Effects on mRNA splicing of mutations in the 3' region of the Saccharomyces cerevisiae actin intron.

Abstract

Point mutations, deletions, and a sequence context change were introduced at positions 3' to the internal conserved TACTAAC sequence-of the Saccharomyces cerevisiae actin intron. In vivo analysis of yeast mRNA splicing suggests that, in contrast to the importance of the polypyrimidine tract in metazoan introns, specific sequences in this region are not required for efficient excision of a yeast intron. However, a double point mutation near the 3' junction (GG/AC) does severely inhibit splicing. Although this mutagenesis of the 3' junction, as well as deletion of most nucleotides between the TACTAAC and the 3' junction, caused only a slight accumulation of primary transcript, the observed accumulation of lariat intermediate by these mutants demonstrates the significance of this region for a step(s) in the splicing process after lariat formation.Nuclear mRNA splicing in both Saccharomyces cerevisiae and higher eucaryotes is a two-stage process; the intermediates are exon 1 and a lariat intron contiguous with exon 2 (the lariat intermediate) (17). Steps in stage 1 include assembly of an RNA-protein complex on the primary transcript (the spliceosome) (3,10,12,29), cleavage at the 5' exon-intron junction, and subsequent formation of a 2'-5' phosphodiester bond between the phosphate at the 5' end of the intron and the ribose 2' hydroxyl of an A nucleotide within the intron. Stage 2 processes include cleavage at the 3' intron-exon junction and ligation of the two exons to produce mature mRNA. A free intron in a branched configuration is an additional product (8,26,32,36,40).Efficient splicing in yeast requires the integrity of the conserved junction sequences, 5' GTAPyGT.... .AG 3' and the internal conserved sequence (ICS), TACTAAC (9,11,14,15,19,20,25,27,30,39). However, the presence of these is not sufficient; these conserved sequences have been found in close proximity and in the correct juxtaposition in at least two transcripts, but are not recognized as defining an intron during posttranscriptional RNA processing (A. Newman and G. Taylor, personal communications). Analysis of intron mutations has demonstrated that there are sequence context and spacing constraints for efficient splicing. Changes in intron sequences adjacent to the 5' junction and ICS can dramatically effect the efficiency of splicing (28), and insertions at various positions within the yeast actin gene have demonstrated that splicing efficiency decreases as the distance increases either between the 5' cap structure and the intron, between the 5' junction and the ICS, or between the ICS and the 3' junction (5, 18). Yeast introns are relatively small (50 to 700 nucleotides) and are present near the 5' end of some primary transcripts.Splicing of mRNA in higher eucaryotes and S. cerevisiae has many common characteristics; however, there are some distinctions. In contrast to the strong conservation of specific intron sequences in yeast, only the GT and AG dinucleotides at the 5' and 3' intron junctions, respectively, are invariant in higher eucaryotes ...

Expression of maize Adh1 intron mutants in tobacco nuclei

“…Neither of these elements appears to be absolutely required for splicing in plant nuclei Filipowicz, 1989, 1991) even though in mammalian systems, branchpoint sequences (Reed and Maniatis, 1988;Wu and Manley, 1989;Zhuang and Weiner, 1989;Zhuang et al, 1989), polypyrimidine tracts (Freyer et a/., 1989;Fu et a/., 1988;Reed, 1989), and downstream exon sequences (Dominski and Kole, 1991 ;Kole, 1986, 1988;Reed and Maniatis, 1986) contribute significantly towards recognition of the 3' splice site. Yeast introns, which also lack polypyrimidine tracts, utilize a highly conserved branchpoint sequence to designate the 3' splice site (Fouser and Friesen, 1987;Langford et a/., 1984;Vijayraghavan et a/., 1986). The absence of these conserved elements in plant introns suggests that 3' splice site selection in plant nuclei follows different rules than in mammalian or yeast systems, possibly being dependent on factors unique to plant introns such as AUrich elements within the introns, or transitions in AUrichness across the intron/exon boundaries.…”

Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing

“…3 ′ SS mutations U301G, A302G, and G303-4C cause a block to exon ligation, resulting in the accumulation of lariat-3 ′ exon intermediate (Fouser and Friesen 1987;Vijayraghavan et al 1989). As for 5 ′ SS and BS ACT1-CUP1 reporter mutations, the Prp8-RP point mutants showed differential copper growth effects in the presence of different 3 ′ SS mutations.…”

Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes

“…In the case of S. cerevisiae, the BS consensus sequence is a highly conserved heptamer composed of "TACTAAC", but with large variability in its location, appearing as far as >100 nt upstream of the 3Јss (Pikielny et al 1983;Langford et al 1984;Fouser and Friesen 1987). Among other hemiascomycetous yeasts, the first two positions of the BS are less conserved (Bon et al 2003).…”