Functional selection of splicing enhancers that stimulate trans-splicing in vitro

Abstract

The role of exonic sequences in naturally occurring trans-splicing has not been explored in detail. Here, we have identified trans-splicing enhancers through the use of an iterative selection scheme. Several classes of enhancer sequences were identified that led to dramatic increases in trans-splicing efficiency. Two sequence families were investigated in detail. These include motifs containing the element ^G/_CGAC^G/_C and also 5′ splice site-like sequences. Distinct elements were tested for their ability to function as splicing enhancers and in competition experiments. In addition, discrete trans-acting factors were identified. This work demonstrates that splicing enhancers are able to effect a large increase in trans-splicing efficiency and that the process of exon definition is able to positively enhance trans-splicing even though the reaction itself is independent of the need for the 5′ end of U1 snRNA. Due to the presence of internal introns in messages that are trans-spliced, the natural arrangement of 5′ splice sites downstream of trans-splicing acceptors may lead to a general promotion of this unusual reaction.