Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Pathway of fungal tRNA splicing and domain organization of the canonical trifunctional tRNA ligase Trl1 versus the monofunctional Mucorales RNA ligase. (A) Intron removal by tRNA splicing endonuclease leaves 2′,3′-cyclic phosphate and 5′-OH ends on the broken tRNA halves. The tRNA exons are then joined by Trl1—a trifunctional tRNA ligase. Trl1 catalyzes two end-healing reactions, performed by a 5′-OH polynucleotide kinase domain and a polynucleotide 2′,3′-CPD domain, to generate the 5′-PO₄ and 3′-OH,2′-PO₄ termini required for sealing by an ATP-dependent RNA ligase domain. The 2′-PO₄ at the resulting splice junction is subsequently removed by the NAD ⁺-dependent RNA 2′-phosphotransferase enzyme Tpt1. (B) Fungal Trl1 consists of N-terminal ligase, central kinase, and C-terminal CPD catalytic modules. Mucorales species have a stand-alone RNA ligase enzyme homologous to the Trl1 ligase domain. Initial searches of Mucorales proteomes did not identify candidate orthologs of the Trl1 kinase and CPD domains.