The Yeast tRNA Splicing Endonuclease: A Tetrameric Enzyme with Two Active Site Subunits Homologous to the Archaeal tRNA Endonucleases

Trotta, Christopher R.; Miao, Feng; Arn, Eric A.; Stevens, Scott W.; Ho, Calvin; Rauhut, Reinhard; Abelson, John

doi:10.1016/s0092-8674(00)80270-6

Cited by 218 publications

(267 citation statements)

References 39 publications

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“…Reactions were incubated for 30 min at 37°C and applied to silica thin layer chromatography plates (J. T. Baker), and products were separated in buffer containing n-propyl alcohol:NH 4 (24). p*-intermediate was made from 12-30 fmol of p*ApA P pA (or p*ApApA P POCH3 ) in the presence of 5 mM NAD, and products were purified by chromatography on silica thin layer plates developed in buffer containing ethanol and 1 M NH 4 OAc, pH 7.2, 7:4.5 v/v, followed by elution in water and drying. Although this procedure yields intermediate that is partially contaminated with some RNA substrate and product (because of the poor separation on these plates), the eluted intermediate lacks NAD and is active (see "Results").…”

Section: Methodsmentioning

confidence: 99%

“…During the first two steps of splicing, this nucleotide bears a 2Ј-3Ј-cyclic phosphate (4,27), and then a 2Ј-phosphate (28) before the half-molecules are joined to generate a splice junction phosphodiester with a 2Ј-phosphate (28). As described here, the 2Ј-phosphate is then modified by addition of an ADP-ribose adduct before removal of the phosphate as ApprϾp (19) and formation of tRNA with a splice junction 2Ј-OH (17).…”

Section: Yeast and Mouse 2ј-phosphotransferases Can Catalyze Step 2 Omentioning

confidence: 99%

“…Splicing in these organisms is initiated by a highly conserved endonuclease that excises the intron (3)(4)(5), followed by joining of the two half-molecules by one of two different ligases (6 -13). In the yeast Saccharomyces cerevisiae, in which the process is best studied, ligation occurs by a four-step reaction, producing a splice junction with a 2Ј-phosphate (14,15).…”

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Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast

Spinelli¹,

Kierzek²,

Turner³

et al. 1999

Journal of Biological Chemistry

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The last step of tRNA splicing in yeast is catalyzed by Tpt1 protein, which transfers the 2 -phosphate from ligated tRNA to NAD to produce ADP-ribose 1؆-2؆-cyclic phosphate (Appr>p). Structural and functional TPT1 homologs are found widely in eukaryotes and, surprisingly, also in Escherichia coli, which does not have this class of tRNA splicing. To understand the possible roles of the Tpt1 enzymes as well as the unusual use of NAD, the reaction mechanism of the E. coli homolog KptA was investigated. We show here that KptA protein removes the 2 -phosphate from RNA via an intermediate in which the phosphate is ADP-ribosylated followed by a presumed transesterification to release the RNA and generate Appr>p. The intermediate was characterized by analysis of its components and their linkages, using various labeled substrates and cofactors. Because the yeast and mouse Tpt1 proteins, like KptA protein, can catalyze the conversion of the KptA-generated intermediate to both product and the original substrate, these enzymes likely use the same reaction mechanism.Step 1 of this reaction is strikingly similar to the ADP-ribosylation of proteins catalyzed by a number of bacterial toxins.tRNA introns occur widely in Eukarya and Archaea (1, 2). Splicing in these organisms is initiated by a highly conserved endonuclease that excises the intron (3-5), followed by joining of the two half-molecules by one of two different ligases (6 -13). In the yeast Saccharomyces cerevisiae, in which the process is best studied, ligation occurs by a four-step reaction, producing a splice junction with a 2Ј-phosphate (14, 15).A single essential gene (TPT1) encodes the 2Ј-phosphotransferase responsible for removal of the splice junction 2Ј-phosphate from ligated tRNA (16,17). This reaction is unusual because the 2Ј-phosphate is transferred to NAD (18), producing mature tRNA and ADP-ribose 1Љ-2Љ cyclic phosphate (ApprϾp) 1 (19). The TPT1 gene product is involved in this step in vivo because a conditional yeast tpt1 mutant, when depleted for the gene product, accumulates at least eight ligated tRNA species bearing a splice junction 2Ј-phosphate (17). This presumably is the essential function of Tpt1 protein. Examination of four of these tRNAs demonstrated that they are also undermodified specifically at the splice junction residue (17).The yeast Tpt1 protein is part of a family of functional 2Ј-phosphotransferases found in Eukarya (Schizosaccharomyces pombe, Candida albicans, Arabidopsis thaliana and Mus musculus), and Eubacteria (Escherichia coli), with other likely members in another bacterial species and several Archaea (46). Expression of the eukaryotic phosphotransferase genes and the E. coli gene (kptA) complements a yeast tpt1 mutant, and the corresponding proteins catalyze the same reaction as the yeast protein, producing ApprϾp from ligated tRNA and NAD. The widespread occurrence of 2Ј-phosphotransferases in Eukarya is consistent with the ubiquitous presence of intron-containing tRNAs and ligases that generate 2Ј-phosphorylated substrates ...

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Section: Methodsmentioning

confidence: 99%

Section: Yeast and Mouse 2ј-phosphotransferases Can Catalyze Step 2 Omentioning

confidence: 99%

mentioning

confidence: 99%

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Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast

Spinelli¹,

Kierzek²,

Turner³

et al. 1999

Journal of Biological Chemistry

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“…In yeast and vertebrates, tRNA splicing is catalyzed by the heterotetrameric tRNA splicing endonuclease (SEN) complex, yielding the 59 half with a 29, 39-cyclic phosphate, the intron with a 59 hydroxyl and 29, 39-cyclic phosphate, and the 39 half with a 59 hydroxyl (Knapp et al 1979;Trotta et al 1997;Paushkin et al 2004). In vertebrates, tRNA splicing occurs in the nucleus (Melton et al 1980;Lund and Dahlberg 1998;Paushkin et al 2004), whereas it is a cytoplasmic process in yeast, since the SEN complex is located on the outer surface of mitochondria (Yoshihisa et al 2003).…”

mentioning

confidence: 99%

“…In vertebrates, tRNA splicing occurs in the nucleus (Melton et al 1980;Lund and Dahlberg 1998;Paushkin et al 2004), whereas it is a cytoplasmic process in yeast, since the SEN complex is located on the outer surface of mitochondria (Yoshihisa et al 2003). Despite the difference in localization, the structure and function of the SEN complex are conserved from yeast to humans (Trotta et al 1997;Paushkin et al 2004).…”

mentioning

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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Sequence analysis of two cosmids from the right arm of theSchizosaccharomyces pombe chromosome II

Lucas¹,

Lyne

Lépingle³

et al. 2000

Yeast

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We report the complete sequence of two cosmids, SPBC19C7 (34815 bp insert, Accession No. AL023859) and SPBC15D4 (33203 bp insert, Accession No. AL031349), localized on chromosome II of the S. pombe genome. Twelve open reading frames (ORFs) were identified in SPBC19C7 and 16 in SPBC5D4. Two known genes were found on each cosmid: cyr1 and uve1 on SPBC19C7, encoding adenylate cyclase and a UV‐endonuclease, respectively, and gpt and pho2 on SPBC15D4, encoding an N‐acetylglucosamine‐1‐phosphate transferase and a4‐nitrophenylphosphatase, respectively. Five ORFs similar to known proteins were found on SPBC19C7, and six on SPBC15D4. They include putative genes for a ubiquitin protein ligase, a prolyl‐tRNA synthetase, a tRNA splicing endonuclease, a voltage‐gated chloride channel, a mannosyl transferase, a kinesin‐like protein, a histone transcriptional regulator, an N‐acetyltransferase, a cystathionine γ‐synthase and a TFIID subunit. Two ORF products of SPBC15D4 do not have clear homologues: one encodes a putative transcriptional regulator with a binuclear zinc domain and the other a protein with six transmembrane domains. Two ORFs from SPBC15D4 are similar to unknown ORFs, one from Saccharomyces cerevisiae and the other from Caenorhabditis elegans. Finally, two ORFs of SPBC19C7 and six of SPBC15D4 correspond to orphan genes. The frequent occurrence of introns and the short and degenerated intron–exon boundaries consensus sequences significantly complicated ORF predictions. Two potential ORF‐free regions spanning several kb were predicted, and a clustering of ORFs transcribed in the same orientation was observed. Copyright © 2000 John Wiley & Sons, Ltd.

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The Yeast tRNA Splicing Endonuclease: A Tetrameric Enzyme with Two Active Site Subunits Homologous to the Archaeal tRNA Endonucleases

Cited by 218 publications

References 39 publications

Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast

Transient ADP-ribosylation of a 2′-Phosphate Implicated in Its Removal from Ligated tRNA during Splicing in Yeast

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

Sequence analysis of two cosmids from the right arm of theSchizosaccharomyces pombe chromosome II

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