Worm Breeder's Gazette 10(1): 67

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.

Trans-splicing in C. elegans

S. Bektesh, K. Van Doren and D. Hirsh

Recently, an alternative form of RNA processing (transsplicing) has 
been shown to occur in C.  elegans (Krause and Hirsh, Cell 49, 753-761,
1987).  In this RNA processing reaction, sequences from one RNA 
molecule are joined in an intermolecular reaction to sequences in a 
second molecule.  mRNAs encoded by three of the four actin genes in C. 
elegans were shown to contain the same 22 nucleotides at their 5' 
ends as a result of transsplicing.  This sequence has been named the 
splice leader.
If the mechanism is trans-splicing, then by analogy with 
trypanosomes, the two precursor RNA molecules would be joined through 
a Y-branch intermediate in an intermolecular reaction.  Our 
experimental results indicate that a Y-branch intermediate is involved 
that can be cleaved with purified debranching enzyme isolated from 
vertebrate cells; therefore, trans-splicing is indeed the mechanism in 
C.  elegans.We were interested in characterizing other mRNAs that 
acquire the splice leader.  A cDNA library was constructed from poly 
A+ mRNA and screened using an oligonucleotide complementary to the 
splice leader.  More than 100 clones were isolated and about two dozen 
have been characterized and sequenced.  In an attempt to identify 
these clones as having homology to known genes, the DNA and deduced 
amino acid sequences were searched against existing databases.  Two 
clones have strong homology with two different ribosomal protein genes 
from rat and yeast and are therefore thought to encode ribosomal 
proteins in the nematode.
A second approach has been to examine published nucleotide sequences 
of C.  elegans genes for the presence of a splice acceptor sequence.  
If such a sequence is present 5' of the initiator ATG codon, and no 
splice donor sequence is found farther upstream, an oligonucleotide 
homologous to a portion of the coding sequence is constructed and used 
for primer extension sequencing of the mRNA.  In this way, the mRNAs 
coding for ubiquitin and glyceraldehyde-3-phosphate dehydrogenase have 
been shown to contain the splice leader at their 5' ends.
Not all mRNAs in C.  elegans acquire the splice leader.  Two 
approaches were taken to determine what proportion of the messages in 
C.  elegans acquire the splice leader.  First, quantitative dot blots 
were done using poly A+ mRNA and oligonucleotide probes.  Following 
hybridization, samples were quantitated in a scintillation counter and 
compared to poly A+ mRNA hybridized to an oligo dT oligonucleotide as 
a measure of all poly A+ mRNAs.  This work suggests that approximately 
10% of the poly A+ mRNAs in the worm acquire the splice leader.  A 
second approach asked what proportion of the translated proteins in an 
in vitro reaction are encoded by mRNAs containing the splice leader at 
their 5' ends.  Poly A+ mRNA was translated in a rabbit reticulocyte 
in vitro translation system either with or without the addition of an 
oligonucleotide complementary to the splice leader.  The products of 
the reactions were separated by two-dimensional gel electrophoresis.  
Approximately 10% of the proteins translated in vitro were arrested 
upon the addition of the oligonucleotide complementary to splice 
leader illustrating that these proteins are the products of mRNAs 
containing the splice leader.
Acquisition of splice leader does not appear to be developmentally 
regulated.  Northern analysis of RNA isolated from oocytes (generously 
provided by Brian Kennedy and Jim McGhee) and from several larval 
stages suggests that the splice leader is present on mRNAs in general, 
and on actin mRNAs in particular, throughout development.
Is this splice leader present in other organisms?  Northern analysis 
of RNAs from other nematodes (C.  elegans var.  Bergerac, C.   
briggsae, Panagrellus revividus, Haemonchus and Ascaris) revealed that 
these worms contain the same or a very closely related splice leader.  
However, Northern analysis of RNAs from yeast, trypanosomes, 
Drosophila, Dictyostelium, Xenopus and human revealed that the 
evolutionary conservation of this splice leader sequence does not 
extend to these organisms.