Intracellular mRNA cleavage by 3' tRNase under the direction of 2'-O-methyl RNA heptamers.
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.
Tamura
Masato
M
Department of Biochemistry, Kagoshima University Dental School, Kagoshima, Kagoshima 890-8544, Japan.
Nashimoto
Chikako
C
Miyake
Noriko
N
Daikuhara
Yasushi
Y
Ochi
Kozo
K
Nashimoto
Masayuki
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004285:Dogs; D015536:Down-Regulation; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012347:RNA, Transfer, Arg / Q000235:genetics; D058727:RNA, Small Untranslated
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012347:RNA, Transfer, Arg; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D004722:Endoribonucleases; C045782:3'-pre-tRNase; D058727:RNA, Small Untranslated