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RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo.

ABSTRACT: 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.

SUBMITTER: Tanaka N

PROVIDER: S-EPMC3162383 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo.

Tanaka Naoko N Meineke Birthe B Shuman Stewart S

The Journal of biological chemistry 20110711 35

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 trl1Δ cells against a fungal ribotoxin that incises the anticodon loop of ...[more]

PMID: 21757685

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Dual functions of yeast tRNA ligase in the unfolded protein response: unconventional cytoplasmic splicing of HAC1 pre-mRNA is not sufficient to release translational attenuation.

Project description:The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1(u) mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1(i) mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1(i) mRNA. Furthermore, the HAC1 5' UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1(u) mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

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tRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay.

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2022-10-10 | GSE190681 | GEO

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RtcB is the RNA ligase component of an Escherichia coli RNA repair operon.

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Project description:Signaling in the ancestral branch of the unfolded protein response (UPR) is initiated by unconventional splicing of HAC1/XBP1 mRNA during endoplasmic reticulum (ER) stress. In mammals, IRE1? has been known to cleave the XBP1 intron. However, the enzyme responsible for ligation of two XBP1 exons remains unknown. Using an XBP1 splicing-based synthetic circuit, we identify RtcB as the primary UPR RNA ligase. In RtcB knockout cells, XBP1 mRNA splicing is defective during ER stress. Genetic rescue and in vitro splicing show that the RNA ligase activity of RtcB is directly required for the splicing of XBP1 mRNA. Taken together, these data demonstrate that RtcB is the long-sought RNA ligase that catalyzes unconventional RNA splicing during the mammalian UPR.

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

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Structures of the noncanonical RNA ligase RtcB reveal the mechanism of histidine guanylylation.

Project description:RtcB is an atypical RNA ligase that joins either 2',3'-cyclic phosphate or 3'-phosphate termini to 5'-hydroxyl termini. In contrast to typical RNA ligases, which rely on ATP and Mg(II), catalysis by RtcB is dependent on GTP and Mn(II) with ligation proceeding through a covalent RtcB-histidine-GMP intermediate. Here, we present three structures of Pyrococcus horikoshii RtcB complexes that capture snapshots along the entire guanylylation pathway. These structures show that prior to binding GTP, a single manganese ion (Mn1) is bound to RtcB. To capture the step immediately preceding RtcB guanylylation, we determined a structure of RtcB in complex with Mn(II) and the unreactive GTP analogue guanosine 5'-(?-thio)triphosphate (GTP?S). This structure shows that Mn1 is poised to stabilize the pentavalent transition state of guanylylation while a second manganese ion (Mn2) is coordinated to a nonbridging oxygen of the ?-phosphoryl group. The pyrophosphate leaving group of GTP?S is oriented apically to His404 with the ?-nitrogen poised for in-line attack on the ?-phosphorus atom. The structure of RtcB in complex with GTP?S also reveals the network of hydrogen bonds that recognize GTP and illuminates the significant conformational changes that accompany the binding of this cofactor. Finally, a structure of the enzymic histidine-GMP intermediate depicts the end of the guanylylation pathway. The ensuing molecular description of the RtcB guanylylation pathway shows that RtcB and classical ATP- and Mg(II)-dependent nucleic acid ligases have converged upon a similar two-metal mechanism for formation of the nucleotidylated enzyme intermediate.

| S-EPMC3670941 | biostudies-literature

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