Transfer RNA cleavages by onconase reveal unusual cleavage sites

Onconase, a protein from amphibian eggs and a homologue of pancreatic ribonuclease (RNase) superfamily, is cytotoxic, exhibits antitumor and antiviral activity, and is in phase III clinical trials. It has been shown to predominantly target cellular tRNA on its entry into mammalian cells (Saxena, S. K., Sirdeshmukh, R., Ardelt, W., Mikulski, S. M., Shogen, K., and Youle, R. J. (2002) J. Biol. Chem. 277, 15142-15146). Cleavage site mapping using natural tRNA substrates, in vitro, revealed predominant cleavage sites at UG and GG residues. Cleavages at UG or the less intense cleavages at CG sites are consistent with the known base specificity of onconase. However, predominance of cleavages at selected G-G bonds is unusual for a homologue of pancreatic RNases. Interestingly, in at least three of the four tRNA substrates studied, the predominant cleavages mapped in the triplet UGG located in the context of the variable loop or the D-arm of the tRNA. The cleavage specificity of onconase observed by us thus indicates another special feature of this enzyme, which may be relevant to its cellular actions.     

Entry into cells and selective degradation of tRNAs by a cytotoxic member of the RNase A family

Onconase (P-30 protein), an enzyme in the ribonuclease A superfamily, exerts cytostatic, cytotoxic, and antiviral activity when added to the medium of growing mammalian cells. We find that onconase enters living mammalian cells and selectively cleaves tRNA with no detectable degradation of rRNA. The RNA specificity of onconase in vitro using reticulocyte lysate and purified RNA substrates indicates that proteins associated with rRNA protect the rRNA from the onconase ribonucleolytic action contributing to the cellular tRNA selectivity of onconase. The onconase-mediated tRNA degradation in cells appears to be accompanied by increased levels of tRNA turnover and induction of tRNA synthesis perhaps in response to the selective toxin-induced loss of tRNA. Degradation products of tRNA(3)(Lys), which acts as a primer for HIV-1 replication, were clearly detected in cells infected with HIV-1 and treated with sublethal concentrations of onconase. However, a new synthesis of tRNA(3)(Lys) also seemed to occur in these cells resulting in plateauing of the steady-state levels of this tRNA. We conclude that the degradation of tRNAs may be a primary factor in the cytotoxic activity of onconase.     

Selection of aminoacyl-tRNAs at sense codons: the size of the tRNA variable loop determines whether the immediate 3' nucleotide to the codon has a context effect.

Codon context can affect translational efficiency by several molecular mechanisms. The base stacking interactions between a codon-anticodon complex and the neighboring nucleotide immediately 3' can facilitate translation by amber suppressors and the tRNA structure is also known to modulate the sensitivity to context. In this study the relative rates of aminoacyl-tRNA selection were measured at four sense codons (UGG, CUC, UUC and UCA), in all four 3' nucleotide contexts, through direct competition with a programmed frameshift at a site derived from the release factor 2 gene. Two codons (UGG and UUC) are read by tRNAs with small variable regions and their rates of aminoacyl-tRNA selection correlated with the potential base stacking strength of the 3' neighboring nucleotide. The other two codons (CUC and UCA) are read by tRNAs with large variable regions and the rate of selection of the aminoacyl-tRNAs in these cases varied little among the four contexts. Re-examination of published data on amber suppression also revealed an inverse correlation between context sensitivity and the size of the variable region. Collectively the data suggest that a large variable loop in a tRNA decreases the influence of the 3' context on tRNA selection, probably by strengthening tRNA-ribosomal interactions.     

Design, characterization and testing of tRNA3Lys-based hammerhead ribozymes

A hammerhead ribozyme targeted against the HIV-1 env coding region was expressed as part of the anticodon loop of human tRNA3Lys without sacrificing tRNA stability or ribozyme catalytic activity. These tRNA-ribozymes were isolated from a library which was designed to contain linkers (sequences connecting the ribozyme to the anticodon loop) of random sequence and variable length. The ribozyme target site was provided in cis during selection and in trans during subsequent characterization. tRNA-ribozymes that possessed ideal combinations of linkers were expected to recognize the cis target site more freely and undergo cleavage. The cleaved molecules were isolated, cloned and characterized. Active tRNA-ribozymes were identified and the structural features conducive to cleavage were defined. The selected tRNA-ribozymes were stable, possessed cleavage rates lower or similar to the linear hammerhead ribozyme, and could be transcribed by an extract containing RNA polymerase III. Retroviral vectors expressing tRNA-ribozymes were tested in a human CD4+ T cell line and were shown to inhibit HIV-1 replication. These tRNA3Lys-based hammerhead ribozymes should therefore prove to be valuable for both basic and applied research. Special application is sought in HIV-1 or HIV-2 gene therapy.     

The structural integrity exerted by N-terminal pyroglutamate is crucial for the cytotoxicity of frog ribonuclease from Rana pipiens

Onconase, a cytotoxic ribonuclease from Rana pipiens, possesses pyroglutamate (Pyr) at the N-terminus and has a substrate preference for uridine–guanine (UG). To identify residues responsible for onconase’s cytotoxicity, we cloned the rpr gene from genomic DNA and expressed it in Escherichia coli BL21(DE3). The recombinant onconase with Met at the N-terminus had reduced thermostability, catalytic activity and antigenicity. Therefore, we developed two methods to produce onconase without Met. One relied on the endogeneous E.coli methionine aminopeptidase and the other relied on the cleavage of a pelB signal peptide. The Pyr1 substitutional variants maintained similar secondary structures to wild-type onconase, but with less thermostability and specific catalytic activity for the innate substrate UG. However, the non-specific catalytic activity for total RNAs varied depending on the relaxation of base specificity. Pyr1 promoted the structural integrity by forming a hydrogen bond network through Lys9 in α1 and Val96 in β6, and participated in catalytic activity by hydrogen bonds to Lys9 and P₁ catalytic phosphate. Residues Thr35 and Asp67 determined B₁ base specificity, and Glu91 determined B₂ base specificity. The cytotoxicity of onconase is largely determined by structural integrity and specific catalytic activity for UG through Pyr1, rather than non-specific activity for total RNAs.     

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.