We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3 0 -processing endoribonuclease (tRNase Z L ) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase Z L in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase Z L method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase Z L directed by sgRNA not owing to a simple antisense effect. We also show that tRNase Z L together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3b by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.
tRNA 3-processing endoribonucleases (tRNase Z, or 3-tRNase; EC 3.1.26.11) are enzymes that remove 3-trailers from pre-tRNAs. An about 12-base-pair stem, a T loop-like structure, and a 3-trailer were considered to be the minimum requirements for recognition by the long form (tRNase ZL) of tRNase Z; tRNase ZL can recognize and cleave a micro-pre-tRNA or a hooker/target RNA complex that resembles a micro-pre-tRNA. We examined four hook RNAs containing systematically weakened T stems for directing target RNA cleavage by tRNase ZL. As expected, the cleavage efficiency decreased with the decrease in T stem stability, and to our surprise, even the hook RNA that forms no T stem-loopdirected slight cleavage of the target RNA, suggesting that the T stem-loop structure is important but dispensable for substrate recognition by tRNase ZL. To analyze the effect of the T loop on substrate recognition, we compared the cleavage reaction for a micro-pre-tRNA with that for a 12-base-pair double-stranded RNA, which is the same as the micro-pre-tRNA except for the lack of the T loop structure. The observed rate constant value for the double-stranded RNA was comparable with that for the micro-pre-tRNA, whereas the K d value for the complex with the double-stranded RNA was much higher than that for the complex with the micro-pretRNA. These results suggest that the T loop structure is not indispensable for the recognition, although the interaction between the T loop and the enzyme exists. Cleavage assays for such double-stranded RNA substrates of various lengths suggested that tRNase ZL can recognize and cleave double-stranded RNA substrates that are longer than 5 base pairs and shorter than 20 base pairs. We also showed that double-stranded RNA is not a substrate for the short form of tRNase Z.The genome of almost every organism encodes the short form (tRNase ZS) 1 and/or the long form (tRNase ZL) of tRNA 3Ј-processing endoribonucleases (tRNase Z, or 3Ј-tRNase; EC 3.1.26.11) (1-6). tRNase ZSs consist of 300 -400 amino acids, whereas tRNase ZLs contain 800 -900 amino acids. These enzymes are responsible for the removal of 3Ј-trailers from pretRNAs (Fig. 1A), which are transcribed as larger forms. In vitro tRNA 3Ј-processing assays have shown that in most cases, the enzymes cleave pre-tRNAs immediately downstream of a discriminator nucleotide, onto which the CCA residues are added to produce mature tRNA in vivo, whereas in some cases, additional cleavages occur 1 nt upstream or 1 or 2 nt downstream (7-14). As an exceptional case, tRNase Z from Thermotoga maritima cleaves pre-tRNAs containing the 74 CCA 76 sequence precisely after the A 76 residue to create the mature CCA 3Ј-termini of tRNA molecules (5).Bacterial and archaeal genomes contain a gene for the short form of tRNase Z only, whereas eukaryote genomes encode either only the long form or both the short and long forms (15). The C-terminal half region of tRNase ZL has high similarity to the whole region of tRNase ZS (1). These regions contain a well conserved histidine motif, whic...
tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.
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