The Impact of mRNA Structure on Guide RNA Targeting in Kinetoplastid RNA Editing

Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5′ end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different “sets” of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a “bank” of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins

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

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistant with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events

mRNA/gRNA rate constants by Surface Plasmon Resonance.

<p>Representative SPR sensograms are shown with line fits. <b>A</b>. CYbU+gCYb-558. <b>B</b>. A6U+gA6-14. <b>C</b>. ND7-550+gND7-550. The association (k_on) and dissociation (k_off) rate constants represent the mean of a minimum of 3 runs (each run utilizing 3 different mRNA concentrations) and are listed with the error in parentheses. RU = resonance units.</p

Predicted secondary structures for the A6P1/gA6-14 and ND7-550/gND7-550 complexes.

<p>Sites where we observed gRNA-directed cleavages C1–C5, are indicated. ES = Editing Site. ES2* indicates the first editing site that is correctly cleaved in the A6P1/gA6-14 interaction. No cleavage at at the first editing site was observed for ND7-550. The gRNA-dependent cleavages for this substrate occurred at sites that are edited in the mature transcript (C2 - ES2, C3 - ES4 and C5 - ES6) and at sites that are not edited in the mature transcript (C1 and C4). The mRNA anchor binding sequence is shown in outline font.</p