title: Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins.
abstract: Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
authors.forename: Xuedong
authors.initials: X
authors.lastname: Kang
authors.forename: Guanghan
authors.initials: G
authors.lastname: Gao
authors.forename: Kestrel
authors.initials: K
authors.lastname: Rogers
authors.forename: Arnold M
authors.initials: AM
authors.lastname: Falick
authors.forename: Sharleen
authors.initials: S
authors.lastname: Zhou
authors.forename: Larry
authors.initials: L
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D047150:Eosinophil Cationic Protein / Q000378:metabolism; D005786:Gene Expression Regulation; D007891:Leishmania / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D017393:RNA Editing; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012313:RNA; D047150:Eosinophil Cationic Protein; D014529:Uridine