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J. Biol. Chem., Vol. 282, Issue 7, 4265-4276, February 16, 2007

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Substrate Determinants for RNA Editing and Editing Complex Interactions at a Site for Full-round U Insertion^*

Catherine Cifuentes-Rojas, Paula Pavia¹, Alfredo Hernandez², Daniel Osterwisch, Concepcion Puerta, and Jorge Cruz-Reyes³

From the Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and Laboratorio of Parasitologia Molecular, Pontificia Universidad Javeriana, Carrera 7a No. 43-82, Ed. 50, Lab 113, Bogotá, Colombia

Multisubunit RNA editing complexes catalyze uridylate insertion/deletion RNA editing directed by complementary guide RNAs (gRNAs). Editing in trypanosome mitochondria is transcript-specific and developmentally controlled, but the molecular mechanisms of substrate specificity remain unknown. Here we used a minimal A6 pre-mRNA/gRNA substrate to define functional determinants for full-round insertion and editing complex interactions at the editing site 2 (ES2). Editing begins with pre-mRNA cleavage within an internal loop flanked by upstream and downstream duplexes with gRNA. We found that substrate recognition around the internal loop is sequence-independent and that completely artificial duplexes spanning a single helical turn are functional. Furthermore, after our report of cross-linking interactions at the deletion ES1 (35), we show for the first time editing complex contacts at an insertion ES. Our studies using site-specific ribose 2' substitutions defined 2'-hydroxyls within the (a) gRNA loop region and (b) flanking helixes that markedly stimulate both pre-mRNA cleavage and editing complex interactions at ES2. Modification of the downstream helix affected scissile bond specificity. Notably, a single 2'-hydroxyl at ES2 is essential for cleavage but dispensable for editing complex cross-linking. This study provides new insights on substrate recognition during full-round editing, including the relevance of secondary structure and the first functional association of specific (pre-mRNA and gRNA) riboses with both endonuclease cleavage and cross-linking activities of editing complexes at an ES. Importantly, most observed cross-linking interactions are both conserved and relatively stable at ES2 and ES1 in hybrid substrates. However, they were also detected as transient low-stability contacts in a non-edited transcript.

Received for publication, June 9, 2006 , and in revised form, December 7, 2006.

^* This work was supported National Institutes of Health Grant GM067130 (to J. C.-R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the Programa de Doctorado Nacionales 2004 from Colciencias, Colombia. Current address: Laboratorio of Parasitologia Molecular, Pontificia Universidad Javeriana. Cra7 3208320-4022, Bogotá, Colombia. Cra. 7a No. 43-82, Ed. 50, Lab 113, Bogotá, Colombia.

² Supported by the National Science Foundation Texas A&M University System Louis Strokes Alliance for Minority Participation Bridge to the Doctorate Fellowship Program.

³ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843. Tel.: 979-458-3374; Fax: 979-862-4718; E-mail: cruzrey{at}tamu.edu.

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