Biochemical and Biophysical Research Communications
A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution
Section snippets
Materials and methods
Cell culture and treatments. The rat neuronal stem cell clone HCN-A94-2 was kindly provided by F.H. Gage (La Jolla, CA) and maintained as described previously [20]. The cells were grown on polyornithine/laminin-coated dishes in DMEM/F-12 (1:1; high glucose) medium containing 1 mM l-glutamine supplemented with 1× N2 supplements (Gibco/BRL, Gaithersburg, MD) and 20 ng/ml FGF-2 (Invitrogen, Carlsbad, CA), without serum at 37 °C under 5% CO2. For long-term primary cultures, 75% of the medium was
Results and discussion
The splicing and processing of native pre-mRNA were analyzed as previously described [17], [18], [19]. Spliceosomes catalyze intron removal in pre-mRNAs by sequential assembly with core spliceosomal components (snRNPs U1, U2, and U4/U6.U5 tri-snRNP) and numerous non-snRNP proteins. The basis for recognition of splicing sites lies in the conserved sequences associated with the splice donor and acceptor sites and the branch point. These sequences were incorporated into the SpRNAi so that it would
Acknowledgements
This work was partially funded by NIH/NCI Grant CA-85722. We thank Drs. Wendy Gilmore and Stanley M. Tahara for their critical reading of the manuscript.
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