Substrate determinants for RNA editing and editing complex interactions at a site for full-round U insertion.
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.
Cifuentes-Rojas
Catherine
C
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and.
Pavia
Paula
P
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and.
Hernandez
Alfredo
A
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and.
Osterwisch
Daniel
D
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and.
Puerta
Concepcion
C
Laboratorio of Parasitologia Molecular, Pontificia Universidad Javeriana, Carrera 7a No. 43-82, Ed. 50, Lab 113, Bogota´, Colombia.
Cruz-Reyes
Jorge
J
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843 and. Electronic address: cruzrey@tamu.edu.
D000818:Animals; D017393:RNA Editing; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism; D017394:RNA, Guide, Kinetoplastida / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*
D012322:RNA Precursors; D012330:RNA, Double-Stranded; D016053:RNA, Protozoan; D017394:RNA, Guide, Kinetoplastida