Highlights d Engineering tRNA-synthetase interactions generates mutually orthogonal PylT/RS pairs d Combination of optimized pairs allows for efficient dual nonsense suppression d Site-specific incorporation of two orthogonal chemical handles in the same protein d Dual-color labeling and crosslinking of surface receptors on live mammalian cells

RtcB enzymes are novel RNA ligases that join 2,3-cyclic phosphate and 5-OH ends. The phylogenetic distribution of RtcB points to its candidacy as a tRNA splicing/repair enzyme. Here we show that Escherichia coli RtcB is competent and sufficient for tRNA splicing in vivo by virtue of its ability to complement growth of yeast cells that lack the endogenous "healing/ sealing-type" tRNA ligase Trl1. RtcB also protects yeast trl1⌬ cells against a fungal ribotoxin that incises the anticodon loop of cellular tRNAs. Moreover, RtcB can replace Trl1 as the catalyst of HAC1 mRNA splicing during the unfolded protein response. Thus, RtcB is a bona fide RNA repair enzyme with broad physiological actions. Biochemical analysis of RtcB highlights the uniqueness of its active site and catalytic mechanism. Our findings draw attention to tRNA ligase as a promising drug target.Escherichia coli RtcB exemplifies a new family of RNA ligases that directly seal 2Ј,3Ј-cyclic phosphate and 5Ј-OH ends (1-3). Direct ligation is thought to be the main pathway of tRNA splicing in animals and archaea (4 -6). By contrast, yeast and plants rely on a different mechanism of tRNA splicing in which the broken 3Ј and 5Ј ends are healed (converted to a 3Ј-OH/2Ј-PO 4 and 5Ј-PO 4 , respectively) and then sealed by a classical ATPdependent RNA ligase (7) (see Fig. 1). RNA ligases of the RtcB family are present in metazoa and protozoa, but not in fungi and plants. RtcB homologs purified from archaeal and mammalian cells can seal broken tRNA halves and are thereby imputed to be the catalysts of archaeal and mammalian tRNA splicing (2, 3). However, this scenario is complicated by the existence of a yeast/plant-like tRNA splicing pathway in mammalian cells (8 -12) and of analogous yeast-like RNA repair enzymes in many archaeal taxa (13-16). Definitive genetic evidence that RtcB is the sole essential agent of the repair phase of mammalian or archaeal tRNA splicing is lacking, and the available genetic evidence concerning the healing-sealing pathway in animals is equivocal. Genetic ablation of a murine homolog of a yeast-like pathway component Tpt1 (the enzyme that removes the 2Ј-phosphate at the splice junction; see Fig. 1) has no discernible phenotype (17), suggesting that the mammalian yeastlike pathway either is functionally redundant with direct ligation or is non-contributory to mammalian tRNA splicing. By contrast, siRNA-directed depletion of the mammalian RNA 5Ј-kinase (an ortholog of the kinase domain of yeast/plant tRNA ligase) elicited a defect in tRNA splicing in vitro (10). However, siRNA-directed depletion of mammalian RtcB was also reported to inhibit ligation of tRNA halves in cell extracts and to delay tRNA splicing in living cells (3). These findings leave unresolved the following key issues: (i) whether RtcB can suffice for tRNA splicing as the only source of tRNA ligase activity in a eukaryal cell and (ii) whether RtcB can perform other RNA repair functions in vivo. Here we address these questions by using budding yeast as a surrogate geneti...

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C‐terminal domain and via an N‐terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N‐terminal WD40 domain to the C‐terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high‐affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.