Worm Breeder's Gazette 14(5): 60 (February 1, 1997)
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.
| 1 | Department of Biology, Indiana University, Bloomington, IN 47405 |
| 2 | Dept of Biochemistry, Biophysics and Genetics, UCSM, 4200 E. 9th Ave, Denver, CO 80262 |
By searching Genbank, we have identified C. elegans cDNAs encoding
proteins known in mammals to be needed for RNA processing: 3' end
formation and splicing. Full length antisense RNAs were generated by in
vitro transcription from plasmids (kindly supplied by Y. Kohara) and
injected into the gonads of adult worms in order to mimic the knockout
phenotype of these genes.
Injection of antisense RNA to the subunits of the 3' end formation
protein, CstF, which binds RNA downstream of the cleavage site and is
thought to be involved in cleavage site selection, resulted in an
embryonic lethal phenotype. This is perhaps not surprising as null
alleles of the Drosophila homolog of the 77K subunit of CstF are also
embryonic lethal (1,2).
To investigate the phenotypes of knockouts of genes involved in
splicing, antisense RNAs were injected into smg-2 animals to prevent
degradation of any resulting incompletely processed RNAs. Injection of
antisense RNA to the integral U1-associated protein, U1C, resulted in a
developmental delay phenotype. In contrast, injection of RNA antisense
to another integral U1 protein, U170K, caused embryonic lethality. The
difference in phenotype between knockouts of these two genes may simply
reflect injection of different concentrations of antisense RNA or
different maternal protein contributions. We have used RT-PCR to look
at processing of the myo-3 gene in these delayed or dead embryos. The
embryos from antisense-injected mothers accumulated large amounts of
incompletely cis-spliced and unprocessed pre-mRNAs suggesting that the
phenotype is indeed due to perturbation of splicing. Interestingly,
only the mRNAs from which both introns had been removed were found to be
trans-spliced, suggesting that either cis-splicing must occur in order
for trans-splicing to occur in vivo or trans-splicing is even more
sensitive to lack of U1 snRNP than is cis-splicing. The latter
explanation would be quite surprising, since trans-splicing in Ascaris
has been shown to be U1 snRNP-independent in vitro (3) (although the
possibility of these proteins serving as components of the SL snRNP has
not been investigated).
We have recently identified two U1A protein homologs arranged in
an operon. The two encoded proteins appear to be quite different from
one another, particularly at key residues within the second RNA binding
domain. Nevertheless, the antisense injection experiments indicate they
are functionally redundant: neither antisense RNA on its own gave a
detectable phenotype, but when both were injected together approximately
half the embryos died and the remainder exhibited delayed development,
hatching 48 hours after laying instead of the usual 12 hours. We have
not yet tested for cis- or trans-splicing defects in these embryos.
Overall, our results demonstrate that genes involved in mRNA
processing are vital to embryonic development and also confirm the power
of antisense technology in predicting the knockout phenotypes of genes.
(1)Mitchelson, A., M. Simonelig, C. Williams and K. O'Hare (1993)
Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic
suppression in Drosophilamelanogaster by suppressor of forked occurs at
the level of RNA stability. Genes Dev 7: 241-9
(2)Takagaki, Y. and J. L. Manley (1994) A polyadenylation factor subunit
is the human homologue of the Drosophila suppressor of forked protein.
Nature 372: 471-474
(3) Hannon, G. J., P. A. Maroney and T. W. Nilsen (1991) U small
nuclear ribonucleoprotein requirements for nematode cis- and
trans-splicing in vitro. J Biol Chem 266: 22792-5