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

Abstract

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.

Inhibition of gene expression in human cells using RNase P-derived ribozymes and external guide sequences

“…Modified EGSs that targeted the 3′ untranslated region of PKC-α were highly potent. No non-specific cleavage, which is usually associated with RNase H-induced reactions, was found, providing direct evidence that RNase P-mediated cleavage induced by EGS is highly specific in targeting its mRNA [38]. In addition, potent downregulation of antiapoptotic protein bcl-xL was observed, further suggesting a general applicability of the EGS technology for anticancer applications [38].…”

“…Second, the sequence specificity of the EGS technology is governed by two different types of interactions between the EGS and the target mRNA: (1) the base-pairing interactions in which the sequence of 12 nucleotides in the EGS hybridizes with the target mRNA, and (2) the interactions between the target mRNA and the other part of the EGS sequence (equivalent to the T-stem and T-loop, and variable regions of a tRNA) which are required for folding of the RNase P-recognizable tertiary structure. Thus, the EGS-based technology is highly specific and does not generate nonspecific "irrelevant cleavage" that is observed in RNase H-mediated cleavage induced by conventional antisense phosphorothioate molecules [36,38]. Third, EGSs exhibit little sign of cytotoxicity because cells expressing these molecules for more than 40 days appear to be normal [36,38,44,81].…”

“…Thus, the EGS-based technology is highly specific and does not generate nonspecific "irrelevant cleavage" that is observed in RNase H-mediated cleavage induced by conventional antisense phosphorothioate molecules [36,38]. Third, EGSs exhibit little sign of cytotoxicity because cells expressing these molecules for more than 40 days appear to be normal [36,38,44,81].…”

“…Using the EGS technology, Ma, Stein, and colleagues have successfully induced RNase Pmediated cleavage of the mRNA that encodes protein kinase C-α (PKC-α) [38]. In their study, EGSs equipped with 2′-O-methyl modification for enhanced stability were exogenously administered into T24 bladder carcinoma cells for specific downregulation of PKC-α expression.…”

“…The enzyme is ubiquitous and essential as it is responsible for processing all tRNA molecules [8][9][10]. In addition, EGS-directed cleavage of target RNA by RNase P is highly specific and does not exhibit significant non-specific cleavage, which is commonly seen with RNase H-mediated cleavage induced by conventional antisense phosphorothioate oligonucleotides [8,38]. Some of the fundamental issues regarding the general efficacy of EGS molecules and how to improve their inhibitory effects (e.g.…”