Published online 23 November 2005
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Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes
Department of Biochemistry and Biophysics, Texas A&M University 2128 TAMU, College Station, TX 77843, USA
*To whom correspondence should be addressed. Tel: +1 979 458 3375; Fax: +1 979 862 4718; Email: cruzrey{at}tamu.edu
Received September 9, 2005. Revised October 17, 2005. Accepted October 17, 2005.
Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.
Present address: Nova-Ocampo, Instituto de Ciencias Agropecuarias, Universidad Autonoma del Estado de Hidalgo. Av. Universidad Km 1, Ex-Hacienda de Aquetzalpa, Rancho Universitario. Tulancingo, Hidalgo, CP 43600, Mexico
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