Intracellular mRNA cleavage by 3' tRNase under the direction of 2'-O-methyl RNA heptamers

Abstract

Mammalian tRNA 3' processing endoribonuclease (3'-tRNase) can cleave any RNA at any site under the direction of small guide RNA (sgRNA) in vitro. sgRNAs can be as short as heptamers, which are much smaller than small interfering RNAs of approximately 21 nt. Together with such flexibility in substrate recognition, the ubiquity and the constitutive expression of 3'-tRNase have suggested that this enzyme can be utilized for specific cleavage of cellular RNAs by introducing appropriate sgRNAs into living cells. Here we demonstrated that the expression of chloramphenicol acetyltransferase can be downregulated by an appropriate sgRNA which is introduced into Madin-Darby canine kidney epithelial cells as an expression plasmid or a synthetic 2'-O-methyl RNA. We also showed that 2'-O-methyl RNA heptamers can attack luciferase mRNAs with a high specificity and induce 3'-tRNase-mediated knock-down of the mRNAs in 293 cells. Furthermore, the MTT cell viability assay suggested that an RNA heptamer can downregulate the endogenous Bcl-2 mRNA in Sarcoma 180 cells. This novel sgRNA/3'-tRNase strategy for destroying specific cellular RNAs may be utilized for therapeutic applications.

Figure 1

sgRNA-directed CAT mRNA knock-down in MDCK cells. (A) A plausible secondary structure of the CAT mRNA–sgCAT complex. An arrow indicates the 3′-tRNase cleavage site expected from the in vitro assay (10). The nucleotide numbers of the mRNA start at the first letter of the initiation codon. (B) Relative CAT protein levels in MDCK cells. The cells were co-transfected with pcDNA3/CAT (0.2 µg/well) and an effector expression plasmid (2 or 4 µg/well) or a 2′-O-methyl RNA (1 or 2 µM). pBluescript SK+ or 2′-O-methyl CAT7, which did not direct CAT mRNA cleavage in vitro, was used as a control. Data are the means ± SD of six independent experiments. (C) Northern blot analysis for CAT mRNA. Total RNA was extracted from the cells co-transfected under the same conditions as above. The level of GAPDH mRNA was used as an internal control.

Figure 1

sgRNA-directed CAT mRNA knock-down in MDCK cells. (A) A plausible secondary structure of the CAT mRNA–sgCAT complex. An arrow indicates the 3′-tRNase cleavage site expected from the in vitro assay (10). The nucleotide numbers of the mRNA start at the first letter of the initiation codon. (B) Relative CAT protein levels in MDCK cells. The cells were co-transfected with pcDNA3/CAT (0.2 µg/well) and an effector expression plasmid (2 or 4 µg/well) or a 2′-O-methyl RNA (1 or 2 µM). pBluescript SK+ or 2′-O-methyl CAT7, which did not direct CAT mRNA cleavage in vitro, was used as a control. Data are the means ± SD of six independent experiments. (C) Northern blot analysis for CAT mRNA. Total RNA was extracted from the cells co-transfected under the same conditions as above. The level of GAPDH mRNA was used as an internal control.

Figure 1

sgRNA-directed CAT mRNA knock-down in MDCK cells. (A) A plausible secondary structure of the CAT mRNA–sgCAT complex. An arrow indicates the 3′-tRNase cleavage site expected from the in vitro assay (10). The nucleotide numbers of the mRNA start at the first letter of the initiation codon. (B) Relative CAT protein levels in MDCK cells. The cells were co-transfected with pcDNA3/CAT (0.2 µg/well) and an effector expression plasmid (2 or 4 µg/well) or a 2′-O-methyl RNA (1 or 2 µM). pBluescript SK+ or 2′-O-methyl CAT7, which did not direct CAT mRNA cleavage in vitro, was used as a control. Data are the means ± SD of six independent experiments. (C) Northern blot analysis for CAT mRNA. Total RNA was extracted from the cells co-transfected under the same conditions as above. The level of GAPDH mRNA was used as an internal control.

Figure 2

2′-O-Methyl heptamer-guided cleavage of modified luciferase mRNAs in 293 cells. (A) The 5′- or 3′-modified luciferase mRNA and secondary structures of the luciferase mRNA–heptamer complexes. The heptamers are shown in lower case letters. Arrows denote the potential 3′-tRNase cleavage sites. (B) Relative luciferase activities. The cells were co-transfected with 1 µg/well of p5LucWT, p5LucM1, p5LucM2, p3LucWT, p3LucM1 or p3LucM2 together with 1 µM CAT7 (as a control), Hep1 or Hep2. (C) Heptamer dose-dependent downregulation of the luciferase activity from p5LucWT (1 µg/well). (D) Time course of the luciferase activity. 293 cells were co-transfected with 1 µg/well of p5LucWT and 1 µM heptamer, and incubated for the indicated time periods. (E) Northern blot analysis for luciferase mRNA. The GAPDH mRNA level was used as an internal control. Data are the means ± SD of six independent experiments.

Figure 2

2′-O-Methyl heptamer-guided cleavage of modified luciferase mRNAs in 293 cells. (A) The 5′- or 3′-modified luciferase mRNA and secondary structures of the luciferase mRNA–heptamer complexes. The heptamers are shown in lower case letters. Arrows denote the potential 3′-tRNase cleavage sites. (B) Relative luciferase activities. The cells were co-transfected with 1 µg/well of p5LucWT, p5LucM1, p5LucM2, p3LucWT, p3LucM1 or p3LucM2 together with 1 µM CAT7 (as a control), Hep1 or Hep2. (C) Heptamer dose-dependent downregulation of the luciferase activity from p5LucWT (1 µg/well). (D) Time course of the luciferase activity. 293 cells were co-transfected with 1 µg/well of p5LucWT and 1 µM heptamer, and incubated for the indicated time periods. (E) Northern blot analysis for luciferase mRNA. The GAPDH mRNA level was used as an internal control. Data are the means ± SD of six independent experiments.

Figure 2

2′-O-Methyl heptamer-guided cleavage of modified luciferase mRNAs in 293 cells. (A) The 5′- or 3′-modified luciferase mRNA and secondary structures of the luciferase mRNA–heptamer complexes. The heptamers are shown in lower case letters. Arrows denote the potential 3′-tRNase cleavage sites. (B) Relative luciferase activities. The cells were co-transfected with 1 µg/well of p5LucWT, p5LucM1, p5LucM2, p3LucWT, p3LucM1 or p3LucM2 together with 1 µM CAT7 (as a control), Hep1 or Hep2. (C) Heptamer dose-dependent downregulation of the luciferase activity from p5LucWT (1 µg/well). (D) Time course of the luciferase activity. 293 cells were co-transfected with 1 µg/well of p5LucWT and 1 µM heptamer, and incubated for the indicated time periods. (E) Northern blot analysis for luciferase mRNA. The GAPDH mRNA level was used as an internal control. Data are the means ± SD of six independent experiments.

Figure 2

2′-O-Methyl heptamer-guided cleavage of modified luciferase mRNAs in 293 cells. (A) The 5′- or 3′-modified luciferase mRNA and secondary structures of the luciferase mRNA–heptamer complexes. The heptamers are shown in lower case letters. Arrows denote the potential 3′-tRNase cleavage sites. (B) Relative luciferase activities. The cells were co-transfected with 1 µg/well of p5LucWT, p5LucM1, p5LucM2, p3LucWT, p3LucM1 or p3LucM2 together with 1 µM CAT7 (as a control), Hep1 or Hep2. (C) Heptamer dose-dependent downregulation of the luciferase activity from p5LucWT (1 µg/well). (D) Time course of the luciferase activity. 293 cells were co-transfected with 1 µg/well of p5LucWT and 1 µM heptamer, and incubated for the indicated time periods. (E) Northern blot analysis for luciferase mRNA. The GAPDH mRNA level was used as an internal control. Data are the means ± SD of six independent experiments.

Figure 2

2′-O-Methyl heptamer-guided cleavage of modified luciferase mRNAs in 293 cells. (A) The 5′- or 3′-modified luciferase mRNA and secondary structures of the luciferase mRNA–heptamer complexes. The heptamers are shown in lower case letters. Arrows denote the potential 3′-tRNase cleavage sites. (B) Relative luciferase activities. The cells were co-transfected with 1 µg/well of p5LucWT, p5LucM1, p5LucM2, p3LucWT, p3LucM1 or p3LucM2 together with 1 µM CAT7 (as a control), Hep1 or Hep2. (C) Heptamer dose-dependent downregulation of the luciferase activity from p5LucWT (1 µg/well). (D) Time course of the luciferase activity. 293 cells were co-transfected with 1 µg/well of p5LucWT and 1 µM heptamer, and incubated for the indicated time periods. (E) Northern blot analysis for luciferase mRNA. The GAPDH mRNA level was used as an internal control. Data are the means ± SD of six independent experiments.

Figure 3

Apoptosis induction by an RNA heptamer. Sarcoma 180 cells were transfected with 1 µM of the 2′-O-methyl heptamer Bclhep or Hep2 (as a control), and incubated with the indicated amount of HGF. Suppression of cell viability was determined by the MTT assay. Data are the means ± SD of six independent experiments.