Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: Structure probing of a gRNA bound to its cognate mRNA

Abstract

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

“…However, the weak binding affinity of some mRNA/ gRNA pairs allows for the presence of unpaired RNAs, preventing accurate structure probing. To counteract this problem, the use of cross-linked RNAs facilitated base pairing of the anchor region and assured folding of the two molecules together (Leung and Koslowsky 2001a;Yu and Koslowsky 2006). 59-Cross-linked RNAs have been shown to support editosome assembly (Leung and Koslowsky 2001a).…”

“…Short (50-70 nucleotides [nt]) guide RNAs (gRNA) with complementarity to segments of pre-edited or partially edited mRNAs provide the sequence information necessary for the precise modifications . Each gRNA has three functionally distinct domains that seem to fold onto a similar structure containing one to two stem-loops (Schmid et al 1995;Yu and Koslowsky 2006). The gRNA anchor domain at the 59-end (4-16 nt) is complementary to the anchor-binding site (ABS) within the mRNA.…”

“…Joining the anchor helix and the U-tail helix is the central portion of the gRNA, or guiding domain, which dictates the type of editing (insertion or deletion) and the number of U's involved in the process. Annealing of the gRNA to the mRNA is proposed to form a threehelical structure that defines the editing site (Leung and Koslowsky 2001a,b;Yu and Koslowsky 2006) and allows editing to proceed mostly from 39 to 59 within the mRNA (Abraham et al 1988;Sturm and Simpson 1990;Koslowsky et al 1991;Maslov and Simpson 1992).…”

“…Only after cross-linking three gRNAs to their respective cognate mRNAs and incorporating the constraints into a computer modeling program were all three pairs shown to fold into a three-helical structure (Leung and Koslowsky 1999) similar to the structure of a fourth mRNA/gRNA pair confirmed previously by S1 protection assay . Through cross-linking and structure probing studies, a secondary structure model has been proposed for a CYb mRNA/gRNA pair (Leung and Koslowsky 2001a,b;Yu and Koslowsky 2006), and it shows the persistence of three helices even after editing of the first sites, raising the importance of this organization.…”

“…It has been suggested that gRNAs and mRNAs are initially bound to protein complexes and then brought together to assemble into a binary complex away from the editosome (Zikova et al 2008) or within the editosome (MadisonAntenucci et al 2002). Alternatively, the mRNA/gRNA complexes could form in the absence of proteins and be the signal for editosome recognition and editing Rusche et al 1997;Leung and Koslowsky 1999;Yu and Koslowsky 2006).…”