A thorough characterization of the splice junction revealed that a 2 0 -phosphomonoester-3 0 ,5 0 -phosphodiester linkage is established in this system [55, 72] . The same has been shown to be true for the yeast tRNA ligase [50] . Yeast (ScTRL1) and plant (exemplified by AtRNL from Arabidopsis thaliana) tRNA ligases-both identified by activity-guided chromatographic purification [50] [51] [52] -are multifunctional enzymes harboring all the activities required to join the 2 0 ,3 0 -cyclic phosphate and 5 0 -hydroxyl termini in three functionally independent domains [74, 75] (Fig. 3) . First, the 2 0 ,3 0 -cyclic phosphate is hydrolyzed by a cyclic phosphodiesterase (CPD) activity to yield a 2 0 -phosphate-3 0hydroxyl terminated 5 0 -exon [50, 76] . In a second step, the 5 0 -hydroxyl at the 3 0 -exon is phosphorylated by a kinase activity [50] . The actual ligation is preceded by ATPdependent adenylation of a lysine located within a KxxG consensus motif in the active site of the nucleotidyl transferase domain [50, 76] . Next, the enzyme transfers the adenyl moiety to the 5 0 -phosphate at the 3 0 -exon [50] . The adenylated RNA is then joined to the 2 0 -phosphate-3 0 -hydroxyl group at the 5 0 -exon with the concomitant release of AMP [51, 52] (Fig. 2B , left box, right branch and Fig. 3 ). Although RNA ligases of bacteriophage T4, fungi, and plants share important mechanistic features and key residues required for catalysis (Fig. 2B , left box and Fig. 3) , their overall sequence similarity is low [52] . However, the presence of the conserved nucleotidyl transferase domain with its ligase motifs in bacteriophage T4, fungal and plant RNA ligases suggests a common evolutionary origin for these enzymes [69] . Despite their relatedness, several differences exist between yeast, plant, and bacteriophage T4 ligases. For example, ligase-and kinase/phosphatase activities reside on separate polypeptides in bacteriophage T4 whereas yeast and plant tRNA ligases are multifunctional proteins harboring all activities required for ligation (Fig. 2B , left box, right branch and Fig. 3 ). Hydrolysis of 2 0 ,3 0 -cyclic phosphates by yeast and plant CPD domains yields 2 0 -phosphate-3 0 -hydroxyl products rather than the 2 0 ,3 0 -cis diol termini generated by T4 Pnkp (Fig. 2B , left box) [50, 55, 72, 76] . This arises from the distinct phosphoesterase domains employed by the bacteriophage T4 (aspartic acid based DxDxT phosphoesterase domain [77] ) and yeast/plant (2H phosphoesterase domain, characterized by two conserved histidines [78] ) ligation pathways [77, 79, 80] (Fig. 3) . In contrast to bacteriophage T4 ligases, the yeast and plant enzymes do not accept 3 0 -phosphate RNA substrates and ligation typically depends on the presence of a 2 0 -phosphate at the substrate termini [79, 81] . Yeast and plant RNA ligases generate splice junctions bearing a 2 0 -phosphate [72] . To convert this noncanonical 2 0 -phosphomonoester-3 0 ,5 0 -phosphodiester linkage into a 3 0 ,5 0 -phosphodiester, an NAD ? -dependent phosphotransferase, termed ScTPT1 in yeast, removes the 2 0phosphate generating ADP-ribose-1 00 ,2 00 -cyclic phosphate (Fig. 2B , left box, right branch) [82] [83] [84] [85] . Yeast and plant tRNA ligases also subtly differ with respect to substrate selection. While yeast tRNA ligase prefers tRNA exon halves over artificial substrates such as, e.g., linear introns [51] , the plant enzyme ligates circular introns as efficiently as tRNA exon halves in direct competition experiments [52] . Although a 5 0 -3 0 RNA ligase activity has been detected in biochemical experiments carried out with HeLa cell extracts [86] , no homologues of known 5 0 -3 0 RNA ligase proteins could be identified in animals [52] . It therefore seems likely that the 5 0 -3 0 RNA ligase in animals has diverged too far from the RNA ligases already known in order to be identified by existing algorithms or represents a completely novel type of enzyme. This view is supported by the recent identification of a 5 0 -3 0 RNA ligase in the cephalochordate Branchiostoma floridae (BfRNL) featuring a stand-alone ligase protein with weak homology to yeast and plant RNA ligases [87] . In this system, the cyclic phosphodiesterase and kinase modules (BfKinase/CPD or BfCLP1) do not reside on the ligase polypeptide (Fig. 2B , left box, right branch and Fig. 3 ). Enzymes catalyzing kinase (HsCLP1) [88] , cyclic phosphodiesterase (HsCNP) [89] [90] [91] , and phosphotransferase (HsTRPT1) [92] activities have been identified in humans. The fact that these enzymes can function in tRNA splicing pathways in vivo [93] [94] [95] has triggered speculation that a yet unidentified 5 0 -3 0 RNA ligase protein might also exist in humans [87] . In support of this assumption, the RNA kinase, HsCLP1, is an integral component of the tRNA endonuclease complex, suggesting the occurrence of coupled endonuclease and kinase reactions in humans [38, 88] . In contrast, tRNA exon half phosphorylation is associated with ligation in S. cerevisiae, where ligase and kinase activities both reside on ScTRL1. However, the relevance of the 5 0 -3 0 RNA ligase mechanism for tRNA splicing in vertebrates is under debate since the 2 0 -phosphotransferase is not essential in mice [96] .