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Published online before print May 11, 2006, 10.1261/rna.2295706
RNA (2006), 12:1219-1228. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society.
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RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei

Anastasia Sacharidou, Catherine Cifuentes-Rojas, Kari Halbig, Alfredo Hernandez, Lawrence J. Dangott, Monica De Nova-Ocampo and Jorge Cruz-Reyes

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA

Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein–RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 5' of the pre-mRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA–protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.

Keywords: Trypanosoma brucei; RNA editing; RNA–protein interactions; editing complexes


Received November 15, 2005 ; accepted April 3, 2006.


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