A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.
Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.
Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 814-0180, Japan. masatora@fukuoka-u.ac.jp
Masatora
M
Fukuda
Kei
K
Kurihara
Yasuyoshi
Y
Tanaka
Masanobu
M
Deshimaru
D001055:Apolipoproteins B / Q000235:genetics; D001483:Base Sequence; D003285:Contractile Proteins / Q000235:genetics; D064448:Filamins; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000235:genetics; D009690:Nucleic Acid Conformation; D059367:RNA Cleavage; D017393:RNA Editing; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*
D001055:Apolipoproteins B; D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins; D016337:RNA, Catalytic; C427204:hammerhead ribozyme