title: Engineering RNA endonucleases with customized sequence specificities.
abstract: Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. Here we engineer artificial site-specific RNA endonucleases to function analogously to DNA restriction enzymes. We combine a general RNA cleavage domain with a series of Pumilio/fem-3-binding factor domains that specifically recognize different 8-nucleotide RNA sequences. The resulting artificial site-specific RNA endonucleases specifically recognize RNA substrates and efficiently cleave near their binding sites. The artificial site-specific RNA endonucleases can be devised to recognize and cleave various RNA target sequences, providing a useful tool to manipulate RNAs in vitro. In addition, we generate designer artificial site-specific RNA endonucleases to specifically silence an endogenous gene in Escherichia coli, as well as a mitochondrial-encoded gene in human cells, suggesting that artificial site-specific RNA endonucleases can serve as a gene-silencing tool with designed specificity.
authors.affiliation: Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: Rajarshi
authors.initials: R
authors.lastname: Choudhury
authors.forename: Yihsuan S
authors.initials: YS
authors.lastname: Tsai
authors.forename: Daniel
authors.initials: D
authors.lastname: Dominguez
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Zefeng
authors.initials: Z
authors.lastname: Wang
mesh_terms: D001665:Binding Sites; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D050259:Genes, Mitochondrial / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D059367:RNA Cleavage
chemical_list: D004722:Endoribonucleases