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.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Koller, U. F. Muller, B. Schmid, A. Missel, V. Kruft, K. Stuart, and H. U. Goringer Trypanosoma brucei gBP21. AN ARGININE-RICH MITOCHONDRIAL PROTEIN THAT BINDS TO GUIDE RNA WITH HIGH AFFINITY J. Biol. Chem., February 7, 1997; 272(6): 3749 - 3757. [Abstract] [Full Text] [PDF]

				R. Aphasizhev and L. Simpson Isolation and Characterization of a U-specific 3'-5'-Exonuclease from Mitochondria of Leishmania tarentolae J. Biol. Chem., June 8, 2001; 276(24): 21280 - 21284. [Abstract] [Full Text] [PDF]