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

ODN-directed accessibility assays.

<p><b>A</b>. Representative images of 8% denaturing polyacrylamide gels. Each reaction contained a pre-hybridized ³²P-labeled mRNA (A6U, CYbU, ND7UHR3, or ND7-550) that was digested with RNase H for 1, 15, and 30 minutes upon addition of a specific ODN (1∶1, 1∶5, 1∶10, or 1∶30 mRNA to ODN ratio). “NO”: no ODN control. The digested products (<) are indicated. <b>B</b>. Percentage of RNase H digestion products. For comparison purposes, the amount of digested A6U shown in each graph was kept constant at 1∶1 ratio. The CYbU mRNA was not included because no digested products were detected. These data are the average of three experiments.</p

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

mRNAs aligned with gRNAs and ODNs.

<p>The mRNA anchor binding site (underlined) is complementary to the gRNA anchor and ODN. Watson-Crick (|), non-Watson-Crick (:) base pairs, mismatches (#) and the first editing site (ES, arrowhead) are indicated. The mRNA sequences continue at the 3′-end, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012235#pone-0012235-g002" target="_blank">figure 2</a>.</p

Solution Structure Probing of ND7-550/gND7-550.

<p>Representative image of a denaturing polyacrylamide gel. The ND7-550 mRNA was 5′-end-labeled and renatured alone or with gND7-550. 0: no enzyme control. 1.5, 3, and 4.5 Units: amount of Mung Bean nuclease. MB and T1 are Mung Bean and RNase T1 digests for sequence mapping. The position of the ABS and the region where the gRNA U-tail is predicted to bind on the mRNA are indicated.</p

Oligodeoxyribonucleotides.

<p>Oligodeoxyribonucleotides.</p