A novel RNA splicing-mediated gene silencing mechanism potential for genome evolution

https://doi.org/10.1016/j.bbrc.2003.09.070Get rights and content

Abstract

Over 90% of the human genome consists of non-protein-coding regions. Introns constitute most of the non-coding regions located in precursor messenger RNAs (pre-mRNAs). During pre-mRNA maturation, the introns are excised out of mRNA and thought to be completely digested prior to translation. If the introns were merely metabolic “leavings,” why would the genome hold such a large amount of extraneous genetic materials? Here we show a novel posttranscriptional gene silencing system identified within mammalian introns. By packaging human spliceosome-recognition sites along with an exonic insert into an artificial intron, we observed that the splicing and processing of such an exon-containing intron in either sense or antisense conformation produced equivalent gene silencing effects, while a palindromic hairpin insert containing both sense and antisense strands resulted in synergistic effects. These findings may explain how cells respond to the presence of transgenic introns that are homologous to pre-existing exons during genomic 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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