Uridylyl insertion/deletion mRNA editing is essential for mitochondrial gene expression in Trypanosoma brucei and governed by multi-protein complexes called editosomes. The final step in each cycle of this post-transcriptional process is that of re-ligating the edited mRNA fragments. The ~20S RNA editing core complex contains two RNA editing ligases, REL1 and REL2, located, respectively, in a deletion and an insertion subcomplex. While REL1 is clearly essential for RNA editing, REL2 knockdown by RNAi has not resulted in a detectable phenotype. To explain these findings, alternative scenarios have been suggested: (a) REL2 is not functional in vivo; (b) REL1 can function in both insertion and deletion editing, whereas REL2 can only function in insertion editing; (c) REL1 has an additional role in repairing erroneously cleaved mRNAs. To further investigate respective functions of the two RELs this study used three complimentary approaches: (i) genetic complementation with chimeric ligase enzymes, (ii) deep sequencing of RNA editing intermediates after ligase inactivation, and (iii) evolutionary analysis. In vivo expression of two chimeric ligases, providing a REL2 catalytic domain at REL1’s position in the deletion subcomplex and a REL1 catalytic domain at REL2’s position in the insertion subcomplex, did not rescue the growth defect caused by REL1 ablation. Although the results were not fully conclusive they suggest that it is the specific catalytic properties of REL1 rather than its position within the deletion subcomplex that makes it essential. In order to identify in vivo substrates of REL1, specific editing intermediates that accumulated after genetic knockdown of REL1 expression were captured by 5’ linker and deep sequenced using Ion Torrent and Illumina technology. Analyses of such unligated editing intermediates with bespoke bioinformatics tools suggest that REL1 functions in deletion editing as expected, but also in the repair of miscleaved mRNAs, implying a novel role for this ligase. Neither role can be fulfilled by REL2, at least not with sufficient efficiency. Sequencing data also suggest that either REL1 is not involved in ligation of addition editing substrates, or that REL2 in this case can fully compensate for loss of REL1. REL1, REL2 and KREPA3 sequences were subjected to analysis using MEGA5 and the HyPhy package available on the Datamonkey adaptive evolution server. Results indicated that all three editosome genes are under much stronger purifying than diversifying selective forces. In general this selection pressure to conserve protein sequence increased from KREPA3 to REL2 to REL1, suggesting a requirement to maintain catalytic function for both ligases. Taken together, these experiments reveal a novel function for REL1 during RNA editing, providing a rationale for its essentiality. Deductively, the results also suggest REL2, which was previously thought to be non-essential, may still be required by the cell at its position in the addition subcomplex. Evolutionary analysis suggests that the RELs and KREPA3 are under the same evolutionary forces to maintain their respective functions in RNA editing.

<p>  </p> <p>Adenosine-to-Inosine (A-to-I) mRNA editing is a post-transcriptional modification of specific sites within the mRNA that has only recently been observed in filamentous fungi. In the wheat scab fungus <em>Fusarium graminearum,</em> this phenomenon has shown to be facilitated by FgTad2 and FgTad3, homologs of Adenine Deaminase Acting on tRNA (ADAT). Interestingly, these two proteins are constitutively expressed in all different life stages<em>, </em>in contrast to only the sexual stage-specific nature of A-to-I mRNA editing in <em>F. graminearum</em>. To understand the molecular mechanisms regulating this process, six candidate co-factor genes were identified which interact with FgTad2 and/or FgTad3, specifically during sexual reproduction. Deletion mutants of four candidate co-factor genes were successfully generated. All four mutants displayed normal asexual development of <em>F. graminearum</em>, but four mutants also altered sexual function. Those four mutant led to formation of morphologically normal perithecia and ascospores, but the perithecia failed to discharge ascospores. More interestingly, in <em>FGSG_10943 </em>deletion mutant, most of these ascospores germinated precociously within the perithecium. I also observed, that among the candidate co-factor genes which are specifically expressed during sexual reproduction, <em>FGSG_10943</em> was significantly upregulated during the later stage of sexual development. This gene is restricted in nature to only a few orders of fungi in the class Sordariomycetes that form dark pigmented ascocarps, particularly Hypocreales and Glomerellales. Taken together, these results indicate that the four candidate co-factor genes are dispensable for vegetative growth of the fungus and involved in ascospore discharge. <em>FGSG_10943</em> appears to be involved in autoinhibition of ascospores inside the perithecia and interact with FgTad2 during sexual reproduction to mediate A-to-I mRNA editing in <em>F. graminearum</em>.</p>