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Growth inhibition by a triple ribozyme targeted to repetitive B2 transcripts
T M Crone1, S L Schalles, C M Benedict
1Departments of Pathology, The Cell and Molecular Biology Program, The Pennsylvania State University, Milton S. Hershey Medical Center, Hershey, PA, USA.
Hepatology (Baltimore, Md.)
|March 30, 1999
Summary
Researchers developed a novel triple ribozyme (TRz) to down-regulate rodent B2 RNAs, which are repetitive DNA sequences. This approach successfully reduced B2 RNA levels and cell growth rates in engineered cells.
Area of Science:
- Molecular Biology
- Genomics
- RNA Therapeutics
Background:
- B2 elements are short, repetitive sequences in rodent genomes, analogous to human Alu sequences.
- Certain B2 subfamilies are transcribed by RNA polymerase III (pol III), with roles potentially in retrotransposition.
- B2 transcripts, normally nuclear, show altered localization in cancer and transformed cells, with unclear significance.
Purpose of the Study:
- To design and validate a novel triple ribozyme (TRz) construct for down-regulating B2 transcripts.
- To investigate the effect of B2 transcript reduction on cell growth and RNA levels.
- To assess the in vivo activity and localization of the designed ribozyme.
Main Methods:
- A triple ribozyme (TRz) construct was designed to target and cleave B2 RNAs.
- Stable transfectant clones were generated from an SV40-immortalized hepatocyte cell line.
- Reverse-transcription polymerase chain reaction (RT-PCR) was used to analyze B2 RNA levels and ribozyme activity/localization.
Main Results:
- The B2-targeted TRz construct demonstrated efficient self-cleavage and liberation of an active ribozyme.
- Transfectant clones exhibited reduced growth rates directly correlated with decreased cytoplasmic B2 RNA levels.
- RT-PCR confirmed efficient in vivo ribozyme self-liberation and showed significant reduction in both nuclear and cytoplasmic B2 RNA.
Conclusions:
- A triple ribozyme (TRz) is an effective tool for down-regulating B2 transcripts in rodent cells.
- Reduction of B2 RNA levels correlates with decreased cell proliferation, suggesting a role in cell growth regulation.
- The study demonstrates the potential of targeted ribozymes for manipulating repetitive element expression and studying their cellular functions.
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