Worm Breeder's Gazette 11(3): 25

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Analysis of the rol-6 Trans-Splice Acceptor Site in vivo

Ruey Fen Liou, Richard Conrad, Jim Kramer and Tom Blumenthal

In the last WBG some of us (Conrad, Thomas, Spieth and Blumenthal) 
reported that a conventional gene (vit-2) can be converted into a 
trans-spliced gene by insertion of all of an intron except the splice 
donor into its 5' untranslated region.  We concluded that the primary 
signal for trans-splicing is the presence of a splice acceptor and 
branch site without a donor site upstream.  In order to learn more 
about the sequence requirements for a trans-splice acceptor, we are 
making mutants of the rol-6 acceptor and testing them in vivo.  The 
experimental system involves injection of a plasmid containing the 2.2 
kb HindIII fragment which carries the dominant roller allele of rol-6.  
We altered several nucleotides in the coding region which result in no 
change in the protein but which allow us to test specifically for the 
RNA product of the transgene.  When this plasmid is injected, rollers 
are obtained and the RNA is found to be trans-spliced exclusively at 
the normal site immediately preceding the AUG at which translation 
initiates.  Most transcripts contain SL1, but surprisingly, some begin 
with SL2.
If the UUCCAG which serves as the splice acceptor is converted to 
UUCCAA, rollers are nevertheless obtained.  Analysis of the RNA from 
these strains revealed that SL1 was spliced onto a different site (
CUUUAG), 20 nucleotides upstream of the normal SL1 acceptor, a site 
which is not used at all when the wild-type acceptor is present.  In 
this mutant, trans-splicing is relatively inefficient (Unspliced 
transcripts are also seen.) and no SL2-spliced products have been 
observed.  We have also tested a few less drastic alterations in the 
splice acceptor site.  One (AUUCAG) still splices solely at the normal 
acceptor, one (UAACAG) splices predominantly at the normal acceptor, 
and one (AAAAAG) splices solely at the upstream acceptor.  All three 
splice at reduced efficiency and only to SL1.  We hypothesize that 
when splicing of the RNA products from the tandem arrays is very 
efficient, which so far is true only with the wild-type splice 
acceptor, the localized SL1 snRNP concentration in the expressing 
cells is reduced, thereby allowing the normally less-favored SL2 snRNP 
to donate its 5' end.
To further test the idea that an acceptor site without a donor site 
upstream is the primary signal for trans-splicing, we inserted a 
consensus donor site (GTAAG) 131 nucleotides upstream of the trans-
splice acceptor.  RNA from transformants containing this construct was 
mostly cis-spliced from this novel donor site to the normal trans-
splice acceptor, but a small amount of SL1 trans-splicing still 
occurred at this site.  Our working hypothesis is that, if the right 
signals were present, cis-splicing, trans-splicing with SL1 and trans-
splicing with SL2 could all compete.  We don't yet know all the 
factors that may mitigate towards cis- or trans-splicing, but the 
quality of the U1 snRNP binding site (the donor site) is certainly 
important.  We are currently trying to locate a signal which biases 
towards an SL1- or SL2-specific splice.