Metzenberg S, Agabian N
Intercampus Program in Molecular Parasitology, University of California-San Francisco 94143-1204, USA.
Mol Biochem Parasitol. 1996 Dec 2;83(1):11-23. doi: 10.1016/s0166-6851(96)02742-9.
In Trypanosoma brucei, pre-mRNAs are joined to a 5' 39 nt spliced leader sequence by trans splicing, a process that has not been well characterized. We have asked whether the 3' splice site regions of human and yeast introns are able to substitute in vivo for the 3' spliced leader acceptor regions of trypanosome pre-mRNA sequences. The ability of heterologous sequences to participate in trans splicing in trypanosomes was assayed by chloramphenicol acetyltransferase (CAT) enzyme activity and/or the detection of spliced CAT mRNA. Four out of the six heterologous 3' splice site regions (human beta-globin intervening sequence (IVS)2, human c-myc IVS2, human factor-VIII IVS1, and yeast actin IVS) functioned as 3' spliced leader acceptor regions in T. brucei, while two did not show significant or detectable levels of CAT activity (human beta-globin IVS1 and human c-myc IVS1). In the case of the human beta-globin IVS1 however, lengthening of the polypyrimidine tract as a result of single purine to pyrimidine transversions produced an active acceptor in which the spliced leader addition site coincides with the 3' splice site of the beta-globin exon 2. These studies indicate that some, but not all 3' acceptor regions in humans can function as spliced leader addition sites in trypansomes.
在布氏锥虫中，前体mRNA通过反式剪接与一个5'端39个核苷酸的剪接前导序列相连，这一过程尚未得到充分表征。我们探究了人类和酵母内含子的3'剪接位点区域是否能够在体内替代锥虫前体mRNA序列的3'剪接前导序列受体区域。通过氯霉素乙酰转移酶（CAT）酶活性和/或检测剪接后的CAT mRNA，来测定异源序列参与锥虫反式剪接的能力。六个异源3'剪接位点区域中的四个（人类β-珠蛋白内含子序列（IVS）2、人类c-myc IVS2、人类因子VIII IVS1和酵母肌动蛋白IVS）在布氏锥虫中发挥了3'剪接前导序列受体区域的功能，而另外两个（人类β-珠蛋白IVS1和人类c-myc IVS1）未显示出显著或可检测到的CAT活性水平。然而，就人类β-珠蛋白IVS1而言，由于单个嘌呤到嘧啶的颠换导致多嘧啶序列延长，产生了一个活性受体，其中剪接前导序列添加位点与β-珠蛋白外显子2的3'剪接位点重合。这些研究表明，人类中的一些但并非所有3'受体区域都可以在锥虫中作为剪接前导序列添加位点发挥作用。
