Monitoring retroviral RNA dimerization in vivo via hammerhead ribozyme cleavage

Abstract

We have used a strategy for colocalization of Psi (Psi)-tethered ribozymes and targets to demonstrate that Psi sequences are capable of specific interaction in the cytoplasm of both packaging and nonpackaging cells. These results indicate that current in vitro dimerization models may have in vivo counterparts. The methodology used may be applied to further genetic analyses on Psi domain interactions in vivo.

FIG. 1

Linear maps of retroviral vectors used in this study. The MoMuLV-based retroviral vector, pLNL-6 (pΨneo) was kindly provided by A. Dusty Miller of the Fred Hutchinson Cancer Research Center, Seattle, Wash. (1), and served as the basic vector for all of the constructs in this study. Target plasmids pΨneo (A) and pΔΨneo (D) contain the neomycin phosphotransferase (neo) gene, allowing selection of transfected cells with G418. Retroviral vectors expressing a ribozyme (pΨhygRbz) (B) or a mutant ribozyme (pΨhyg*Rbz) (C) were derived from pLNL-6 by replacement of the neo gene with hygromycin phosphotransferase (hygro)-Rbz or hygro-*Rbz constructs. Specifically, a 1.1-kb BamHI fragment containing the hygro gene was subcloned upstream of either a functional or a mutant ribozyme gene in pBluescript. The hygro-ribozyme segments were inserted into the BclI and HindIII sites of pLNL-6 (B and C). This functionally destroyed both the BclI and BamHI sites in the LTR-hygromycin-LTR (LHL) vectors.

FIG. 2

Pairing of the anti-neo ribozyme with its target in LNL-6. The ribozyme cleavage site in the neo transcript is between nucleotides 2391 and 2392 in the pLNL-6 sequence and is approximately 1,800 nucleotides downstream of the start point of transcription. Since the ribozyme cloning site in the pΨhygRbz vector was constrained to a site just downstream of the hygro coding region, the neo target site was chosen to position it at the same relative distance from the messenger cap site as the ribozyme, to enhance the probability of the ribozyme pairing arms interacting with the target sequence in the dimerized RNAs. The ribozyme has asymmetric base pairing, with 6 bp in stem I and 32 bp in stem III. The long stem III facilitates the ribozyme and target association, whereas once cleavage has taken place, the short stem I facilitates rapid product dissociation and possibly hastens the ultimate degradation of the cleaved RNA (16). To control for ribozyme versus antisense activity, we generated a mutant, inactive version of the anti-neo ribozyme and inserted it in place of the functional ribozyme in pΨhyg*Rbz (Fig. 1C).

FIG. 3

Ribozyme-mediated inhibition of G418-resistant transfectants in cultured PA317 Cells. The columns show the average numbers of transformed colonies obtained after G418 plus hygromycin selection from three separate experiments; the error bars indicate the data range.

FIG. 4

Ψ-mediated colocalization of ribozyme and target RNAs in PA317 packaging cells. Each column shows the average number of colonies for duplicate transfections obtained under the indicated selection conditions; the error bars show the data range.

FIG. 5

Ribozyme activity assay of Ψ-mediated colocalization in PA317 packaging cells. In the negative control (no plasmid), all cells died within 1 week. Each column shows the average number of colonies for duplicate transfections obtained under the indicated selection conditions; the error bars show the data range.

FIG. 6

Ψ-mediated colocalization of RNAs in 293 nonpackaging cells. Each column shows the average number of colonies for duplicate transfections obtained under the indicated selection conditions; the error bars show the data range. (A) Ribozyme-specific inhibition of target expression; (B) Ψ-dependent inhibition of target expression.

FIG. 6

Ψ-mediated colocalization of RNAs in 293 nonpackaging cells. Each column shows the average number of colonies for duplicate transfections obtained under the indicated selection conditions; the error bars show the data range. (A) Ribozyme-specific inhibition of target expression; (B) Ψ-dependent inhibition of target expression.