure were carried out. Treatment of RNase T with the sulfhydryl-modifying agent 5,5'-dithiobis-(2-nitrobenzoic acid) leads not only to inactivation, but also to monomerization of the protein. Similarly, specifically converting Cys-168 to either serine or asparagine leads to loss of activity and to monomer formation at 37 degrees C. However, at 10 degrees C the serine mutant remains as a dimer and retains full RNase T activity, whereas the asparagine derivative shows only a low level of activity and of dimer formation. These data show a strong correlation between activity and the dimer form of RNase T. The importance of dimer formation was also shown in vivo using genetic studies. An inactive mutant of RNase T, termed HA2, which exists as a dimer at 37 degrees C in vitro, completely suppresses endogenous RNase T activity in vivo and in vitro when introduced into a RNase T+ cell on a multicopy phagemid, most likely as a consequence of inactive heterodimer formation. Introduction of the HA2 gene on a single-copy plasmid, as expected, leads to a proportionally smaller effect on endogenous activity. The dominant negative effect displayed by the HA2 protein can be relieved by an additional mutation in HA2 RNase T that abolishes its ability to dimerize. An inactive mutant asparagine derivative of Cys-168, which also does not dimerize, also shows little of the dominant negative phenotype. Thus, these data demonstrate that RNase T dimerizes in vivo, that the dimer form is required for RNase T activity, and that Cys-168 is needed for dimerization of the enzyme.

Cysteine↗
PubMed1997

Single-strand-preferring RNases degrade double-stranded RNAs by destabilizing its secondary structure.

To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

Animals↗
PubMed1998

Localization of a molecular form of interferon-regulated RNase L in the cytoskeleton.

RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involved in the molecular mechanism of interferon (IFN) action. Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regulation of RNA stability in IFN-treated or virus-infected mammalian cells. Knowledge of RNase location within cells may provide additional information about its function. Previous work located RNase as a detergent-soluble molecule in nuclei and cytoplasm. In this study, we demonstrate that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton. A cellular fractionation procedure was used to prepare the cytoskeleton, which was shown to contain 2-5A binding activity not due to cytoplasmic contaminants. In contrast to the cytoplasmic fraction, which contained RNase L with a 2-5A-accessible site, the insoluble RNase molecular form of the cytoskeleton could not be assayed by the classic radiobinding method or the covalent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2-5A site. The 2-5A binding site present in the cytoskeleton was completely masked and not directly accessible to its 2-5A activator. This particular molecular form of RNase can be detected after a specific denaturing-renaturing treatment of the cytoskeleton, which separates the RNase from cytoskeletal proteins, unmasking the 2-5A site. The cytoskeletal RNase was no longer present at this site when cells were stimulated for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data suggest the existence of a pathway that targets the RNase to another subcellular location. To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein kinase C (PKC) to catalyze significant phosphorylation of the RNase.

Adenine Nucleotides↗
PubMed2006

Style-by-style analysis of two sporadic self-compatible Solanum chacoense lines supports a primary role for S-RNases in determining pollen rejection thresholds.

A method for the quantification of S-RNase levels in single styles of self-incompatible Solanum chacoense was developed and applied toward an experimental determination of the S-RNase threshold required for pollen rejection. It was found that, when single style values are averaged, accumulated levels of the S(11)- and S(12)-RNases can differ up to 10-fold within a genotype, while accumulated levels of the S(12)-RNase can differ by over 3-fold when different genotypes are compared. Surprisingly, the amount of S(12)-RNase accumulated in different styles of the same plant can differ by over 20-fold. A low level of 160 ng S-RNase in individual styles of fully incompatible plants, and a high value of 68 ng in a sporadic self-compatible (SSC) line during a bout of complete compatibility was measured, suggesting that these values bracket the threshold level of S-RNase needed for pollen rejection. Remarkably, correlations of S-RNase values to average fruit sets in different plant lines displaying sporadic self-compatibility (SSC) to different extents as well as to fruit set in immature flowers, are all consistent with a threshold value of 80 ng S(12)-RNase. Taken together, these results suggest that S-RNase levels alone are the principal determinant of the incompatibility phenotype. Interestingly, while the S-RNase threshold required for rejection of S(12)-pollen from a given genetic background is the same in styles of different genetic backgrounds, it is different when pollen donors of different genetic backgrounds are used. These results reveal a previously unsuspected level of complexity in the incompatibility reaction.

Flowers↗
PubMed1997

Identification of stylar RNases associated with gametophytic self-incompatibility in almond (Prunus dulcis).

Stylar proteins of 13 almond (Prunus dulcis) cultivars with known S-genotypes were surveyed by IEF and 2D-PAGE combined with immunoblot and N-terminal amino acid sequence analyses to identify S-RNases associated with gametophytic self-incompatibility (SI) in this plant species. RNase activities corresponding to Sa and Sb, two of the four S-alleles tested, were identified by IEF and RNase activity staining. The Sa-RNase band reacted with the anti-S4-serum prepared from Japanese pear (Pyrus serotina); no reaction with the antiserum was observed with the Sb-RNase band. When the Sa-RNase band was excised from an IEF gel stained for RNase activity, subjected to SDS-PAGE, and detected by immunoblotting, it appeared that this band consisted of a single protein that reacted with the anti-S4-serum with M(r) of about 28 kDa. With 2D-PAGE and silver staining of the stylar extracts, all four S-proteins could be successfully distinguished from each other in the highly basic zone of the gel. Although Sb-, Sc-, and Sd-proteins had roughly the same M(r) of about 30 kDa, the Sc-protein seemed to be slightly smaller than the Sb-protein and slightly larger than the Sd-protein. In 2D-PAGE profiles as well, the Sa-protein had M(r) of about 28 kDa, apparently smaller than the other three proteins. A bud sport, in which one of the two S-alleles of the original cultivar is impaired, was visualized as a loss of Sc-protein, which is consistent with the previous pollination study. All four S-proteins reacted with the anti-S4-serum, probably because of the differing conformations of these S-proteins in the IEF and 2D-PAGE gels. The Sa-protein in 2D-PAGE appeared to be identical to Sa-RNase in IEF; both had the same M(r) and were reactive with the anti-S4-serum. N-terminal amino acid sequence analysis of the four S-proteins revealed that they were highly homologous to each other and similar to the S-RNases of Malus, Pyrus, Scrophulariaceae, and Solanaceae. Taken together, RNases in the style are strongly suggested to be associated with the gametophytic SI of almond. This is the first report identifying and characterizing S-RNase in almond.

Amino Acid Sequence↗
PubMed2002

A tobacco S-like RNase inhibits hyphal elongation of plant pathogens.

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Amino Acid Substitution↗