Abstract
Most eukaryotic box C/D small nucleolar (sno) or Cajal body–specific RNAs guide base pairing with target RNAs and direct site-specific 2′-O-methylation. We designed an artificial C/D RNA to target the branch point adenosine of ACT1 pre-mRNA to block its splicing. Saccharomyces cerevisiae expressing this guide RNA gene controlled by a GAL1 promoter grew normally on dextrose but not on galactose medium. The pre-mRNA was specifically 2′-O-methylated, prohibiting maturation of ACT1 mRNA. Targeting other adenosines in this region while maintaining almost identical complementarity did not affect ACT1 mRNA level or cell growth, suggesting that targeting the branch-point adenosine was truly 2′-O-methylation–specific rather than an antisense effect; moreover, only the 3′-most branch site adenosine served as the branch point. We targeted other essential intron-containing genes, and observed a similar phenotype. We demonstrated that a Box C/D RNA can guide modification at the pre-mRNA branch point, thus silencing its expression and inducing cell death.
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Acknowledgements
We thank members of the Yu laboratory for valuable discussions. This work was supported by grants GM62937 and GM078223 from the US National Institutes of Health.
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Zhao, X., Yu, YT. Targeted pre-mRNA modification for gene silencing and regulation. Nat Methods 5, 95–100 (2008). https://doi.org/10.1038/nmeth1142
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DOI: https://doi.org/10.1038/nmeth1142
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