Mechanism of action of a yeast RNA ligase in tRNA splicing

Greer, Chris L.; Peebles, Craig L.; Gegenheimer, Peter; Abelson, John

doi:10.1016/0092-8674(83)90473-7

Cited by 303 publications

(235 citation statements)

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“…The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6,7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).…”

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confidence: 99%

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

Chakravarty

Subbotin²,

Chait³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

105

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A cherished tenet of nucleic acid enzymology holds that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′ phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A(5′)pp(5′)N-in the case of polynucleotide ligases. RtcB exemplifies a family of RNA ligases implicated in tRNA splicing and repair. Unlike classic ligases, RtcB seals broken RNAs with 3′-phosphate and 5′-OH ends. Here we show that RtcB executes a three-step ligation pathway entailing (i) reaction of His337 of the enzyme with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate; (ii) transfer of guanylate to a polynucleotide 3′-phosphate to form a polynucleotide-(3′)pp(5′)G intermediate; and (iii) attack of a 5′-OH on the -N(3′)pp(5′)G end to form the splice junction. RtcB is structurally sui generis, and its chemical mechanism is unique. The wide distribution of RtcB proteins in bacteria, archaea, and metazoa raises the prospect of an alternative enzymology based on covalently activated 3′ ends. RNA or A(5′)pp(5′)DNA; and (iii) ligase catalyzes attack by a polynucleotide 3′-OH on A(5′)pp(5′)RNA/DNA to form a 3′-5′ phosphodiester bond and release AMP (1). The salient principle of classic ligase catalysis is that the high energy of the ATP phosphoanhydride bond is transferred via the enzyme to the polynucleotide 5′ end, thereby activating the 5′ end for phosphodiester synthesis, with AMP as a favorable leaving group. The shared chemical mechanism of classic RNA and DNA ligases is reflected in their conserved core tertiary structures and active sites (1).Classic ATP-dependent RNA ligases are encoded by diverse taxa in all three phylogenetic domains. In bacteria, fungi, and plants they function as components of multienzyme RNA repair pathways that heal and seal broken RNAs with 2′,3′ cyclic phosphate and 5′-OH ends (2-5). In the healing phase, the 2′,3′ cyclic phosphate end is hydrolyzed by a phosphoesterase enzyme to a 3′-OH, and the 5′-OH end is phosphorylated by a polynucleotide kinase enzyme to yield a 5′-monophosphate. The resulting 3′-OH and 5′-phosphate ends are then suitable for ATP-dependent sealing by RNA ligase. This "healing-and-sealing" pathway is responsible for tRNA splicing in fungi and plants (6, 7), for mRNA splicing in the fungal unfolded protein response (8), and for tRNA restriction repair during bacteriophage infection of Escherichia coli (9).An alternative "direct ligation" pathway for joining 2′,3′ cyclic phosphate and 5′-OH ends during mammalian tRNA splicing was discovered nearly 30 y ago (10-12), when it was shown that the 2′,3′ cyclic phosphate of the cleaved pre-tRNA is incorporated at the splice junction in the mature tRNA. (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 seali...

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confidence: 99%

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

Chakravarty

Subbotin²,

Chait³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

105

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“…In yeast and plants, after splicing, the 59 and 39 halves are ligated through a series of enzymatic reactions that require three activities of the tRNA ligase Rlg1 (Greer et al 1983;Phizicky et al 1986;Englert and Beier 2005): (1) a cyclic phosphodiesterase (CPDase) activity that opens the 29, 39 cyclic phosphate of the 59 exon, (2) a polynucleotide kinase activity that phosphorylates the 59 end of the 39 exon, and (3) an ATP-dependent ligase activity that joins the two exons together. Finally, the 29-phosphate remaining at the ligation junction is removed by the phosphotransferase Tpt1 (Culver et al 1993) to form the mature tRNA.…”

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confidence: 99%

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

Hopper

2014

Genes Dev.

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In eukaryotes and archaea, tRNA splicing generates free intron molecules. Although~600,000 introns are produced per generation in yeast, they are barely detectable in cells, indicating efficient turnover of introns. Through a genome-wide search for genes involved in tRNA biology in yeast, we uncovered the mechanism for intron turnover. This process requires healing of the 59 termini of linear introns by the tRNA ligase Rlg1 and destruction by the cytoplasmic tRNA quality control 59-to-39 exonuclease Xrn1, which has specificity for RNAs with 59 monophosphate.

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“…Joining of the exons involves a ligase that generates a mature sized tRNA bearing a splice junction 2Ј-phosphate (9). The ligase from yeast catalyzes four distinct chemical steps to effect ligation: the 2Ј-3Ј cyclic phosphate at the end of the 5Ј exon is opened to a 2Ј-phosphate by a cyclic phosphodiesterase activity; the 5Ј-OH at the beginning of the 3Ј exon is phosphorylated by a polynucleotide kinase activity in the presence of GTP; the 5Ј-phosphate is activated by adenylylation from ATP; and then ligation occurs with loss of the adenylate moiety (9 -11).…”

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confidence: 99%

A 2′-Phosphotransferase Implicated in tRNA Splicing Is Essential in Saccharomyces cerevisiae

Culver

McCraith

Consaul

et al. 1997

Journal of Biological Chemistry

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The last step of tRNA splicing in the yeast Saccharomyces cerevisiae is catalyzed by an NAD-dependent 2 -phosphotransferase, which transfers the splice junction 2 -phosphate from ligated tRNA to NAD to produce ADPribose 1؆-2؆ cyclic phosphate. We have purified the phosphotransferase about 28,000-fold from yeast extracts and cloned its structural gene by reverse genetics. tRNA splicing is essential in both the yeast Saccharomyces cerevisiae and humans, since both organisms contain tRNA gene families whose members all contain intervening sequences. Yeast has 10 such intron-containing tRNA gene families (of the approximately 45 total tRNA gene families) (see Ref. 1 for review), and humans have at least one intron-containing tRNA gene family (2). Since all known eukaryotic nuclear-encoded tRNA Tyr genes contain introns, it is likely that tRNA splicing is essential in all eukaryotes for processing of tRNA genes.tRNA introns are invariably located 1 base 3Ј of the anticodon, and this location is critical for the the first step of splicing. In both Xenopus and yeast the endonuclease binds the mature domain of the precursor tRNA, measures the length of the anticodon stem to locate the intron (3, 4), and excises it if the structure at the 3Ј splice site is correct (5, 6). The products of the reaction are exons bearing 2Ј-3Ј cyclic phosphates and 5Ј-hydroxyl groups at their ends, as shown in Fig. 1 (7, 8).Joining of the exons involves a ligase that generates a mature sized tRNA bearing a splice junction 2Ј-phosphate (9). The ligase from yeast catalyzes four distinct chemical steps to effect ligation: the 2Ј-3Ј cyclic phosphate at the end of the 5Ј exon is opened to a 2Ј-phosphate by a cyclic phosphodiesterase activity; the 5Ј-OH at the beginning of the 3Ј exon is phosphorylated by a polynucleotide kinase activity in the presence of GTP; the 5Ј-phosphate is activated by adenylylation from ATP; and then ligation occurs with loss of the adenylate moiety (9 -11). The result of ligation is a mature sized tRNA bearing a splice junction 2Ј-phosphate (see Fig. 1). A ligase present in wheat germ (12, 13), Chlamydomonas (14), and humans (15) also generates splice junctions with a 2Ј-phosphate, and the wheat germ protein is very similar in catalytic activities to the yeast enzyme (16,17). Since removal of the 2Ј-terminal phosphate prevents the yeast ligase from working in vitro (10, 18), the 2Ј-phosphate is likely formed at the splice junction when this ligase acts in vivo. The yeast ligase is known to be responsible for tRNA splicing in yeast, since conditional ligase mutants accumulate unligated tRNA exons under nonpermissive conditions (19). However, a second ligase, which uses a completely different chemical reaction and does not generate a splice junction 2Ј-phosphate, has been implicated in tRNA splicing in humans in vitro (20,21) and in Xenopus oocytes in vivo (22).Removal of the splice junction 2Ј-phosphate occurs by a highly unusual reaction: a 2Ј-phosphotransferase transfers the splice junction phosphate to NAD, forming th...

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Mechanism of action of a yeast RNA ligase in tRNA splicing

Cited by 303 publications

References 22 publications

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

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

Healing for destruction: tRNA intron degradation in yeast is a two-step cytoplasmic process catalyzed by tRNA ligase Rlg1 and 5′-to-3′ exonuclease Xrn1

A 2′-Phosphotransferase Implicated in tRNA Splicing Is Essential in Saccharomyces cerevisiae

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