Nature Methods
- 5, 95 - 100 (2008)
Published online: 9 December 2007; | doi:10.1038/nmeth1142
Targeted pre-mRNA modification for gene silencing and regulationXinliang Zhao & Yi-Tao Yu
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Correspondence should be addressed to Yi-Tao Yu yitao_yu@urmc.rochester.edu 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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