Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase.
RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.
Piller
K J
KJ
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Rusché
L N
LN
Sollner-Webb
B
B
D000818:Animals; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004722:Endoribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D017393:RNA Editing; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D016053:RNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D016053:RNA, Protozoan; D004722:Endoribonucleases; D011130:RNA Ligase (ATP)