C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification

Abstract

A hairpin ribozyme, RzCR2A, directed against position 323 of the hepatitis C virus 5'-untranslated region (HCV 5'-UTR) was used to establish and validate a novel method for the detection of cellular target molecules for hairpin ribozymes, termed C-SPACE (cleavage-specific amplification of cDNA ends). For C-SPACE, HeLa mRNA containing the transcript of interest was subjected to in vitro cleavage by RzCR2A in parallel with a control ribozyme, followed by reverse transcription using a modified SMART cDNA amplification method and cleavage-specific PCR analysis. C-SPACE allowed identification of the RzCR2A target transcript from a mixture containing the entire cellular mRNA while only requiring knowledge of the ribozyme binding sequence for amplification. In a similar approach, C-SPACE was used successfully to identify human 20S proteasome alpha-subunit PSMA7 mRNA as the cellular target RNA of Rz3'X, a ribozyme originally designed to cleave the negative strand HCV 3'-UTR. Rz3'X was found to substantially inhibit HCV internal ribosome entry site (IRES) activity and PSMA7 was subsequently confirmed to be involved in HCV IRES-mediated translation. Thereby, C-SPACE was validated as a powerful tool to rapidly identify unknown target RNAs recognized and cleaved by hairpin ribozymes.

Figure 1

In vitro cleavage of long or short HCV RNA transcripts by RzCR2A and RzCR13. To analyze ribozyme activity in vitro, long and short RNA transcripts (sub) comprising recognition sites for hairpin ribozymes RzCR2A and RzCR13 were generated as schematically depicted (A). Ribozymes RzCR2A, RzCR13 as well as two control ribozymes (40 nM) were incubated separately with long (200 nM) (B and C) or short (200 nM) substrate (D) at 37°C for 3 h. Reactions were resolved on 5% (long substrate) or 15% (short substrate) denaturing polyacrylamide/7 M urea gels. Uncleaved substrate RNA and the 5′- and 3′-cleavage products are indicated. The 3′-fragment of CR2A short substrate is not visible on the gel since it does not contain radiolabeled rUTP (D).

Figure 2

Schematic representation of C-SPACE for ribozyme-mediated target gene identification. HeLa mRNA containing the transcript of interest is subjected to in vitro cleavage by a particular hairpin ribozyme along with a control ribozyme reaction. Ribozyme-mediated cleavage is followed by reverse transcription using a modified SMART cDNA amplification method (Clontech) and cleavage-specific PCR analysis (for details see text).

Figure 3

Optimization of C-SPACE using RzCR2A-mediated cleavage of HeLa mRNA. (A) mRNA was obtained from a HeLa cell line stably expressing a bi-cistronic hygromycin–HCV 5′-UTR–core RNA transcript. Several oligonucleotide primers were used for amplification of C-SPACE products derived from cleavage by RzCR2A or the control RzBR1. The RzCR2A recognition sequence within the HCV 5′-UTR is underlined. The cleavage site located 5′ of the GUC triplet (bold) is indicated by an arrow and G residues resulting from dC-tailing by reverse transcriptase are shaded. (B) C-SPACE amplification products were resolved on a 2% agarose gel and combinations of 5′- and 3′-oligonucleotides used for PCR are indicated. Control amplification for detection of uncleaved HCV substrate was performed with primers CR2A and TAG (lanes 2 and 6) or with CR2A and M2 (not shown). A 100 bp DNA ladder (Gibco BRL) is loaded to the left. Specific amplification was achieved using the 5′-primer SMCR2A5/6 with the 3′-universal TAG oligonucleotide (lane 4).