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Targeted pre-mRNA modification for gene silencing and regulation

Nature Methods volume 5, pages 95–100 (2008)Cite this article

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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Figure 1: RNA-guided RNA 2′-O-methylation.
Figure 2: Box C/D RNA targeting ACT1 pre-mRNA impairs cell growth.
Figure 3: Act-BP-guide directs site-specific 2′-O-methylation.
Figure 4: Levels of ACT1 mRNA and ACT1 pre-mRNA in cells expressing or not expressing Act-BP-Guide.
Figure 5: Targeted 2′-O-methylation on other intron-containing genes.

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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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  1. Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, 14642, New York, USA

    Xinliang Zhao & Yi-Tao Yu

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Correspondence to Yi-Tao Yu.

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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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