We have generated several deletions within the intrqn of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses. Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing. In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences. Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.The protein-coding sequences of many eucaryotic genes are interrupted by noncoding sequences, called introns, which are removed from the primary RNA transcripts by RNA splicing (1, 4,27). In the yeast Saccharomyces cerevyisiae, highly conserved sequences at the 5' splice site (GTATGT), 3' splice site (PyAG), and internal branch acceptor site (TACTAAC) have been documented as structural requirements for correct and efficient removal of introns from mRNA precursors (9,12,18,19,30,31,35). These highly conserved elements hatve been subject to intense study (7,11,19,24,28,38), whereas only little is known about the nonconserved sequences in yeast introns. Cellini et al. reported that a 15-base-pair (bp) deletion between the internal branch acceptor site and the 3' splice site of the yeast actin intron has no effect on splicing (6); a deletion of 170 nucleotides which leaves intact the first 13 nucleotides at the 5' splice junction and most of the 3' part of this intron also does not adversely affect splicing (12). On the other hand, splicing of a transcript with a shortened 81-nucleotidelong intron in a truncated yeast-acanthamoeba hybrid construct is impaired (19). In addition, certain deletions in the nonconserved regions adjacent to consensus sequences in the yeast rp5l gene intron reportedly do result in an accumulation of unspliced mRNA precursor, although the deletions do not affect the steady-state level of mRNA (29).The object of interest in this study was the 309-bp intron which interrupts the protein-coding sequences of the S. cerevisiae actin gene (13,26). To test whether nonconserved sequences in the actin intron are absolutely necessary for accurate, efficient splicing in vivo and whether there is a critical minimum length requirement for this intron, we introduced several deletions into specific regions. Into one of the shortened introns, which was not excised, several different sequences of similar length were inserted; since splicing was restored we concluded that the primary structure of the deleted sequence is not necessary, but the spacing it provides is required, for efficient splicing. Our findings especially led us to the conclusion that there is an essential minimum spacing requirement between the 5' splice site and the internal ...
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