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Gametophytic self-incompatibility in Lycium parishii (Solanaceae): allelic diversity, genealogical structure, and patterns of molecular evolution at the S-RNase locus.

We characterized allelic diversity at the locus controlling self-incompatibility (SI) for a population of Lycium parishii (Solanaceae) from Organ Pipe National Monument, Arizona. Twenty-four partial sequences of S-RNase alleles were recovered from 25 individuals. Estimates of allelic diversity range from 23 to 27 alleles and, consistent with expectations for SI, individuals are heterozygous. We compare S-RNase diversity, patterns of molecular evolution, and the genealogical structure of alleles from L. parishii to a previously studied population of its congener L. andersonii. Gametophytic SI is well characterized for Solanaceae and although balancing selection is hypothesized to be responsible for high levels of allelic divergence, the pattern of selection varies depending on the portion of the gene considered. Site-specific models investigating patterns of selection for L. parishii and L. andersonii indicate that positive selection occurs in those regions of the S-RNase gene hypothesized as important to the recognition response, whereas positive selection was not detected for any position within regions previously characterized as conserved. A 10-species genealogy including S-RNases from a pair of congeners from each of five genera in Solanaceae reveals extensive transgeneric evolution of L. parishii S-RNases. Further, within Lycium, the Dn/Ds ratios for pairs of closely related alleles for intraspecific versus interspecific comparisons were not significantly different, suggesting that the S-RNase diversity recovered in these two species was present prior to the speciation event separating them. Despite this, two S-RNases from L. parishii are identical to two previously reported alleles for L. andersonii, suggesting gene flow between these species.

Base Sequence↗

Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III--the effect of dsRNA binding on gene expression.

RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency. Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III. The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates. Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele. Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lambdaN gene translation by competing for the RNA-binding site of the wild-type endonuclease. The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns. Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner. In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers. Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

Alleles↗

Exploring the minimal substrate requirements for trans-cleavage by RNase P holoenzymes from Escherichia coli and Bacillus subtilis.

We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.

Bacillus subtilis↗

The quaternary structure of RNase G from Escherichia coli.

RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.

Cysteine↗

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.

3T3 Cells↗

RNase L inhibitor (RLI) antisense constructions block partially the down regulation of the 2-5A/RNase L pathway in encephalomyocarditis-virus-(EMCV)-infected cells.

The interferon-(IFN)-inducible 2',5'-oligoadenylate (2-5A)/endoribonuclease L (RNase L) pathway plays a major role in the antiviral and antiproliferative effects of IFN. The 2-5A/RNase L pathway appears to be regulated by the cell-growth status or viral infection. Viruses, and picornaviruses in particular, have evolved strategies to escape the 2-5A/RNase L-pathway-associated antiviral activity. We have recently cloned a cDNA coding for RLI, a RNase-L-specific protein inhibitor. Its regulated expression by viral infection could provide a new strategy to modulate the 2-5A/RNase L pathway. Since RNase L had been shown to be down regulated upon encephalomyocarditis (EMCV) infection, we stably transfected HeLa cells with a RLI antisense cDNA expressing vector. Four independent clones named VAS1, VAS2, VAS3 and VAS4 and one clone transfected with the empty vector (VV) as control, were analyzed. The level of RLI was decreased by 20% for VAS1, 25% for VAS2, 75% for VAS3 and 50% for VAS4. The inactivation of RNase L observed during EMCV infection was decreased in these clones as compared to control HeLa cells. Here again the results vary between the four clones. The maximum inhibition of RNase L (90%) was observed in control cells and in VAS1 while 48% inhibition was observed in VAS4 and 25% in VAS3. The reversal in RNase L inhibition thus reflects closely the resulting RLI level, in keeping with a major role of RLI in EMCV-induced down regulation of 2-5A-binding activity of RNase L. Moreover, cells expressing a low level of RLI (VAS3 and VAS 4) are partially resistant to EMCV infection.

ATP-Binding Cassette Transporters↗

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

Acetylglucosamine↗

Selective abolition of pancreatic RNase binding to its inhibitor protein.

We have modified RNase inhibitor (RI) protein so that it no longer detectably binds pancreatic RNases but retains near-native affinity for human angiogenin (ANG). The K(i) value for RNase A is increased by a factor of >10(8), from 36 fM to >4 microM, and the selectivity factor for ANG is now >10(9). This dramatic change was achieved by remodeling the human RI loop segment Cys-408 -Leu-409 -Gly-410, which makes minor interactions with pancreatic RNase but does not contact ANG. The modifications selected were designed to sterically hinder docking of the undesired ligand. Three of the variants tested (C408W, G410W, and C408W/G410W) bind RNase A with almost the same avidity as WT RI. However, combination of the 408/410 double Trp replacement with deletion of the intervening residue, Leu-409, was sufficient to abolish inhibition of RNase A and human pancreatic RNase. The K(i) value for ANG with the deletion variant is 1.1 fM, only 2-fold higher than with WT RI. This variant may have potential utility both as an anticancer drug targeting ANG and as a tool for the investigation of the biological function of ANG. More generally, these findings demonstrate that a protein-protein interaction can be effectively and specifically disrupted by redesigning an interface region that makes no major energetic contribution to complex stability. This finding, in turn, may have implications for the development of small molecules that modulate protein-protein interactions.

Base Sequence↗

A transcriptional signaling pathway in the IFN system mediated by 2'-5'-oligoadenylate activation of RNase L.

Virus replication in higher vertebrates is restrained by IFNs that cause cells to transcribe genes encoding antiviral proteins, such as 2'-5' oligoadenylate synthetases. 2'-5' oligoadenylate synthetase is stimulated by dsRNA to produce 5'-phosphorylated, 2'-5'-linked oligoadenylates (2-5A), whose function is to activate RNase L. Although RNase L is required for a complete IFN antiviral response and mutations in the RNase L gene (RNASEL or HPC1) increase prostate cancer rates, it is unknown how 2-5A affects these biological endpoints through its receptor, RNase L. Presently, we show that 2-5A activation of RNase L produces a remarkable stimulation of transcription (>/=20-fold) for genes that suppress virus replication and prostate cancer. Unexpectedly, exposure of DU145 prostate cancer cells to physiologic levels of 2-5A (0.1 muM) induced approximately twice as many RNA species as it down-regulated. Among the 2-5A-induced genes are several IFN-stimulated genes, including IFN-inducible transcript 1/P56, IFN-inducible transcript 2/P54, IL-8, and IFN-stimulated gene 15. 2-5A also potently elevated RNA for macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1, a TGF-beta superfamily member implicated as an apoptotic suppressor of prostate cancer. Transcriptional signaling to the macrophage inhibitory cytokine-1/nonsteroidal antiinflammatory drug-activated gene-1 promoter by 2-5A was deficient in HeLa cells expressing a nuclease-dead mutant of RNase L and was dependent on the mitogen-activated protein kinases c-Jun N-terminal kinase and extracellular signal-regulated kinase, both of which were activated in response to 2-5A treatments. Because 2-5A and RNase L participate in defenses against viral infections and prostate cancer, our findings have implications for basic cellular mechanisms that control major pathogenic processes.

Adenine Nucleotides↗

Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P.

Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5' termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein-protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae.

Base Sequence↗

High-resolution structure of RNase P protein from Thermotoga maritima.

The structure of RNase P protein from the hyperthermophilic bacterium Thermotoga maritima was determined at 1.2-A resolution by using x-ray crystallography. This protein structure is from an ancestral-type RNase P and bears remarkable similarity to the recently determined structures of RNase P proteins from bacteria that have the distinct, Bacillus type of RNase P. These two types of protein span the extent of bacterial RNase P diversity, so the results generalize the structure of the bacterial RNase P protein. The broad phylogenetic conservation of structure and distribution of potential RNA-binding elements in the RNase P proteins indicate that all of these homologous proteins bind to their cognate RNAs primarily by interaction with the phylogenetically conserved core of the RNA. The protein is found to dimerize through an extensive, well-ordered interface. This dimerization may reflect a mechanism of thermal stability of the protein before assembly with the RNA moiety of the holoenzyme.

Crystallography, X-Ray↗

The catalytic domain of RNase E shows inherent 3' to 5' directionality in cleavage site selection.

RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2'-O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5'-monophosphate RNA termini but has an inherent mode of cleavage in the 3' to 5' direction. Target sequences made uncleavable by the introduction of 2'-O-methyl-modified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5' to, but not 3' to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3' to 5' "scanning" mechanism and imply that anchoring of the enzyme to the 5'-monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.

Base Sequence↗

Role of mammalian RNase inhibitor in cell-free protein synthesis.

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.

Animals↗

Identification of a precursor molecular for the RNA moiety of the processing enzyme RNase P.

A precursor molecule for 10Sb (M1) RNA, the RNA moiety of the RNA processing enzyme ribonuclease P (EC 3.1.26.5), is accumulated transiently in an Escherichia coli strain containing a plasmid that carries the 10Sb RNA gene. The same RNA precursor molecule is accumulated, in relatively large quantities, in a temperature-sensitive RNase E- mutant at the nonpermissive temperature. The RNA precursor includes 10Sb RNA and an extra 3' fragment that contains a termination stem and loop. It can be processed in vitro to a molecule the size of 10Sb RNA. None of the four endoribonucleases of E. coli--RNase III, RNase E, RNase F, or RNase P--takes part in this cleavage reaction. Therefore, we suggest that the processing of the precursor-10Sb RNA to 10Sb RNA is carried out by a thus-far unidentified endoribonuclease. The accumulation of a RNA molecule in a RNase E- mutant that does not contain a cleavage site for RNase E has been encountered previously and can be explained by assuming the existence of a RNA processing complex in the E. coli cell.

Base Sequence↗

RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA.

RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript. In mutants deficient in RNase III, no species cleaved by RNase III are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely. Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA. Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth. Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase III were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced.

Base Sequence↗

RNase III stimulates the translation of the cIII gene of bacteriophage lambda.

The bacteriophage lambda cIII gene product regulates the lysogenic pathway by stabilizing the lambda cII regulatory protein. Our results show that the expression of the lambda cIII gene is subject to specific requirements. Tests of a set of cIII-lacZ gene and operon fusions reveal that a sequence upstream of the cIII ribosome binding site is needed for cIII translation. The sequence contains an inefficient RNase III processing site. Furthermore, expression of cIII is drastically reduced in cells lacking RNase III. We have isolated a phage carrying a mutation (r1), which lies in the upstream sequence, that leads to a reduction in cIII translation and inactivates the RNase III processing site. The r1 mutant is nevertheless still dependent on RNase III for cIII translation; r1 reduces cIII translation by a factor of 3 in wild-type cells and by a factor of approximately equal to 30 in an RNase III mutant host. We propose that RNase III stimulates cIII translation by binding to the upstream sequence and thereby exposing the cIII ribosome binding site. This stimulation does not involve RNA cleavage. Consistent with this hypothesis is our finding that, in vitro, unprocessed cIII mRNA is translated, whereas RNase III-cleaved cIII mRNA is not.

Bacteriophage lambda↗

Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed.

Amino Acid Sequence↗

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution.

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

Animals↗