Human and fungal 3′ splice sites are used by Trypanosoma brucei for trans splicing

Abstract

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 β-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 β-globin IVS1 and human c-myc IVS1). In the case of the human β-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 β-globin exon 2. These studies indicate that some, but not all 3′ acceptor regions in humans can function as spliced leader addition sites in trypanosomes.