Construction and characterization of lck- and fyn-specific tRNA: ribozyme chimeras.
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.
Baier
G
G
Division of Cell Biology, La Jolla Institute for Allergy and Immunology, CA 92037.
Coggeshall
K M
KM
Baier-Bitterlich
G
G
Giampa
L
L
Telford
D
D
Herbert
E
E
Shih
W
W
Altman
A
A
D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015536:Down-Regulation; D005822:Genetic Vectors; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics*; D012190:Retroviridae; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000378:metabolism
D004274:DNA, Recombinant; D011518:Proto-Oncogene Proteins; D016337:RNA, Catalytic; D012358:RNA, Transfer, Met; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn