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