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PubMed2003

Implications for RNase L in prostate cancer biology.

Recently, the interferon (IFN) antiviral pathways and prostate cancer genetics and have surprisingly converged on a single-strand specific, regulated endoribonuclease. Genetics studies from several laboratories in the U.S., Finland, and Israel, support the recent identification of the RNase L gene, RNASEL, as a strong candidate for the long sought after hereditary prostate cancer 1 (HPC1) allele. Results from these studies suggest that mutations in RNASEL predispose men to an increased incidence of prostate cancer, which in some cases reflect more aggressive disease and/or decreased age of onset compared with non-RNASEL linked cases. RNase L is a uniquely regulated endoribonuclease that requires 5'-triphosphorylated, 2',5'-linked oligoadenylates (2-5A) for its activity. The presence of both germline mutations in RNASEL segregating with disease within HPC-affected families and loss of heterozygosity (LOH) in tumor tissues suggest a novel role for the regulated endoribonuclease in the pathogenesis of prostate cancer. The association of mutations in RNASEL with prostate cancer cases further suggests a relationship between innate immunity and tumor suppression. It is proposed here that RNase L functions in counteracting prostate cancer by virtue of its ability to degrade RNA, thus initiating a cellular stress response that leads to apoptosis. This monograph reviews the biochemistry and genetics of RNase L as it relates to the pathobiology of prostate cancer and considers implications for future screening and therapy of this disease.

2',5'-Oligoadenylate Synthetase↗
PubMed2003

Targeting degradation of RNA by RNase H using DNA hairpins.

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.

Base Sequence↗
PubMed2004

RNase T1 variant RV cleaves single-stranded RNA after purines due to specific recognition by the Asn46 side chain amide.

Attempts to alter the guanine specificity of ribonuclease T1 (RNase T1) by rational or random mutagenesis have failed so far. The RNase T1 variant RV (Lys41Glu, Tyr42Phe, Asn43Arg, Tyr45Trp, and Glu46Asn) designed by combination of a random and a rational mutagenesis approach, however, exhibits a stronger preference toward adenosine residues than wild-type RNase T1. Steady state kinetics of the cleavage reaction of the two dinucleoside phosphate substrates adenylyl-3',5'-cytidine and guanylyl-3',5'-cytidine revealed that the ApC/GpC ratio of the specificity coefficient (k(cat)/K(m)) was increased approximately 7250-fold compared to that of the wild-type. The crystal structure of the nucleotide-free RV variant has been refined in space group P6(1) to a crystallographic R-factor of 19.9% at 1.7 A resolution. The primary recognition site of the RV variant adopts a similar conformation as already known from crystal structures of RNase T1 not complexed to any nucleotide. Noteworthy is a high flexibility of Trp45 and Asn46 within the three individual molecules in the asymmetric unit. In addition to the kinetic studies, these data indicate the participation of Asn46 in the specific recognition of the base and therefore a specific binding of adenosine.

Adenine↗
PubMed1998

Role of base G-2 of pre-tRNAfMet in cleavage site selection by Escherichia coli RNase P in vitro.

In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described. A total of 0. 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture. In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P. Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo [Meinnel and Blanquet (1995) J. Biol. Chem. 270, 15906-15914] were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction. The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction. It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P. In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet.

Base Composition↗
PubMed2003

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Arabinose↗
PubMed2006

Activation of murine RNase L by isopolar 2'-phosphonate analogues of 2',5' oligoadenylates.

To determine the influence of methylene group insertion in the internucleotide linkage on the binding process of 2',5'-oligoadenylates to RNase L, a series of 2'-phosphonate-modified trimers and tetramers were synthesized from appropriate monomeric units and evaluated for their ability to bind to murine RNase L. Tetramers pAAXA modified by ribo-, arabino-, or xylo-2'-phosphonate unit X in the third position were capable of binding to RNase L in nanomolar concentrations. The replacement of the first residue (pXAAA), or both the first and the third residues (pXAXA), was also tolerated by the enzyme. In contrast, in all cases, the replacement of the second residue (pAXAA) resulted in the significant decrease of binding ability. Additionally, no more than two phosphonate modifications in the tetramer were allowed to retain the binding affinity to the enzyme. Although all three tetramers pAAXA were found to be potent enzyme binders, only tetramers modified by ribo- and xylo-2'-phosphonate unit X activated the RNase L-catalyzed cleavage of the RNA substrate. Surprisingly, tetramer pAAXA, modified by arabino-2'-phosphonate unit X, did not activate the enzyme and can be considered a potent antagonist. In comparison with their natural counterpart, the phosphonate analogues of the pA4 exhibit superior resistance toward nucleases present in the murine spleen homogenate.

Adenine Nucleotides↗
PubMed2001

Genotype-dependent differences in S12-RNase expression lead to sporadic self-compatibility.

Sporadic self-compatibility, the occasional fruit formation after otherwise incompatible pollinations, has been observed in some S12-containing genotypes of Solanum chacoense but not in others. We have sequenced this S12 allele and analyzed its expression in four different genotypes. The S12-RNase levels were generally less abundant than those of other S-RNases present in the same plants. In addition, two-fold and five-fold differences in the amount of S12-RNase and S12 RNA, respectively, were observed among the genotypes analyzed. A comparison with the genetic data showed that genotypes with the highest levels were fully and permanently self-incompatible, whereas those with the lowest levels were those in which sporadic self-compatibility had been observed. The mature protein contains four potential glycosylation sites and genotype-specific differences in the pattern of glycosylation are also observed. Our results suggest the presence of modifier genes which affect, in a genotype-dependent manner, the level of expression and the post-translational modification of the S12-RNase.

Alleles↗
PubMed1998

Further characterization of human RNase MRP/RNase P and related autoantibodies.

We characterized a panel of human RNase MRP/RNase P autoantibodies by immunoprecipitation, immunodepletion, immunoaffinity purification and immunoblotting. We report on the protein spectrum that is recognized by RNase MRP/RNase P autoantibodies. We also describe another, related patient serum that based on these assays does not immunoprecipitate RNase P/MRP/Th40. This autoantibody 'KC', however, coimmunoprecipitates the RNase MRP/RNase P associated RNAs from HeLa and La9 cell extracts as shown by nuclease protection experiments.

Antibody Specificity↗
PubMed2004

Isothermal titration calorimetric study of RNase-A kinetics (cCMP --> 3'-CMP) involving end-product inhibition.

PURPOSE: Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (KM and kcat) of the hydrolysis of cCMP by RNase-A, a reaction that includes end-product competitive inhibition by 3'-CMP. METHODS: The heat generated from injection of 9-15 microl cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na+ acetate buffer (pH 5.5; 37 degrees C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1)/deltaH(app) x d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (Kp) and enthalpy of the inhibitor binding interaction was obtained from the saturation data of 60 sequential injections of 3'-CMP (1.2 mM) into 0.05 mM RNase-A. The data of the plot of -d[cCMP]/dt against [cCMP] were fitted to kinetic equations incorporating Kp to yield KM and kcat. RESULTS: DeltaH(app) for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters KM = 105.3 microM, 121.6 microM, and 131.3 microM; kcat = 1.63 s(-1), 1.56 s(-1), and 1.71 s(-1). The end-product bound with 1:1 stoichiometry and Kp = 53.2 microM. CONCLUSIONS: The combination of progress curve analysis and ITC allowed rapid and facile measurement of the kinetic parameters for catalytic conversion of cCMP to 3'-CMP by RNase-A, a reaction complicated by end-product inhibition.

Animals↗
PubMed1995

Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops.

The enzyme RNase E (ref. 1) cuts RNA at specific sites within single-stranded segments. The role of adjacent regions of secondary structure in such cleavages is controversial. Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I. These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites. Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access. Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Base Sequence↗
PubMed2000

Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences.

Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.

3' Untranslated Regions↗
PubMed2005

Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z.

Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds.

Allosteric Regulation↗
PubMed2007

RNase MRP RNA and human genetic diseases.

RNase MRP RNA is the RNA subunit of the RNase mitochondrial RNA processing (MRP) enzyme complex that is involved in multiple cellular RNA processing events. Mutations on RNase MRP RNA gene (RMRP) cause a recessively inherited developmental disorder, cartilage-hair hypoplasia (CHH). The relationship of the genotype (RMRP mutation), RNA processing deficiency of the RNase MRP complex, and the phenotype of CHH and other skeletal dysplasias is yet to be explored.

Base Sequence↗
PubMed2006

Evidence that substrate-specific effects of C5 protein lead to uniformity in binding and catalysis by RNase P.

The ribonucleoprotein enzyme RNase P processes all pre-tRNAs, yet some substrates apparently lack consensus elements for recognition. Here, we compare binding affinities and cleavage rates of Escherichia coli pre-tRNAs that exhibit the largest variation from consensus recognition sequences. These results reveal that the affinities of both consensus and nonconsensus substrates for the RNase P holoenzyme are essentially uniform. Comparative analyses of pre-tRNA and tRNA binding to the RNase P holoenzyme and P RNA alone reveal differential contributions of the protein subunit to 5' leader and tRNA affinity. Additionally, these studies reveal that uniform binding results from variations in the energetic contribution of the 5' leader, which serve to compensate for weaker tRNA interactions. Furthermore, kinetic analyses reveal uniformity in the rates of substrate cleavage that result from dramatic (> 900-fold) contributions of the protein subunit to catalysis for some nonconsensus pre-tRNAs. Together, these data suggest that an important biological function of RNase P protein is to offset differences in pre-tRNA structure such that binding and catalysis are uniform.

5' Flanking Region↗
PubMed2006

Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences.

We show that structural type A and B bacterial ribonuclease P (RNase P) RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Our findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not crucial for RNase P function in vivo, at least under standard growth conditions. The discrimination of mature tRNA by RNase P, so far thought to prevent product inhibition of the enzyme in the presence of a large cellular excess of mature tRNA relative to the precursor form, is apparently not crucial for RNase P function in vivo.

Bacillus subtilis↗
PubMed2005

Probing the structure of Saccharomyces cerevisiae RNase MRP.

In yeast, RNase MRP (mitochondrial RNA processing), a ribonucleoprotein precursor rRNA processing enzyme, possesses one putatively catalytic RNA and ten protein subunits and is highly related to RNase P. Structural analysis of the MRP RNA provides data that closely match a previous secondary-structure model derived from phylogenetic analysis, with the exception of an additional stem. This stem occupies an equivalent position to the P7 stem of RNase P RNA and its inclusion confers on MRP RNA a greater similarity to the core P RNA structure. In vivo studies indicate that the P7-like stem can form, but is not a part of, the active enzyme structure. Stem formation would increase RNA stability in the absence of proteins and our alternative structure may be a valid intermediate species in RNase MRP assembly. Further ongoing studies of this enzyme reveal an extensive network of interactions between subunits and a probable central role for the Pop1, Pop4 and Pop7 subunits.

Base Pairing↗
PubMed2000

Bacterial cell death induced by human pro-apoptotic Bax is blocked by an RNase E mutant that functions in an anti-oxidant pathway.

BACKGROUND: Bax is a member of the Bcl-2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis-inducing activity in the mammalian cells. RESULTS: We isolated a Bax-resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat-resistant, suppressor of Bax-1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N- and C-terminal halves. The introduction of the truncated 5' end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat- and Bax-resistance was not the catalytic site for the endoribonuclease activity of RNase E. CONCLUSIONS: The N-terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti-oxidant pathway and that a mutant RNase E has a novel function as an anti-oxidant.

Apoptosis↗
PubMed2001

Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli.

BACKGROUND: Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5' end-processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism. RESULTS: We found that rng:cat mutant strains overproduced a protein of about 100 kDa. N-terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng:cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin-chase experiment revealed that the half-life of adhE mRNA was 2.5-fold longer in the rng:cat disruptant than in the wild-type. CONCLUSION: These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.

Alcohol Dehydrogenase↗