A pre-tRNA carrying intron features typical of Archaea is spliced in yeast

Segni, Gianfranco Di; Borghese, Lodovica; Sebastiani, Silvia; Tocchini‐Valentini, Glauco P.

doi:10.1261/rna.7138805

Cited by 13 publications

(14 citation statements)

References 31 publications

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“…In either case, it is the tRNA-splicing endonuclease that recognizes the splice sites (11). Previous work from Tocchini-Valentini and colleagues (12,13) has demonstrated that mature domain-independent splicing can occur in yeast or Xenopus if a sequence that will form a BHB structure is inserted into a model pre-tRNA or mRNA. Moreover, an exogenous archaeal endonuclease expressed endogenously in mammalian cells can splice a BHB structure inserted into a reporter mRNA (14).…”

mentioning

confidence: 99%

Long-distance splicing

Anderson

Staley²

2008

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

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With the draft sequence of the human genome came the surprise that there were fewer genes than imagined. From where does complexity spring if not from the number of genes in an organism? RNA splicing provides at least part of the answer. Pre-mRNA splicing by alternative pathways is well known to expand an organism's protein diversity by generating distinct protein isoforms. Beyond cis-splicing at a single locus (Fig. 1a), there is evidence for specialized cis-splicing that results from read-through transcription of adjacent loci followed by splicing to generate transcription-induced chimeras (TICs) from two genes (Fig. 1b) (1, 2), as in the TNSF12/TNSF13 chimera expressed in human T cells (3). In contrast to these cis-splicing events, trans-splicing joins exons from separate pre-mRNA transcripts. These transcripts can be encoded by different DNA strands at the same locus, as in trans-splicing of the mod(mdg4) gene in Drosophila, or by different alleles at the same locus, as for the lola gene, also in Drosophila (Fig. 1c) (4). All of these RNA splicing events involve transcripts from the same general region of the genome. In the work by Di Segni et al. in this issue of PNAS (5), the authors provide evidence suggesting yet another pathway to increase protein diversity, a pathway that involves cis-splicing of a single mRNA or trans-splicing of distinct mRNAs from distant genes by the tRNA-splicing machinery ( Fig. 1 d and e).Whereas splicing of nuclear pre-mRNA generally requires an RNA-protein machine, termed the spliceosome, splicing of eukaryotic tRNAs requires only proteins-an endonuclease, a ligase, and a phosphotransferase. These enzymes catalyze tRNA splicing in four steps (6, 7). First, the tRNA endonuclease cleaves both exon-intron junctions. This cleavage produces two half-tRNAs as well as a linear intron and at the cleavage sites, free hydroxyls at the cleaved 5Ј ends and 2Ј,3Ј-cyclic phosphates at the cleaved 3Ј ends. The next three steps are catalyzed by the tRNA ligase and involve the polynucleotide kinase, cyclic phosphodiesterase, adenylate synthetase, and ligase activities of this enzyme (7). First, the 5Ј-OH is phosphorylated and the 3Ј-cyclic phosphate is opened to form a 3Ј-OH and 2Ј-phosphate. Then, the two tRNA halves are ligated and, at least in vitro, the excised tRNA intron is also ligated, circularizing the intron (8). Finally, the 2Ј-phosphate is cleaved from the ribose. Just as pre-mRNA splicing generally occurs in the nucleus, pre-tRNA splicing in higher eukaryotes occurs in the nucleus, but pretRNA splicing in budding yeast occurs in the cytoplasm (9).How does the pre-tRNA splicing pathway recognize a substrate? In general, eukaryotic pre-tRNA is recognized for splicing, not by its intron sequences, as is largely the case in pre-mRNA splicing, but by the structure of the mature tRNA domain itself (10). In contrast, archaeal pre-tRNAs form a structure at the splice sites called a bulge-helix-bulge (BHB), which is required for recognition by the tRNAsplicing machinery. In either case, i...

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confidence: 99%

Long-distance splicing

Anderson

Staley²

2008

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

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“…1A, hybrid precursor 1; Thompson et al 1989). The BHB structure is typical of archaeal tRNA introns, and we have already demonstrated in vitro and in vivo that it is recognized and efficiently spliced by the eukaryal tRNAsplicing endonuclease (Fabbri et al 1998;Fruscoloni et al 2001;Di Segni et al 2005). It is conceivable that the BHB structure facilitates the folding of the tRNA body.…”

Section: Resultsmentioning

confidence: 99%

Splicing of mRNA mediated by tRNA sequences in mouse cells

Zamboni

Scarabino²,

Tocchini‐Valentini³

2009

RNA

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tRNA splicing is essential for the formation of tRNAs and therefore for gene expression. A circularly permuted sequence of an amber-suppressor pre-tRNA gene was inserted into the sequence encoding the mouse NEMO protein. We demonstrated that, in mouse cells, the hybrid pre-tRNA/pre-mRNAs can be spliced precisely at the sites of the pre-tRNA intron. This splicing reaction produces functional tRNAs that suppress amber codons as well as translatable mRNAs that sustain the NF-kB activation pathway. The RNA molecules extracted from mouse cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then applied the Archaea-express technology, in which an archaeal RNA endonuclease is expressed in mouse cells. We show that both the endogenous eukaryal endonuclease and the archaeal one cleave the hybrid pre-tRNA/pre-mRNAs in the same manner with an additive effect.

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“…In addition, an artificial CCA sequence was added at the tRNA 3Ј-end. A mature tRNA can be obtained from this precursor if the tRNA splicing endonuclease cleaves at the pretRNA splicing sites, the cut ends are ligated, and a cleavage by RNase P occurs, leaving a mature 5Ј-end and a 3Ј-terminal CCA in the tRNA (20,22). Very recently, a similar gene organization has been found in a red alga (23).…”

Section: A Pretrna Sequence Can Be Removed From the Middle Of A Premrnamentioning

confidence: 97%

“…In a previous study, we have shown that long noncanonical pretRNA molecules, containing the BHB structure, are correctly cleaved and ligated in S. cerevisiae, producing functional tRNAs (20). Here, we addressed the question whether the yeast tRNA splicing system can specifically splice mRNA sequences.…”

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confidence: 99%

Cis- andtrans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells

Segni

Gastaldi

Tocchini‐Valentini

2008

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

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The formation of chimeric mRNAs is a strategy used by human cells to increase the complexity of their proteome, as revealed by the ENCODE project. Here, we use Saccharomyces cerevisiae to show a way by which trans-spliced mRNAs can be generated. We demonstrate that a pretRNA inserted into a premRNA context directs the splicing reaction precisely to the sites of the tRNA intron. A suppressor pretRNA gene was inserted, in cis, into the sequence encoding the third cytoplasmic loop of the Ste2 or Ste3 G proteincoupled receptor. The hybrid RNAs are spliced at the specific pretRNA splicing sites, releasing both functional tRNAs that suppress nonsense mutations and translatable mRNAs that activate the signal transduction pathway. The RNA molecules extracted from yeast cells were amplified by RT-PCR, and their sequences were determined, confirming the identity of the splice junctions. We then constructed two fusions between the premRNA sequence (STE2 or STE3) and the 5-or 3-pretRNA half, so that the two hybrid RNAs can associate with each other, in trans, through their tRNA halves. Splicing occurs at the predicted pretRNA sites, producing a chimeric STE3-STE2 receptor mRNA. RNA trans-splicing mediated by tRNA sequences, therefore, is a mechanism capable of producing new kinds of RNAs, which could code for novel proteins.ENCODE ͉ G protein-coupled receptors ͉ genomics ͉ tRNA endonuclease I n higher eukaryotes, most genes contain one or more introns, which are removed by the spliceosomal complex in a regulated manner. When multiple introns are present, alternative splicing provides a way to increase the number of mRNAs from each gene and hence to enhance protein diversity. In lower eukaryotes, like the yeast Saccharomyces cerevisiae, only Ϸ250 genes of Ͼ6,000 contain an intron, and for most of them, a single intron is present. Splice-site sequences in yeast introns are characterized by a strict consensus, and alternative splicing is rare (1, 2).The formation of chimeric mRNAs constitutes another strategy that eukaryotic cells can use to increase proteome complexity. This phenomenon is much more widely spread than previously thought. Recent studies have shown that many of the human genes analyzed in the ENCODE project use exons lying outside their canonical boundaries (3, 4).Chimeric mRNAs can be generated by several mechanisms. In one way, an exon of one gene is fused to an exon of a different gene in a reaction mediated by the spliceosome complex. This fusion can occur in cis, when the two exons derive from two adjacent cotranscribed genes (5, 6), or in trans, when the two exons come from different premRNA transcripts (7,8). Another way for the generation of chimeric transcripts requires the use of a tRNA-splicing endonuclease. We have shown that an endonuclease of archaeal origin can carry out trans-and cis-splicing of mRNA in cultured mouse cells (9).The expression of tRNA genes in most organisms requires the accurate removal of intervening sequences. In Bacteria, pretRNA splicing is autocatalytic, whereas in Archaea a...

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A pre-tRNA carrying intron features typical of Archaea is spliced in yeast

Cited by 13 publications

References 31 publications

Long-distance splicing

Long-distance splicing

Splicing of mRNA mediated by tRNA sequences in mouse cells

Cis- andtrans-splicing of mRNAs mediated by tRNA sequences in eukaryotic cells

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