A functionally divergent paralog of the tRNA splicing ligase required for cell division but not tRNA maturation in Trypanosoma brucei

In eukaryotes a varying number of tRNAs undergo splicing, rendering them functional for protein synthesis; tRNA splicing is thus essential. Trypanosoma brucei ( T. brucei ) has a single intron-containing, tRNA Tyr , still requiring a fully functional splicing machinery. Splicing involves intron cleavage by the splicing-specific tRNA endonuclease complex, followed by exon ligation, which in some eukaryotes (including T. brucei ) is performed by the multi-domain enzyme tRNA Ligase (TRL1). Using phylogenetic comparative analysis, we and others showed the existence of a paralog of TbTRL1, which lacks the conserved ligase residues but maintains apparently intact kinase and phosphodiesterase domains. Here, we demonstrate that down regulation of the paralogous protein (we termed TbKCPD) by RNAi causes a growth defect, which cannot be rescued by providing a spliced version of tRNA Tyr . We also tested the functionality of the kinase and phosphodiesterase domains by genetic rescue with a recoded copy of the gene impervious to RNAi. We show that both domains are essential for viability. Global proteomics comparing RNAi induced to control cells unexpectedly revealed effects on proteins primarily involved in flagellar biogenesis and in line with this observation, we also show that TbKCPD leads to defects in cell division; an essential function that goes beyond splicing.