Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells

Abstract

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

Figure 1

Secondary structures of mature tRNAs and tRNA-driven ribozymes. (A) Comparison of pre-tRNA^Val with mature tRNA^Val. The wild-type tRNA is synthesized as a precursor that undergoes a series of maturation steps. (B) Secondary structures of the various tRNA-Rz that were efficiently exported to the cytoplasm. Artificial sequences in the linker region are indicated by lowercase letters. The ribozyme sequence is shown in red and the substrate-binding sites in orange. (C) Secondary structure of tBR-Rz and (D) Rz-N, which accumulated in the nucleus. The sequence underlined in purple is the sequence complementary to the sequences of a probe used for in situ hybridization in Figure 3A.

Figure 2

The intracellular localization of pol III transcripts. (A) Steady-state levels of expression and intracellular localization of tRNA-Rz. Northern blotting analysis was performed with total RNA from intracellular fractions (N, nuclear; C, cytoplasmic). (B) Cell images after treatment for separation of the cytoplasmic fraction. We confirmed that, under our conditions for treatment with digitonin, only cell membranes were disrupted and nuclear membranes remained intact. (C) Northern blots showing the kinetics of the export of tRNA-Rz at various times after transfection. As a control, intracellular U6 snRNA, which remains in the nucleus, was also analyzed.

Figure 3

The efficient transport of the tRNA-Rz in mammalian cells. (A) Analysis by in situ hybridization of tRNA-Rz in HeLa S3 cells. (B) Microinjection analysis of tRNA-Rz in HeLa S3 cells. FITC-labeled tRNA-Rz was injected into nuclei of HeLa cells with excess amount of wild-type tRNA^Val (bottom).

Figure 4

Demonstration of the integrity of exported tRNA-Rz isolated from the cytoplasm. (A) Cleavage activity of the ribozyme (tRNA^Val-R32) extracted from the cytoplasmic fraction. The ³²P-labeled substrate RNA (S11) was cleaved only by RNA extracted from the cytoplasmic fraction of cells that had been transfected with the plasmid encoding the ribozyme that cleaved S11. Other ribozymes (tBR-Rz and tRNA CPP Rz) were not designed to cleave S11. (B) Determination of the 5′ end of tRNA-Rz that had been exported to the cytoplasm. Reverse transcription was performed with cytoplasmic RNAs and the 5′-³²P-labeled ribozyme-specific primer (RT). As size markers (M), tRNA-Rz RNAs that had correctly processed 5′ ends and 5′-extended ends were prepared by T7 transcription.

Figure 5

Determination of the sequences of the 3′ ends of various tRNA-driven ribozyme transcripts. (A) Schematic representation of the experimental procedure for cloning each tRNA-Rz. We isolated tRNA-Rz from HeLa cells and ligated them to a DNA adaptor that had a 3′ amino end to avoid self-ligation of adaptors. The ligated products were amplified by RT–PCR with 5′ and 3′ primers. (B) Sequences of the cDNAs that corresponded to the 3′ ends of various tRNA-Rz. Dotted lines indicate deletions. Underlining indicates mutations. The sequences of the 3′ portions of genes for ribozymes on the expression plasmids were as follows: Rz2, 5′-TCGGAAACGGTTTTTTTCTATCGCGTC-3′; Rz-BR, 5′-TTCGGTCCG CTTTTTTTGGCTGCAGCG-3′; Rz-N, 5′-ACTCGAGCGCTTTTTTTCTATCGCGTC-3′. Continual Ts are those of the terminator sequence. Bold letters in the table correspond to those terminator Ts in figure legend. Some T residues attach to transcribed ribozymes as shown in (B).

Figure 6

Comparison of in vitro and intracellular activities of the nuclear- or cytoplasmic-localizing ribozyme in HeLa cells. (A) Activities of the tRNA^Val HIV Rz2 and tBR-Rz in vitro. The typical gel image of time courses of the cleavage reactions and the calculated k_obs values for both ribozymes are shown. (B) Activities of the tRNA^Val-HIV Rz2 and tBR-Rz in vivo. Although the tRNA^Val HIV Rz2 was efficiently transported into the cytoplasm, the tBR-Rz remained in the nucleus. The results shown are the averages of results from four sets of experiments.