Barrett N L, Li X, Carmichael G G
Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA.
Nucleic Acids Res. 1995 Dec 11;23(23):4812-7. doi: 10.1093/nar/23.23.4812.
We have examined the influence of splicing signals on the stability of polyoma virus late RNAs in the nucleus. Late primary transcripts contain a single 5' splice site and three alternative 3' splice sites. In earlier work we showed that the presence of introns was not required for late RNA accumulation, however, the 5' splice site was essential, as removal of only the 5' splice site was sufficient to destabilize late RNAs up to 100-fold when compared with early RNAs. A complementary clone which retained the 5' splice site but which carried small deletions of all late region 3' splice sites produced wild-type levels of unspliced late RNA. In order to extend this work we have constructed additional types of mutants. Point mutations in the 5' splice site confirmed its importance for RNA stability. Other mutants included constructs in which the spacing between the 5' splice site and the late promoter was altered and 5' splice site insertion mutants where a 58 bp fragment containing the 5' splice site sequence was inserted separately at various restriction sites in the late region. Both types of mutants lacked all of the late 3' splice sites and had only a single 5' splice site. RNase protection analyses of late and early RNAs from these constructs revealed that moving the 5' splice site away from the late promoter (or from its normal context) destabilized late RNAs > 10-fold relative to the wild-type. We conclude that both 5' splice site integrity and its proximity to the late promoter play important roles in the nuclear stability of polyoma virus late RNAs.
我们研究了剪接信号对多瘤病毒晚期RNA在细胞核中稳定性的影响。晚期初级转录本包含一个单一的5'剪接位点和三个可变的3'剪接位点。在早期的工作中我们表明，晚期RNA积累并不需要内含子的存在，然而，5'剪接位点是必不可少的，因为仅去除5'剪接位点就足以使晚期RNA与早期RNA相比不稳定达100倍。一个保留5'剪接位点但对所有晚期区域3'剪接位点进行小缺失的互补克隆产生了野生型水平的未剪接晚期RNA。为了扩展这项工作，我们构建了其他类型的突变体。5'剪接位点的点突变证实了其对RNA稳定性的重要性。其他突变体包括改变5'剪接位点与晚期启动子之间间距的构建体以及5'剪接位点插入突变体，其中包含5'剪接位点序列的58 bp片段分别插入晚期区域的各个限制性位点。这两种类型的突变体都缺乏所有晚期3'剪接位点，并且只有一个单一的5'剪接位点。对这些构建体的晚期和早期RNA进行的核糖核酸酶保护分析表明，将5'剪接位点从晚期启动子移开（或从其正常环境中移开）会使晚期RNA相对于野生型不稳定10倍以上。我们得出结论，5'剪接位点的完整性及其与晚期启动子的接近程度在多瘤病毒晚期RNA的核稳定性中都起着重要作用。
