Worm Breeder's Gazette 14(3): 12 (June 1, 1996)

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.

Further adventures with antisense RNA: A tool for studying zygotic gene expression, too.

Patricia E. Kuwabara

MRC-Laboratory of Molecular Biology, Hills Road, Cambridge, England UK CB2 2QH

        Guo and Kemphues reported on the first successful use of in
vitro synthesised antisense RNA to phenocopy a maternal-effect embryonic
mutant phenotype, that of the  par-1 gene (Cell, 81: 611-620, 1995). 
More recently, a number of other labs have demonstrated the general
usefulness of this technique in generating phenocopies of other
maternal-effect mutations.  
        Here, it is reported that 1) Antisense RNA can phenocopy zygotic
mutations, which have effects at different developmental stages 2)
Antisense RNA can phenocopy predicted loss-of-function phenotypes of  C.
briggsae homologues of  C. elegans genes 3) The severity of phenocopies
produced by antisense RNA is influenced by position and 4) Antisense RNA
directed against an intron sequence fails to produce a mutant phenocopy.
These experiments were performed using the sex-determining gene  tra-2,
which promotes hermaphrodite development in XX animals; XX  tra-2(lf)
mutants are transformed into pseudomales. Klass et al. (Dev. Biol. 69:
329-335,1976) have shown that wild-type  tra-2 activity is required
throughout larval development, because animals carrying the 
tra-2(b202ts) allele develop as intersexes with variably masculinised
somatic gonad, tail, and germ line depending on the timing of
temperature shifts.  No maternal-effects are associated with  tra-2
         The  tra-2 antisense RNA studies were initiated to show that
the Cb-tra-2 homologue promotes hermaphrodite sex determination in  C.
briggsae. This approach was adopted because the sequences of Cb-TRA-2A
and Ce-TRA-2A are extremely diverged (only 43% identical) and it was not
possible to show that transgenic Cb-tra-2 has cross-species feminising
activity in C. elegans.  The progeny of C. briggsae hermaphrodites
injected with antisense RNA (~1 kb) corresponding to a 3' region of the
4.7 kb  Cb-tra-2 mRNA showed masculinisation of the somatic gonad,
vulva, and tail (truncated spike), including the absence of a vulva. 
Those animals injected with 5' antisense RNA (~1 kb) also produced
masculinised progeny, however the degree of masculinisation was more
extensive. For example, some animals injected with 5' tra-2 antisense
RNA developed male tails with a fan and ray, whereas those injected with
3' tra-2 antisense showed only truncation of the tail spike.
        Studies similar to those described above were also performed
using Ce-tra-2 to gain more information about the general utility and
properties of antisense RNA, because  Ce-tra-2 is a large genetically
well characterised gene whose wild-type activity is required throughout
larval development and in a variety of tissues.  As summarised in the
figure below, antisense RNA was synthesised against four non-overlapping
regions (labeled A-D) of the 4.7 kb (and 1.8 kb)  tra-2 mRNA and the
progeny of injected mothers were examined.  Although this study is
complicated by the presence of at least 2  tra-2 mRNAs (4.7 and 1.8 kb),
it was previously shown that the 4.7 kb  tra-2 mRNA encodes the primary
somatic feminising activity of the  tra-2 locus.  As shown, 
there is a strong correlation between the 5' - 3' location of the
sequences chosen for generating antisense RNA and the extent of somatic
masculinisation.  Region A antisense RNA injections produced
pseudomales, similar in phenotype to XX  tra-2(lf) mutants.  Region B
and C antisense RNA produced intersex animals, and Region D antisense
RNA produced animals with a female soma and male germ line (Mog
        Finally, given the rapid progress of the Sequencing Consortium
in generating genomic C. elegans gene sequences, it was of interest to
determine whether antisense RNA directed against intron sequences could
also produce a mutant phenocopy.  In the case of  Ce-tra-2, antisense
RNA (~600 nt) directed against a  tra-2 intron sequence failed to
produce somatically masculinised progeny in 9/9 injected animals. This
result suggests that antisense RNA is likely to disrupt wild-type gene
activity at the post-transcriptional level.
        To conclude, these results indicate that antisense RNA can
phenocopy not only maternal-effect mutations, but also zygotic mutations
that may have their effects at different stages of larval development. 
It is surprising that  tra-2 antisense RNA shows such perdurance. 
However, as the antisense RNAs used in these experiments were
synthesised without capping, which is a modification of the Guo and
Kemphues protocol, it is possible that uncapped RNAs might escape
degradation - a hypothesis that remains to be tested. These experiments
also suggest that it is preferable to synthesise antisense RNA
corresponding to either a 5' region of an mRNA or to use exon, but not
intron sequences when using genomic templates.  
        Thanks to Tim Schedl and Allen Jones for sharing their
unpublished antisense RNA results.