Archaeal 3′-phosphate RNA splicing ligase characterization identifies the missing component in tRNA maturation

Abstract

Intron removal from tRNA precursors involves cleavage by a tRNA splicing endonuclease to yield tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate. A tRNA ligase then incorporates this phosphate into the internucleotide bond that joins the two halves. Although this 3′-P RNA splicing ligase activity was detected almost three decades ago in extracts from animal and later archaeal cells, the protein responsible was not yet identified. Here we report the purification of this ligase from Methanopyrus kandleri cells, and its assignment to the still uncharacterized RtcB protein family. Studies with recombinant Pyrobaculum aerophilum RtcB showed that the enzyme is able to join spliced tRNA halves to mature-sized tRNAs where the joining phosphodiester linkage contains the phosphate originally present in the 2′,3′-cyclic phosphate. The data confirm RtcB as the archaeal RNA 3′-P ligase. Structural genomics efforts previously yielded a crystal structure of the Pyrococcus horikoshii RtcB protein containing a new protein fold and a conserved putative Zn 2þ binding cleft. This structure guided our mutational analysis of the P. aerophilum enzyme. Mutations of highly conserved residues in the cleft (C100A, H205A, H236A) rendered the enzyme inactive suggesting these residues to be part of the active site of the P. aerophilum ligase. There is no significant sequence similarity between the active sites of P. aerophilum ligase and that of T4 RNA ligase, nor ligases from plants and fungi. RtcB sequence conservation in archaea and in eukaryotes implicates eukaryotic RtcB as the long-sought animal 3′-P RNA ligase.ligation | tRNA biosynthesis | RNA processing E ukaryotes and archaea contain a number of intron-containing tRNA genes. After their transcription the intron is cleaved from the precursor tRNA by the splicing endonuclease. The resulting tRNA halves are then joined by a tRNA ligase to form mature-sized tRNA (1). The cleavage by the tRNA splicing endonuclease leaves the 5′ exon with a 2′,3′-cyclic phosphate terminus and the 3′ exon with a 5′-hydroxyl group (2). The wellknown multifunctional yeast tRNA ligase, a class I 5′-P RNA ligase (RNL) (3), is unable to directly join these ends together (4). Instead, the class I 5′-P RNL uses its 2′,3′-cyclic-3′-phophodiesterase and 5′-RNA polynucleotide kinase activities to yield a 2′-phosphate-3′-hydroxyl and a 5′-phosphate, which the yeast enzyme then joins via formation of a 2′-phosphate-3′,5′-phosphodiester bond in an ATP-dependent reaction. In plants, mature tRNAs are formed in a similar manner by the class II 5′-P RNL (5, 6).Although the 5′-P ligation pathway is known to exist in animals, almost 30 years ago a different RNA ligase activity was discovered in HeLa cell extracts (7). This 3′-P RNL ligates tRNA 3′-halves beginning with a 5′-hydroxyl, and 5′-halves ending in a 2′,3′-cyclic phosphate by incorporating this phosphate into the internucleotide bond that joins the two halves. Attempts to purify this activity to homogeneity were unsuccess...

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase

“…RtcB proteins seem to function in most, if not all, archaea as GTP-dependent tRNA-splicing ligases and join spliced tRNA molecules halves to form mature-sized tRNAs molecules e.g., in archaeal precursor tRNA-Trp. 21 The observation that the genomes of some archaea contain 2 open reading frames, previously predicted to function as DNA ligases, led to the discovery of a putative second family of archaeal RNA ligases. 22,23 The founding member of this putative RNA ligase family is the Pyrococcus abyssi reading frame Pab1020.…”

RNA ligase RtcB splices 3′-phosphate and 5′-OH ends via covalent RtcB-(histidinyl)-GMP and polynucleotide-(3′)pp(5′)G intermediates

“…(The junction phosphate in yeast tRNA splicing derives from the γ-phosphate of the NTP substrate for the 5′ kinase step.) Direct ligation languished until 2011, when three laboratories identified bacterial, archaeal, and mammalian RtcB proteins as RNA ligase enzymes capable of sealing 2′,3′ cyclic phosphate and 5′-OH ends (13)(14)(15). In support of a bona fide RNA repair function for RtcB in vivo, it was shown that E. coli RtcB is competent and sufficient for eukaryal tRNA splicing, by virtue of its ability to complement growth of yeast cells that lack the endogenous healing-and-sealing-type tRNA ligation system (16).…”

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

“…T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1)(2)(3)(4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5)(6)(7).…”