Jan Wrzesinski

Identification of the magnesium, europium and lead binding sites in E. coli and lupine tRNAPhe by specific metal ion‐induced cleavages

The Pb, Eu and Mg‐induced cleavages in E. coli and lupine tRNAPhe have been characterized and compared with those found in yeast tRNAPhe. The pattern of lupine tRNAPhe hydrolysis closely resembles that of yeast tRNAPhe, while several major differences occur in the specificity and efficiency of the E. coli tRNAPhe hydrolysis. The latter tRNA is cleaved with much lower yield in the D‐loop, and interestingly, cleavage is also detected in the variable region, that is highly resistant to hydrolysis in eukaryotic tRNAs. The possible location of tight Pb, Eu and Mg binding sites in E. coli tRNAPhe is discussed on the basis of the specific hydrolysis data.

Specific RNA cleavages induced by manganese ions.

The specificity and efficiency of manganese ion‐induced RNA hydrolysis was studied with several tRNA molecules. In case of yeast tRNAPhe, the main cleavage occurs at p16 and minor cuts at p17–18, p20–21, p34 and p36–37. The major Mn(II)‐induced cut in yeast elongator tRNAMet is also located in the D‐loop at p16 and it is stronger than that observed in tRNAPhe. In initiator tRNAMet from yeast two strong Mn(II) cleavages of equal intensity occur at p16 and p17. This is in contrast with single, much weaker cuts induced in the D‐loop of that tRNA by Mg(II), Eu(III) and Pb(II) ions. Interestingly, in case of yeast tRNAGlu the main cleavage caused by Mn(II), Mg(II) and Pb(II) ions occurs in the anticodon loop. The involvement of hypermodified base mnm5s2U in this cleavage was ruled out based on results obtained with in vitro transcript of yeast tRNAGlu anticodon arm. Mutation of a single base A37G in the anticodon loop of the transcript drastically reduced the specificity of Mn(II)‐induced hydrolysis.

Altered expression of porcine Piwi genes and piRNA during development.

Three Sus scrofa Piwi genes (Piwil1, Piwil2 and Piwil4) encoding proteins of 861, 985 and 853 aminoacids, respectively, were cloned and sequenced. Alignment of the Piwi proteins showed the high identity between Sus scrofa and Homo sapiens. Relative transcript abundance of porcine Piwil1, Piwil2 and Piwil4 genes in testes, ovaries and oocytes derived from sexually immature and mature animals was examined using Real-Time PCR. Expression of the three Piwi mRNAs was proved to be tissue specific and restricted exclusively to the gonads. In testes of adult pigs the highest relative transcript abundance was observed for the Sus scrofa Piwil1 gene. On the other hand, in testes of neonatal pigs the Piwil1 transcript level was over 2–fold reduced while the level of Piwil2 transcript was higher. As regards the expression of the Piwil4 transcript, its level was 34-fold elevated in testes of neonatal piglet when compared to adult male. In ovaries of prepubertal and pubertal female pigs transcript abundance of the three Piwi genes was significantly reduced in comparison with testes. However, similarly to testes, in ovaries of neonatal pigs the Piwil2 gene was characterized by the highest relative transcript abundance among the three Piwi genes analysed. In prepubertal and pubertal oocytes Piwil1 transcript was the most abundant whereas the expression of Piwil4 was undetectable. We also demonstrated that expression of piRNA occurs preferentially in the gonads of adult male and female pigs. Moreover, a piRNA subset isolated from ovaries was 2–3 nucleotides longer than the piRNA from testes.

Structural basis for recognition of Co2+ by RNA aptamers

Co2+ binding RNA aptamers were chosen as research models to reveal the structural basis underlying the recognition of Co2+ by RNA, with the application of two distinct methods. Using the nucleotide analog interference mapping assay, we found strong interference effects after incorporation of the 7‐deaza guanosine phosphorotioate analog into the RNA chain at equivalent positions G27 and G28 in aptamer no. 18 and G25 and G26 in aptamer no. 20. The results obtained by nucleotide analog interference mapping suggest that these guanine bases are crucial for the creation of Co2+ binding sites and that they appear to be involved in the coordination of the ion to the exposed N7 atom of the tandem guanines. Additionally, most 7‐deaza guanosine phosphorotioate and 7‐deaza adenosine phosphorotioate interferences were located in the common motifs: loop E‐like in aptamer no. 18 and kissing dimer in aptamer no. 20. We also found that purine rich stretches containing guanines with the highest interference values were the targets for hybridization of 6‐mers, which are members of the semi‐random oligodeoxyribonucleotide library in both aptamers. It transpired that DNA oligomer directed RNase H digestions are sensitive to Co2+ and, at an elevated metal ion concentration, the hybridization of oligomers to aptamer targets is inhibited, probably due to higher stability and complexity of the RNA structure.

Interaction of aminoglycosides and their copper(II) complexes with nucleic acids: implication to the toxicity of these drugs

Cupric complexes of eight aminoglycosidic antibiotics were screened for their specific behavior towards tRNAPhe, both in oxidative and neutral surrounding. Without H2O2, the cleavage efficiency was dependent on the resultant charge of the molecule. A comparative assay using tRNAPhe devoid of the natural hypermodification in the anticodon loop proved that hypermodification is indispensable for site recognition and subsequent cleavage. The intensity of single and double strand scissions in plasmid DNA also proceeded in a charge-dependent manner. Unlike free antibiotics, their cupric complexes in the presence of H2O2, facilitated plasmid linearisation and degradation. The participation of ROS in those processes was confirmed using NDMA as a reporter molecule, whose consumption was influenced by the protonation state of the complex.

Selection of RNA Oligonucleotides That Can Modulate Human Dicer Activity In Vitro

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.

Effect of modified nucleotides on structure of yeast tRNAPhe comparative studies by metal ion-induced hydrolysis and nuclease mapping

Structural differences between native yeast tRNA(Phe), its in vitro transcript and the U8G mutant have been investigated using metal ion-induced hydrolysis and nuclease digestion. Differences in the solution structure of the molecules involve four regions: the D- and T-loops, the variable region and the anticodon loop. Efficiency of the Pb(II); Eu(II)-, Mn(II)- and Mg(II)-induced hydrolysis at the main cleavage sites in the D-loop is significantly reduced for unmodified tRNAs. Moreover, only the in vitro transcripts are susceptible for cleavage in the T-loop and entire anticodon loop. Other changes in the transcript molecule involve 50-fold enhancement of S1 nuclease digestion at p36, weak cleavages in the D-loop and lack of some digestion sites in the T-loop. The nuclease V1 digestion patterns are very similar for studied molecules. Changes in the pattern of hydrolysis of the D-loop caused by mutation of the conservative base U8 to G are detected by metal-induced hydrolysis only. Our results indicate clearly that metal ions and enzymatic probes monitor different features of RNA structure and their combined use is highly advantageous in studying subtle structural changes in tRNA.

High affinity of copper(II) towards amoxicillin, apramycin and ristomycin. Effect of these complexes on the catalytic activity of HDV ribozyme.

Three representatives of the distinct antibiotics groups: amoxicillin, apramycin and ristomycin A were studied regarding their impact on hepatitis D virus (HDV) ribozyme both in the metal-free form and complexed with copper(II) ions. Hence the Cu(II)-ristomycin A complex has been characterized by means of NMR, EPR, CD and UV-visible spectroscopic techniques and its binding pattern has been compared with the coordination modes estimated previously for Cu(II)-amoxicillin and Cu(II)-apramycin complexes. It has thus been found that all three antibiotics bind the Cu(II) ion in a very similar manner, engaging two nitrogen and two oxygen donors into coordination with the square planar symmetry in physiological conditions. All three tested antibiotics were able to inhibit the HDV ribozyme catalysis. However, in the presence of the complexes, the catalytic reactions were almost completely inhibited. It was important therefore to check whether the complexes used in lower concentrations could inhibit the HDV ribozyme catalytic activity, thus creating opportunities for their practical application. It turned out that the complexes used in the concentrations of 50μM influenced the catalysis much less effectively comparing to the 200 micromolar concentration. The kobs values were lower than those observed in the control reaction, in the absence of potential inhibitors: 2-fold for amoxicillin, ristomycin A and 3.3-fold for apramycin, respectively.

Phosphate residues of antigenomic HDV ribozyme important for catalysis that are revealed by phosphorothioate modification

The aim of our study was to obtain new details on the role of phosphate residues in antigenomic hepatitis delta virus (HDV) ribozyme for the development of metal ion binding sites and their participation in the cleavage mechanism. In these studies, the wild-type ribozyme and four in vitro selected variants, R37, R20, R25 and R51, were used. The application of nucleotide phosphorothioates and the NAIM (nucleotide analog interference mapping) technique for the cis-acting ribozymes, wild-type, R25 and R51, revealed the importance of the J4/2 and P1.1 regions in the catalysis. Interestingly, in the wild-type ribozyme, the largest interference effects were observed close to catalytic C76 in the presence of Ca2+, while in the case of Mg2+ were in the structurally important helix P1.1. The results obtained for R25 and R51 suggest different coordination of the divalent ions to the phosphate residues within the ribozyme catalytic core. Additionally, replacing the non-bridging oxygen atoms on sulfur in a phosphate group at the cleavage site in trans-acting ribozyme variants showed that interactions between pro-RP and pro-SP oxygen atoms, and catalytic metal ions, had moderate effects on the cleavage reaction. In the wild-type ribozyme, the ratio of SP/RP isomer cleavage rates decreased from 25 for Mg2+-induced cleavage to ca. 4 when thiophilic Mn2+ or Cd2+ were added; thus a “rescue effect” was observed. Interestingly, the R37, R20, R25 and R51 ribozymes showed a reduced RP/SP ratio of cleavage rates and much smaller “rescue effects”. This suggests that the binding of divalent metal ions in the vicinity of the phosphate group at the cleavage site is very sensitive to the overall ribozyme structure.

Capreomycin and hygromycin B modulate the catalytic activity of the delta ribozyme in a manner that depends on the protonation and complexation with Cu2+ ions of these antibiotics

Catalytic RNA molecules (ribozymes) have often been used for the testing of interactions of antibiotics with ribonucleic acids. We showed that the impact of capreomycin and hygromycin B on delta ribozyme catalysis might change dramatically, from stimulation to inhibition, depending on conditions. In order to evaluate possible mechanisms of modulation of the ribozyme catalytic activity we used our earlier data on species distribution for protonated forms of capreomycin and hygromycin B and their complexes with Cu(2+) ions at different pH values. We proposed that, upon inhibition, the protonated amino group of capreomycin was located in the ribozyme catalytic cleft interfering with binding catalytic Mg(2+). Such a mechanism was also supported by the results of ribozyme inhibition with capreomycin complexed with Cu(2+). The effects of stimulation of the delta ribozyme activity by capreomycin and hygromycin B were less pronounced than inhibition. Possibly, the amino functions of these antibiotics might be involved in a general acid-base catalysis performed by the ribozyme, acting as proton acceptors/donors.