Functional selection of splicing enhancers that stimulate trans-splicing in vitro.
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/C)GAC(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.
Boukis
L A
LA
Center for RNA Molecular Biology, Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Bruzik
J P
JP
D000818:Animals; D017164:Ascaris lumbricoides / Q000235:genetics; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D005091:Exons; D066298:In Vitro Techniques; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism
D012322:RNA Precursors; D012333:RNA, Messenger