Lariat structures are in vivo intermediates in yeast pre-mRNA splicing

Domdey, Horst; Apostol, Barbara L.; Lin, Ren‐Jang; Newman, Andrew; Brody, Edward N.; Abelson, John

doi:10.1016/0092-8674(84)90468-9

Cited by 384 publications

(225 citation statements)

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“…In Saccharomyces cerevisiae, when the A at position six is mutated, splicing is not completely prevented (12,50). This A has been shown to be the branch point to which the first G of the 5' splice junction is linked to form a lariat (9). The B-4G (A--G) mutation in the 5'-CTAAT-3' sequence of our intron also did not prevent splicing; however, splicing efficiency was drastically reduced, although cells containing this mutation were able to grow without supplementing uracil.…”

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confidence: 82%

“…Recently it has been shown that this sequence can serve in S. pombe as a branch sequence, forming a lariat with the A residue (30). In Saccharomyces cerevisiae, the highly conserved 5'-TAC TAAC-3' serves as a branch sequence (9,26,34). This sequence is found in all Saccharomyces cerevisiae introns and occurs 10 to 57 nucleotides upstream of the 3' splice site.…”

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confidence: 99%

“…In the following steps, the 3' splice site is cleaved and exon 1 and exon 2 are linked together. The intron is released as a free lariat (9,37,42,44).There is evidence that at least five different small ribonucleoprotein particles (snRNPs) are involved in this process (29). These trans-acting factors are involved in the recognition of introns by interacting with specific cis-acting signals such as the 5' splice site, branch sequence, and 3' splice site of the precursor RNA (3, 4, 7).…”

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confidence: 99%

“…In the following steps, the 3' splice site is cleaved and exon 1 and exon 2 are linked together. The intron is released as a free lariat (9,37,42,44).…”

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confidence: 99%

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Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Gatermann¹,

Hoffmann²,

Rosenberg³

et al. 1989

Mol. Cell. Biol.

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Insertion of a 36-base-pair (bp) synthetic oligonucleotide comprising the sequence 5'-GTAGGT(19N)CTAAT (4N)AG-3' into several different positions within the coding region of the naturally intronless ura4 gene of Schizosaccharomyces pombe leads to an efficiently spliced gene producing a functional product. This suggests that the proper signals within an intron are sufficient to initiate and complete a splicing event independent of the location of the intron in the gene. Point mutations in the 5' junction (5'-GTAGGT-3') and in the putative branch sequence (5'-CTAAT-3') affect splicing efficiency significantly. A G-to-A transition at the first nucleotide at the 5' splice junction (5'-ATAGGT-3') abolishes the use of the authentic splice junction and leads to the increased use of an alternative splice site. No functional product is produced from this transcript. An A-to-G transition of the second A in the putative branch sequence (5'-CTAGT-3') lowers the splicing efficiency drastically, but still results in a functional gene product. Furthermore, extension of the 36-bp intron to introns more than 180 bp in size abolishes splicing, suggesting that the splicing apparatus might be restricted to very short introns. We discuss the possibility that S. pombe introns represent a simple type of eucaryotic intron.Intervening sequences appear to be spliced from nuclear pre-mRNA by a common mechanism. The precursor RNA of eucaryotic cells is assembled into a spliceosome (splicing complex) in which the actual splicing event takes place (5,7,15,23). Splicing can be viewed as a stepwise dynamic process which includes recognition of the intron, assembly of the spliceosome, and the actual splicing events (3, 13, 39). During this process, the precursor RNA is converted into an intermediate lariat structure by linking the first guanosine residue of the intron to an adenosine residue within the intron via a 2'-5' phosphodiester bond; the exon 1-intron junction is simultaneously cleaved. In the following steps, the 3' splice site is cleaved and exon 1 and exon 2 are linked together. The intron is released as a free lariat (9,37,42,44).There is evidence that at least five different small ribonucleoprotein particles (snRNPs) are involved in this process (29). These trans-acting factors are involved in the recognition of introns by interacting with specific cis-acting signals such as the 5' splice site, branch sequence, and 3' splice site of the precursor RNA (3, 4, 7). In addition, specific snRNPsnRNP interactions also seem to play a role in the spliceosome assembly and in the splicing process itself (4, 24). However, the precise functions of each of the snRNPs are not yet fully understood (6,7,27,36). Single proteins are also involved in the splicing process, although little is known about their functions (28).It is interesting that the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe appear to have introns which differ from one another and from those in higher eucaryotes. In contrast to the introns i...

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confidence: 82%

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confidence: 99%

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confidence: 99%

“…In the following steps, the 3' splice site is cleaved and exon 1 and exon 2 are linked together. The intron is released as a free lariat (9,37,42,44).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Gatermann¹,

Hoffmann²,

Rosenberg³

et al. 1989

Mol. Cell. Biol.

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“…A characteristic of S. cerevisiue introns is the presence of the 3' internal consensus sequence TACTAAC, which has recently been shown to act as the lariat branch point in intron processing [20,211. By contrast, the internal intron consensus based on 13 S. ponzbe introns (including those of CDC17) is limited to the much less stringent sequence CTRAY (Table 1) …”

Section: Discussionmentioning

confidence: 99%

Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe

Barker

White

Johnston

1987

European Journal of Biochemistry

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We have sequenced a 4200-base-pair fragment of Schizosaccharomyces pombe DNA which encompasses the entire DNA ligase gene, CDC17. S1 mapping has enabled us to identify two small introns (40 and 62 nucleotides) at the 5' end of the coding region of the gene and their 3' internal conserved sequences match the CTRAY consensus found in other S. pombe introns. The major transcription initiation and 3' polyadenylation sites have been mapped and are preceded by higher eukaryotic-like TATA and AATAAA sequences respectively. Furthermore, the CDCl7 mRNA carries a poly(A) tail whose length (approximately 250 nucleotides) is typical of that found in higher eukaryotic mRNAs, and is in contrast to the much shorter polyadenylated sequences found for the mRNAs of the budding yeast, Saccharomyces cerevisiae. The deduced amino acid sequence of the S. pombe DNA ligase predicts a protein of 86182 daltons, and an overall 53% homology with the snme enzyme from S. cerevisiae. In particular, a stretch of 24 amino acids with 100% sequence homology spans the putative ATP-binding region which is also conserved in T4 and T7 bacteriophage DNA ligases.The cellular function of DNA ligase is to seal nicks in double-stranded DNA, and this enzymic activity is required predominantly during DNA replication for the joining of newly synthesised Okazaki fragments. Yeast cells appear to contain a single species of nuclear DNA ligase, and conditional temperature-sensitive mutants with a defect in this enzyme arrest in medial nuclear division with a typical cdc (cell division cycle) phenotype when transferred to the non-permissive temperature [l]. By functional complementation of such mutants we have been able to clone both the CDCY gene from Succharomyces cerevisiae [2], and the analogous DNA ligase gene, CDC17, from the fission yeast, Schizosaccharomyces pombe [3].Experiments with synchronous cultures of S. cerevisiae have shown that the CDC9 gene is periodically transcribed in the mitotic cell cycle just prior to the onset of DNA synthesis [4, 51. We have recently published the sequence of CDC9 [6], and drawn attention to repeated sequence elements in the upstream regulatory region which may be analogous in function to repeated sequences found upstream of the periodically expressed S. cerevisiae HO gene [7]. We have also identified a domain of the DNA ligase protein which appears to be conserved in both T4 and T7 bacteriophage DNA ligases and which could be the site of ATP binding [S].Budding and fission yeasts are distantly related from an evolutionary point of view, and there are fundamental differences in the organisation of their respective cell cycles [9]. For this reason, and as part of a comparative molecular study of these two yeasts we have determined the structure and sequence of the S. pombe DNA ligase gene, CDC17. The deduced amino acid sequence of CDCl7 shows greater than 50% overall homology with that of CDC9, and significantly,

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RNA Splicing and Genes

Sharp¹

1988

JAMA

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The splicing of long transcripts of RNA (copied from DNA in the cell nucleus) into smaller, specific mRNA (ready for export to the protein-producing machinery in the cytoplasm) is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing the 5' and 3' splice sites. This complex contains the evolutionarily highly conserved small nuclear RNAs (snRNAs) U2, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRNAs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

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Lariat structures are in vivo intermediates in yeast pre-mRNA splicing

Cited by 384 publications

References 28 publications

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Introduction of functional artificial introns into the naturally intronless ura4 gene of Schizosaccharomyces pombe.

Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe

RNA Splicing and Genes

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