McCullough A J, Schuler M A
Department of Plant Biology, University of Illinois, Urbana 61801-3838.
Mol Cell Biol. 1993 Dec;13(12):7689-97. doi: 10.1128/mcb.13.12.7689-7697.1993.
cis-spliced nuclear pre-mRNA introns found in a variety of organisms, including Tetrahymena thermophila, Drosophila melanogaster, Caenorhabditis elegans, and plants, are significantly richer in adenosine and uridine residues than their flanking exons are. The functional significance of this intronic AU richness, however, has been demonstrated only in plant nuclei. In these nuclei, 5' and 3' splice sites are selected in part by their positions relative to AU-rich elements spread throughout the length of an intron. Because of this position-dependent selection scheme, a 5' splice site at the normal (+1) exon-intron boundary having only three contiguous consensus nucleotides can compete effectively with an enhanced exonic site (-57E) having nine consensus nucleotides and outcompete an enhanced site (+106E) embedded within the AU-rich intron. To determine whether transitions from AU-poor exonic sequences to AU-rich intronic sequences influence 5' splice site selection in other organisms, alleles of the pea rbcS3A1 intron were expressed in Drosophila Schneider 2 cells, and their splicing patterns were compared with those in tobacco nuclei. We demonstrate that this heterologous transcript can be accurately spliced in transfected Drosophila nuclei and that a +1 G-to-A knockout mutation at the normal splice site activates the same three cryptic 5' splice sites as in tobacco. Enhancement of the exonic (-57) and intronic (+106) sites to consensus splice sites indicates that potential splice sites located in the upstream exon or at the 5' exon-intron boundary are preferred in Drosophila cells over those embedded within AU-rich intronic sequences. In contrast to tobacco, in which the activities of two competing 5' splice sites upstream of the AU-rich intron are modulated by their proximity to the AU transition point, D. melanogaster utilizes the upstream site which has a higher proportion of consensus nucleotides. The enhanced version of the cryptic intronic site is efficiently selected in D. melanogaster when the normal +1 site is weakened or discrete AU-rich elements upstream of the +106E site are disrupted. Selection of this internal site in tobacco requires more drastic disruption of these motifs. We conclude that 5' splice site selection in Drosophila nuclei is influenced by the intrinsic strengths of competing sites and by the presence of AU-rich intronic elements but to a different extent than in tobacco.
在多种生物体中发现的顺式剪接核前体mRNA内含子，包括嗜热栖热菌、黑腹果蝇、秀丽隐杆线虫和植物，其腺苷和尿苷残基比其侧翼外显子丰富得多。然而，这种内含子AU丰富性的功能意义仅在植物细胞核中得到证实。在这些细胞核中，5'和3'剪接位点部分是根据它们相对于遍布内含子长度的富含AU元件的位置来选择的。由于这种位置依赖性选择方案，位于正常（+1）外显子-内含子边界的只有三个连续共有核苷酸的5'剪接位点可以有效地与具有九个共有核苷酸的增强外显子位点（-57E）竞争，并胜过嵌入富含AU内含子中的增强位点（+106E）。为了确定从富含AU的外显子序列到富含AU的内含子序列的转变是否会影响其他生物体中的5'剪接位点选择，将豌豆rbcS3A1内含子的等位基因在果蝇Schneider 2细胞中表达，并将其剪接模式与烟草细胞核中的进行比较。我们证明，这种异源转录本可以在转染的果蝇细胞核中准确剪接，并且正常剪接位点处的+1 G到A敲除突变会激活与烟草中相同的三个隐蔽5'剪接位点。将外显子（-57）和内含子（+106）位点增强为共有剪接位点表明，位于上游外显子或5'外显子-内含子边界的潜在剪接位点在果蝇细胞中比嵌入富含AU内含子序列中的位点更受青睐。与烟草不同，在烟草中，富含AU内含子上游的两个竞争5'剪接位点的活性受它们与AU转变点的接近程度调节，黑腹果蝇利用具有更高比例共有核苷酸的上游位点。当正常的+1位点被削弱或+106E位点上游离散的富含AU元件被破坏时，果蝇中隐蔽内含子位点的增强版本会被有效选择。在烟草中选择这个内部位点需要更剧烈地破坏这些基序。我们得出结论，果蝇细胞核中的5'剪接位点选择受竞争位点的内在强度和富含AU内含子元件的存在影响，但程度与烟草不同。
