Intron encoded sequences necessary for trans splicing in transiently transfected Brugia malayi.

Abstract

Trans splicing is a process found in a variety of organisms, including Leishmania [1], trypanosomes, a number of parasitic nematodes [2–6] and the free-living nematode Caenorhabditis elegans [7]. In nematodes, this process involves the addition of a 22 nt sequence, known as the spliced leader (SL), to the 5′ end of the nascent mRNAs [8]. In vitro biochemical systems employing nuclear extracts have been used to extensively dissect the trans splicing pathway in the intestinal parasite Ascaris suum [8–12]. These studies have resulted in the identification of a number of trans acting factors that are involved in trans splicing [11–13] and have also succeeded in identifying the structural factors in the SL pre-RNA that are necessary for correct processing of the nascent transcript [8]. Recently, a transient transfection system for the parasitic nematode Brugia malayi was employed to map the promoter domains in the sequences present upstream of the gene for the heat shock protein 70 (HSP70) homologue of B. malayi. The native HSP70 message is trans spliced in vivo, with the SL added at a site that is located 47 nt upstream of the translational start [14]. Surprisingly, mRNAs transcribed from B. malayi embryos transfected with a synthetic transgene consisting of 659 nt upstream of the HSP70 ORF (including the native SL addition site) fused to a luciferase reporter gene were not trans spliced [15]. However, transgenes consisting of in frame fusions of the HSP70 659 nt upstream domain, exon1, intron 1 and part of exon 2 were correctly cis and trans spliced [15]. Furthermore, subsequent experiments employing a similar construct in which most of exon 1