title: Mechanism of U insertion RNA editing in trypanosome mitochondria: the bimodal TUTase activity of the core complex.
abstract: Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC. In addition, the U insertion activity exhibited by RET2 as an RECC subunit was compared with characteristics of the monomeric protein. We show that interaction of RET2 with RECC is accomplished via a protein-protein contact between its middle domain and a structural subunit, MP81. The recombinant RET2 catalyzes a faithful editing on gapped (precleaved) double-stranded RNA substrates, and this reaction requires an internal monophosphate group at the 5' end of the mRNA 3' cleavage fragment. However, RET2 processivity is limited to insertion of three Us. Incorporation into the RECC voids the internal phosphate requirement and allows filling of longer gaps similar to those observed in vivo. Remarkably, monomeric and RECC-embedded enzymes display a similar bimodal activity: the distributive insertion of a single uracil is followed by a processive extension limited by the number of guiding nucleotides. Based on the RNA substrate specificity of RET2 and the purine-rich nature of U insertion sites, we propose that the distributive +1 insertion creates a substrate for the processive gap-filling reaction. Upon base-pairing of the +1 extended 5' cleavage fragment with a guiding nucleotide, this substrate is recognized by RET2 in a different mode compared to the product of the initial nucleolytic cleavage. Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of California Irvine School of Medicine, B240 Medical Sciences I, Irvine, CA 92697, USA.
authors.forename: Gene-Errol
authors.initials: GE
authors.lastname: Ringpis
authors.forename: Inna
authors.initials: I
authors.lastname: Aphasizheva
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Wang
authors.forename: Lan
authors.initials: L
authors.lastname: Huang
authors.forename: Richard H
authors.initials: RH
authors.lastname: Lathrop
authors.forename: G Wesley
authors.initials: GW
authors.lastname: Hatfield
authors.forename: Ruslan
authors.initials: R
authors.lastname: Aphasizhev
mesh_terms: D054629:Genome, Mitochondrial; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D017393:RNA Editing; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D017394:RNA, Guide, Kinetoplastida; D012333:RNA, Messenger / Q000378:metabolism; D016053:RNA, Protozoan / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D017394:RNA, Guide, Kinetoplastida