Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing

Fouser, Lynette A.; Friesen, James D.

doi:10.1016/0092-8674(86)90540-4

Cited by 144 publications

(140 citation statements)

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“…Because the DNA sequence upstream of the mature protein failed to reveal an in-frame ATG that could initiate translation of the protein, the methionine that initiates translation of the COXSb gene product (and hence the length and composition of the Vb leader peptide) was initially unclear. However, because this region contains the sequence TACTAAC, one of the highly conserved sequences found in all yeast introns (19,28,44), we presumed that the COX5b mRNA was spliced and that splicing was involved in the generation of a translational initiation codon. The existence and boundaries of the intron were confirmed experimentally (described below).…”

Section: Resultsmentioning

confidence: 99%

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

Cumsky¹,

Trueblood²,

Ko³

et al. 1987

Mol. Cell. Biol.

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In Saccharomyces cerevisiae, subunit V of the inner mitochondrial membrane protein complex cytochrome c oxidase is encoded by two Cytochrome c oxidase is a key enzyme in the regulation of cellular energy production in eucaryotes (18). As a member of the electron transport chain of the inner mitochondrial membrane, this enzyme catalyzes the reduction of oxygen in a reaction that is essentially irreversible (57, 59). Since this is the only irreversible reaction in the mitochondrial electron transport chain, it is equivalent to the committed step in a metabolic pathway. As such it is an important control point in the regulation of electron flow through the chain (18). At present, it is unclear how eucaryotic cells alter their cytochrome c oxidase activity levels in response to changes in energy demand. Is it by changing (increasing or decreasing) the number of assembled holoenzyme molecules in the inner membrane? Is it by modulating the activity of individual enzyme molecules? Or is it by the assembly of isologous forms of subunits, which have different effects on catalysis, into holoenzyme molecules (4,12,23,24,26)?To study these and related questions, we are examining various aspects of the biogenesis of cytochrome c oxidase in Saccharomyces cerevisiae. This enzyme consists of nine different polypeptide subunits (46); subunits I, II, and III are encoded by mitochondrial DNA, whereas subunits IV, V, VI, VII, Vlla, and VIII are encoded by nuclear DNA. The structural genes encoding all but one of these subunits (subunit VII) have been identified and characterized (6, 11-13, 20, 25, 30, 33, 42, 51, 53, 60, 61). Subunits I, II, and III as well as subunits IV, VI, and VIIa are each encoded by a unique gene. In contrast, subunit V is encoded by a small gene family composed of two nonidentical genes, COXSa and COXSb (12).Previously, we isolated and partially characterized both COXS genes (12, 13) and showed that the protein product of either can assemble into holocytochrome c oxidase. Subsequently, two other laboratories also isolated and sequenced * Corresponding author. (25,51). However, because only partial nucleotide sequences for COXSa and COXSb have been available until now the relation between COXSa and COX5b and this COXS gene has been unclear. In studies described here we demonstrate that COXSa and this recently described COXS gene are identical. We also demonstrate that COXSa and COXSb exist in single copy and are markedly different. They encode proteins that differ in 55 of 154 amino acids, their 5'-and 3'-flanking regions lack homology, and one of them, COXSb, possesses an intron that interrupts its initiation codon. In the accompanying paper (54) we describe the effects of null mutations in COX5a and COXSb on in vivo cytochrome c oxidase activity, cell respiration, and growth on nonfermentable carbon sources. We also demonstrate that these two genes encode interchangeable mature protein subunits that are expressed at markedly different levels. MATERIALS AND METHODSPlasmids, strains, and growth media. The...

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Section: Resultsmentioning

confidence: 99%

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

Cumsky¹,

Trueblood²,

Ko³

et al. 1987

Mol. Cell. Biol.

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“…It has been reported for the yeast Saccharomyces cerevisiae and for higher eucaryotes that changes of the invariant first G of the 5' splice junction lead to the accumulation of lariat structures which are not further processed (12,34,46). Mutations of the first nucleotide of the 5' splice junction in Saccharomyces cerevisiae clearly lead in vivo to the accumulation of splicing intermediates, and no mature message can be detected (12,34). For mutations in the highly conserved branch sequence 5'-TACTAAC-3', conflicting results have been reported.…”

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confidence: 99%

“…For mutations in the highly conserved branch sequence 5'-TACTAAC-3', conflicting results have been reported. 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).…”

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confidence: 99%

“…To analyze whether the mutations 5'-1A and B-4G accumulate splicing intermediates such as lariat structures, as reported for the yeast Saccharomyces cerevisiae (12,34,50), we performed primer extension analyses using a 17-nucleotide synthetic primer which hybridizes in exon 2, 20 nucleotides downstream of the 3' splice site. In both cases, we detected accumulation of pre-mRNA; however, we could not detect a primer extension product which mapped to the branch point, which would indicate accumulation of lariat structures (results not shown).…”

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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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“…It has long been known that mutating the 59 splice sites of nuclear premessenger RNAs can lead to the activation of cryptic 59 junctions that are ignored by the splicing machinery under normal circumstances (reviewed in Black, 1995)+ Although the factors that promote the use of most cryptic 59 splice sites have not been identified, a few examples have been reported in which activation was clearly due to base pairing with a specific snRNA+ The first of these was discovered through a genetic screen in Saccharomyces cerevisiae that sought extragenic mutations that allowed removal of an intron containing a Gϩ1A change at the natural 59 splice site (Newman & Norman, 1991)+ Splicing occurred not at the mutant junction, but rather at a cryptic 59 splice site which bore little resemblance to the S. cerevisiae consensus+ Cloning of the suppressor gene revealed that activation was due to a mutation in U5 snRNA that allowed base pairing with nucleotides just upstream from the cryptic 59 junction (Newman & Norman, 1991+ Similarly, mutant U5 snRNAs activated nonconsensus 59 splice sites via base pairing to nucleotides upstream from the scissile bond in mammalian cells (Cortes et al+, 1993)+ Other cryptic 59 splice sites were shown to be activated through a distinct mechanism: pairing between nucleotides downstream from the scissile bond and a highly conserved sequence in U6 snRNA+ Early studies had shown that mutating position ϩ5 of the 59 splice site in S. cerevisiae led to the accumulation of lariat intermediates resulting from transesterification at an aberrant upstream site (Jacquier et al+, 1985;Parker & Guthrie, 1985;Fouser & Friesen, 1986), but the molecular mechanism remained a mystery for several years+ In the wake of crosslinking data that placed U6 snRNA in proximity to the 59 splice site (Sawa & Abelson, 1992;Sawa & Shimura, 1992;Sontheimer & Steitz, 1993), compensatory base analysis was used to demonstrate that these aberrant 59 splice sites were activated via base pairing to U6 snRNA (Kandels- Lewis & Séraphin, 1993;Lesser & Guthrie, 1993)+ Like the U5/ exon interaction, the U6/intron interaction is important for splicing in mammals as well as yeast (e+g+, Crispino & Sharp, 1995;Hwang & Cohen, 1996)+ A third snRNA implicated in the selection of natural 59 splice sites is U1, and its potential role in activation of cryptic 59 splice sites has also been investigated+ These studies revealed, first, that RNA fragments containing three cryptic 59 junctions found in human b-globin can be coprecipitated with the U1 snRNP (Chabot & Steitz, 1987)+ Second, improving the complementarity of one of these to the 59 end of U1 snRNA increased its use (Nelson & Green, 1990)+ Third, mutating the first G of the large rabbit b-globin intron shifted the site of the first transest...…”

Section: Introductionmentioning

confidence: 99%

Activation of a cryptic 5′ splice site by U1 snRNA

Alvarez

Wise

2001

RNA

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In the course of analyzing 59 splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 59 junction containing a nonconsensus nucleotide at position 12. An even more unusual feature of this cryptic 59 junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 59 splice site participates in activation of the cryptic 59 splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 59 junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 59 splice site even in the presence of a wild-type sequence at the natural 59 junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 59 splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 59 junction.

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Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing

Cited by 144 publications

References 47 publications

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

Structural analysis of two genes encoding divergent forms of yeast cytochrome c oxidase subunit V.

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

Activation of a cryptic 5′ splice site by U1 snRNA

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