Worm Breeder's Gazette 13(4): 46 (October 1, 1994)

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.

Mono- and Polycistronic pre-mRNA Splicing in a C. elegans embryo extract

Scott Kuersten and Tom Blumenthal

Department of Biology, Indiana University, Bloomington, IN 47405

Our lab has been interested in the signals that specify SL1 and SL2
trans-splicing.  Rick Conrad has shown previously that SL1 is specified by
an AU-rich outron, a functional 3' splice site and the absence of a 5'
splice site.  In addition, we have shown that SL2 trans-splicing is
specific to downstream genes in polycistronic transcripts.  Recently we
have begun to study these RNA processing events in vitro using a crude
whole cell extract that is similar to Tim Nilsen's Ascaris extract, but
derived from C.elegans embryos.  When this extract is incubated with a
monocistronic substrate containing the outron, the first two exons and the
first intron of the rol-6 gene, a trans-spliced product is observed by
RT-PCR.  As expected, this product is strictly dependent on the addition of
ATP to the splicing reaction.  In addition, the added RNA gets
predominantly or entirely SL1, as rol-6 does in vivo, and trans-splicing
occurs at the natural trans-splice site.

One hypothesis  that is readily testable in vitro is that SL1 splicing is 
directed by the presence of the 5' cap near the trans-splice site. 
Conversly, SL2 could be spliced to downstream genes because they are left
uncapped after separation from the upstream gene by cleavage and
polyadenylation.  To test this idea, monocistronic pre-mRNA was
synthesized with and without a 5' cap, incubated with extract, and the
processed products tested for SL specificity using RT/PCR. The results show
that in vitro the presence or absence of the cap does not influence SL
specificity.  Both capped and uncapped transcripts received only SL1.

The extract did not originally show any cis-splicing activity.  However, we
found that addition of both creatine phosphate and creatine phosphokinase
to regenerate ATP not only improved the efficiency of trans-splicing, it
also resulted in accumulation of a product of the size predicted for the
trans-spliced product in which the intron had been removed.  This may
indicate a higher ATP requirement for cis- than for trans-splicing.
Furthermore, it was also found that arresting embryogenesis at the 200-cell
stage, using the FUDR procedure of Stroeher et al. (Develop. Biol. 163:
367, 1994), produced extract with a marked improvement in both cis and

When a polycistronic pre-mRNA was tested, the downstream gene received
both SL1 and SL2 (as assayed by RT/PCR).  Thus, preliminary experiments
suggest that this extract is capable of all three types of pre-mRNA splicing
that occur in C. elegans: Cis-splicing, SL1 trans-splicing at outrons and both
SL1 and SL2 trans-splicing at downstream trans-splice sites in
polycistronic pre-mRNAs.  Current experiments are directed at increasing
the efficiency of the extracts to allow us to measure the amount of
splicing using a non-PCR-based assay.  In vivo some downstream genes in
operons appear to receive exclusively SL2, while others receive a mixture
of the two SL's.  Although we have seen only the latter in vitro, we hope
to use the in vitro splicing system to learn the mechanism of
trans-splicing specificity.