Insertion of Part of an Intron into the 5′ Untranslated Region of a Caenorhabditis elegans Gene Converts It into a Zraws-Spliced Gene

Abstract

In nematodes, the RNA products of some genes are trans-spliced to a 22-nucleotide spliced leader (SL), while the RNA products of other genes are not. In Caenorhabditis elegans, there are two SLs, SL1 and SL2, donated by two distinct small nuclear ribonucleoprotein particles in a process functionally quite similar to nuclear intron removal. We demonstrate here that it is possible to convert a non-trans-spliced gene into a trans-spliced gene by placement of an intron missing only the 5′ splice site into the 5′ untranslated region. Stable transgenic strains were isolated expressing a gene in which 69 nucleotides of a vit-5 intron, including the 3′ splice site, were inserted into the 5′ untranslated region of a vit-2/vit-6 fusion gene. The RNA product of this gene was examined by primer extension and PCR amplification. Although the vit-2/vit-6 transgene product is not normally trans-spliced, the majority of transcripts from this altered gene were trans-spliced to SL1. We termed the region of a trans-spliced mRNA precursor between the 5′ end and the first 3′ splice site an “outron.” Our results suggest that if a transcript begins with intronlike sequence followed by a 3′ splice site, this alone may constitute an outron and be sufficient to demarcate a transcript as a trans-splice acceptor. These findings leave open the possibility that specific sequences are required to increase the efficiency of trans-splicing.