Targeted pre-mRNA modification for gene silencing and regulation.
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.
Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Xinliang
X
Zhao
Yi-Tao
YT
Yu
D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D020868:Gene Silencing / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012333:RNA, Messenger