Authors
L Brouck1; D Licko1; L Jeacock1; Z Nare1; M Sardis1; J Smith2; E Gluenz3; AG Cook4; A Schnaufer1; 1 Institute of Immunology & Infection Research, University of Edinburgh, UK; 2 Institute of Infection, Immunity and Inflammation, University of Glasgow, UK; 3 Institute of Cell Biology, University of Bern, Switzerland; 4 Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, UK Discussion
Uridine insertion/deletion mRNA editing is essential for mitochondrial gene expression in kinetoplastids. This intricate process is directed by small guide RNAs (gRNAs) that provide the genetic information to generate mature, translation-competent mRNAs. Editing involves several macromolecule complexes that anneal gRNA and mRNA, cut the mRNA at specific sites, add or remove uridine nucleotides, and re-ligate the edited mRNA fragments. For each round of editing, this final step is performed by two RNA editing ligases, REL1 and REL2. While REL1 is essential for the survival of the sleeping sickness parasite Trypanosoma brucei, REL2 knockdown by RNAi has not produced any detectable phenotype. This is unexpected given that both ligases are active in vitro and that strong purifying selection is acting on both ligase genes, as indicated by their low dN/dS ratios. These observations suggest conserved function over evolutionary time.
By combining genomics and structure-function analyses, we aim to elucidate the respective roles of REL1 and REL2 in RNA editing. RNA sequencing of editing intermediates in T. brucei REL1 knockdown cells revealed the particular importance of this ligase in sealing two types of sites: i) those where editing usually stalls, potentially due to structural barriers, and ii) ‘non-canonical’ sites, where the mRNA appears to have been cleaved erroneously, suggesting a potential repair function. Moreover, a comparison of substrate requirements using recombinant proteins showed that REL1 is more tolerant than REL2 towards nucleotide mismatches in the gRNA-mRNA anchor duplex. This difference between the two ligases may stem from their highly divergent interdomain region, which is predicted to interact with the RNA substrates. We also generated REL2 null mutants in promastigote Leishmania mexicana using CRISPR-Cas9, confirming the non-essential nature of this enzyme across trypanosomatids and providing new opportunities to investigate the function of this ligase. REL1, on the other hand, could only be deleted from the L. mexicana genome in the presence of an ectopic REL1 gene copy.
In summary, our data confirm functional divergence of the two REL paralogs across trypanosomatid parasites and suggest a structural basis for differences in substrate specificity. 
