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Construction and characterization of lck- and fyn-specific tRNA: ribozyme chimeras
G Baier1, K M Coggeshall, G Baier-Bitterlich
1Division of Cell Biology, La Jolla Institute for Allergy and Immunology, CA 92037.
Abstract:
Two src-family protein tyrosine kinases (PTKs), p56lck, and p59fyn, are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative contribution to these events is not clearly defined. Here, we have explored the potential of catalytic RNA molecules, or ribozymes, as tools for selectively inhibiting expression of the corresponding target genes in T cells. Several lck- or fyn-specific hammerhead ribozymes were synthesized, cloned into a bacterial transcription vector, and found to display specific catalytic activity in vitro. In order to achieve stable high-level ribozyme expression in intact cells, selected ribozymes were subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA(met) transcription unit. Upon retroviral transduction of a human leukemic T cell line (Jurkat), two out of four chimeric tRNA:ribozymes, fyn-1 and lck-1, were stably expressed at levels of approximately 10,000 or approximately 25,000 copies/cell, respectively. Ribozyme expression was associated with a reduction of up to 80% (lck) or 61% (fyn) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not reduced, suggesting a post-transcriptional compensatory mechanism that increases translation or stability of the p56lck and/or p59fyn proteins.
Insights
Ribozymes were developed to inhibit lck and fyn gene expression in T cells, reducing target mRNA levels significantly. However, protein expression remained unchanged, suggesting compensatory mechanisms.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Src-family protein tyrosine kinases (PTKs), including p56lck and p59fyn, are crucial for T cell receptor (TCR)/CD3 signaling.
- The precise roles and relative contributions of p56lck and p59fyn in T cell activation are not fully elucidated.
Purpose of the Study:
- To investigate the use of catalytic RNA molecules (ribozymes) for selective gene silencing of lck and fyn in T cells.
- To assess the efficacy of ribozymes in reducing target gene expression at both mRNA and protein levels.
Main Methods:
- Design and synthesis of hammerhead ribozymes specific for lck or fyn mRNA.
- Cloning of ribozymes into retroviral vectors for stable expression in Jurkat T cells.
- Quantification of ribozyme expression, target mRNA reduction, and target protein levels.
Main Results:
- Specific catalytic activity of synthesized ribozymes was confirmed in vitro.
- Stable expression of tRNA:ribozymes (fyn-1 and lck-1) was achieved in Jurkat T cells.
- Ribozyme expression led to significant reduction in endogenous lck mRNA (up to 80%) and fyn mRNA (up to 61%).
Conclusions:
- Ribozymes can effectively reduce target mRNA levels for lck and fyn in T cells.
- Despite mRNA reduction, p56lck and p59fyn protein levels were not decreased, indicating post-transcriptional regulation.
- Further research is needed to understand compensatory mechanisms affecting protein expression post-ribozyme treatment.