A functional gene discovery in the Fas-mediated pathway to apoptosis by analysis of transiently expressed randomized hybrid-ribozyme libraries

Abstract

The sequence of much of the human genome is now available and the next goal is to identify functional genes and to clarify their roles. We have recently developed a novel system for isolation of genes in the Fas- and TNF-alpha-mediated pathways to apoptosis using poly(A)-connected hammerhead ribozyme libraries with randomized substrate-binding arms at both the 5' and 3' ends of ribozymes. The transcripts of these hybrid ribozymes have a poly(A) motif that can recruit RNA helicases and, thus, they can effectively attack target sites. In the previous studies, hybrid ribozymes were stably expressed. In order to save selection times, in this study we adopted transiently expressed hybrid ribozymes. In the case of Fas-mediated apoptosis, when we transiently introduced these hybrid-ribozyme libraries into Fas-expressing HeLa cells, we were able to isolate surviving clones that were resistant to or exhibited a delay in Fas-mediated apoptosis. We identified many pro-apoptotic genes and novel genes using this strategy with these transiently expressed hybrid-ribozyme libraries. In contrast, we identified significantly smaller numbers of candidate genes using conventional ribozyme libraries that were expressed transiently. Thus, when changes of a particular phenotype occur within a short period of time, our gene discovery system based on transiently expressed hybrid-ribozyme libraries should also be useful for the rapid identification of functional genes in the post-genome era.

Figure 1

The rationale for the system for discovery of genes that act in Fas-mediated apoptosis using poly(A)-connected ribozyme libraries. (A) Construction of plasmids that contained randomized ribozyme (Rz) libraries with a poly(A) motif. Amp^r, ampicillin; Puro^r, puromycin; Terminator, terminator of a pol III transcription. (B) Schematic diagram of the gene discovery system.

Figure 1

The rationale for the system for discovery of genes that act in Fas-mediated apoptosis using poly(A)-connected ribozyme libraries. (A) Construction of plasmids that contained randomized ribozyme (Rz) libraries with a poly(A) motif. Amp^r, ampicillin; Puro^r, puromycin; Terminator, terminator of a pol III transcription. (B) Schematic diagram of the gene discovery system.

Figure 2

The number of surviving clones after treatment of HeLa-Fas cells with Fas-specific antibody (α-Fas) in each selection cycle. (A) Expression of the various ribozymes in HeLa-Fas cells, as detected by RT–PCR. (B) The number of surviving clones after treatment of HeLa-Fas cells. Pink bars indicate a poly (A)-connected ribozyme library and light green bars indicate results for the conventional ribozyme library.

Figure 3

The determination of the levels of target genes expression. (A) The levels of expression of target genes in cells that expressed poly(A)-connected (A60) or unconnected Bik-ribozyme, or CAD-ribozyme. Bik mRNA and CAD mRNA were detected by RT–PCR with primers specific for the respective genes (see Materials and Methods). CBP is an endogenous control. The levels of expression of all target genes that were identified using the gene discovery system in cells that expressed a poly(A)-connected ribozyme. WT, wild-type. (B) Target mRNAs were detected by RT–PCR with up primers specific for the respective genes and oligo dT down primer. UG, unknown gene.

Figure 3

The determination of the levels of target genes expression. (A) The levels of expression of target genes in cells that expressed poly(A)-connected (A60) or unconnected Bik-ribozyme, or CAD-ribozyme. Bik mRNA and CAD mRNA were detected by RT–PCR with primers specific for the respective genes (see Materials and Methods). CBP is an endogenous control. The levels of expression of all target genes that were identified using the gene discovery system in cells that expressed a poly(A)-connected ribozyme. WT, wild-type. (B) Target mRNAs were detected by RT–PCR with up primers specific for the respective genes and oligo dT down primer. UG, unknown gene.

Figure 4

The levels of apoptotic functions of target genes. (A) The extent of apoptosis (%) 36 h after treatment with Fas-specific antibody in populations of cells that expressed poly(A)-connected or unconnected ribozymes, or an inactive ribozyme directed against the gene for Bik or the gene for CAD. Values are means with SD of results from three replicates in each case. WT, wild-type. (B) The extent of apoptosis (%) 36 h after treatment with α-Fas in populations of cells that expressed poly(A)-connected ribozymes directed against each gene. Values are means with SD of results from three replicates in each case. UG, unknown gene.

Figure 4

The levels of apoptotic functions of target genes. (A) The extent of apoptosis (%) 36 h after treatment with Fas-specific antibody in populations of cells that expressed poly(A)-connected or unconnected ribozymes, or an inactive ribozyme directed against the gene for Bik or the gene for CAD. Values are means with SD of results from three replicates in each case. WT, wild-type. (B) The extent of apoptosis (%) 36 h after treatment with α-Fas in populations of cells that expressed poly(A)-connected ribozymes directed against each gene. Values are means with SD of results from three replicates in each case. UG, unknown gene.