Worm Breeder's Gazette 10(2): 9

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.

C. elegans Trans-spliced Leader Exists as an Sm Precipitable Particle

Kevin Van Doren, James Bruzik, Joan Steitz and David Hirsh

Maturation of some mRNAs in C.  elegans involves the acquisition of 
a 22 nt leader spliced on the 5' end.  This is believed to occur 
through a trans-splicing mechanism.  The discovery of trans-splicing 
on mRNAs that also mature through cis-splicing of introns raises 
interesting questions.  What is the machinery responsible for trans-
splicing?  Are the same components used in both cis- and trans-
splicing?  Are there components unique to trans-splicing and to cis-
splicing?  How are the two mechanisms kept separate from one another 
to prevent splicing of the leader sequence to an inappropriate 3' 
splice acceptor site within the body of an mRNA?
We began to address some of these questions by examining the spliced 
leader precursor (preSL).  Computer analysis of the preSL sequence 
generated a striking secondary structure.  PreSL RNA is folded so that 
the 22 nt that comprise the spliced leader form a stem-loop structure 
with the cleavage point being the first base pair of the stem 
following the loop.  Also striking was a sequence with perfect 
conservation to the consensus Sm binding site (AA(U3-6)GG) found in 
snRNAs.  This suggested that preSL might be found in an snRNP-like 
particle.  To test the functionality of the Sm binding sequence, we 
assayed material that was Sm precipitable.  Cell free extracts were 
prepared from sodium hypochlorite treated embryos.  PreSL RNA is 
precipitable by four different patient sera recognizing snRNPs.  
However, a monoclonal antibody directed against the Sm determinant 
fails to precipitate preSL RNA as does patient sera specific for U1 or 
U2 snRNPs.
PreSL can also associate with proteins necessary to form an Sm 
precipitable particle in a heterologous system.  Using an in vitro 
transcribed preSL RNA and either Hela nuclear or cytoplasmic extracts, 
we have found that the preSL RNA becomes precipitable by the patient 
sera described above.
Using monoclonal antibodies generated against m(3) (2,2,7)G we have 
shown that the preSL RNA has a trimethyl cap, another feature 
characteristic of eukaryotic snRNAs.
PreSL RNA in C.  elegans has features similar to the snRNAs of 
eukaryotes that participate in cis-splicing.  It is bound by Sm 
proteins and possesses a trimethyl cap structure.  PreSL RNA is unique 
in that it contains the 5' exon to be spliced to the pre-mRNA during 
the trans-splicing reaction.  We suggest that preSL may serve two 
functions during trans-splicing.  First, it provides the 5' exon to be 
spliced to the pre-mRNA.  In addition, preSL may substitute for U1 in 
the trans-splicing reaction.