Worm Breeder's Gazette 10(1): 71

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.

A RNA Duplex Denaturase Activity in C. elegans

M. Krause and H. Weintraub

An increasingly popular method to identify and study gene function 
is anti-sense mediated inhibition of gene expression.  Inhibition by 
anti-sense RNA presumably results from the annealing of a 
complementary RNA to its target message.  Anti-sense mediated 
inhibition has been used successfully in cultured cell lines, 
Dictyostelium, Drosophila and Xenopus oocytes.  Surprisingly, attempts 
to inhibit gene expression in Xenopus embryos were not successful.  It 
has since been shown that there is a developmentally regulated 
activity that denatures RNA:RNA duplexes in Xenopus (Bass and 
Weintraub, Cell 48, 1986).  The denaturing activity in Xenopus is 
found in embryos but not oocytes and therefore could account for the 
difference in anti-sense inhibition at these developmental stages.  
However, it should be noted that there is no direct evidence that this 
denaturase is relevant to previous failures of anti-sense inhibition 
in Xenopus embryos.
Because of our interest in employing anti-sense methodologies to the 
study of early development in C.  elegans, we were curious to see if C.
elegans also had a RNA:RNA duplex denaturase activity.  Following 
the protocols utilized for Xenopus, we made S-100 extracts from worms 
and assayed them for RNA duplex unwinding activity.  We find that C.  
elegans has such a denaturase and that it appears to be 
developmentally regulated.  S-100 extracts from either embryos, L1s or 
L2-L3s show the denaturase is abundant in embryos and L1s and 
relatively absent from L2s and L3s.  These developmental differences 
hold whether comparing microgram equivalent, cell equivalent or 
nuclear equivalent amounts of extract protein.
As with Xenopus, it is unclear if the worm RNA duplex denaturase is 
relevant to anti-sense experiments in C.  elegans.  If the denaturase 
becomes problematic, alternative approaches may circumvent the 
unwinding activity.  For example, it is not known if the unwinding 
activity is localized in the cell (eg.  nuclear vs.  cytoplasmic).  
Preliminary evidence from Xenopus embryos suggests a nuclear 
localization for the denaturase.  If the worm denaturase is nuclear, 
then cytoplasmic injections of anti-sense RNA could conceivably 
inhibit expression at the translational level.  In any event, it would 
be prudent to demonstrate that presumptive sense and anti-sense 
duplexes are stable and persistent before evaluating the success or 
failure of anti-sense RNAs in C.  elegans.As a footnote, a recent 
variation to the anti-sense theme has emerged in which modified 
oligodeoxynucleotides are used as the antisense substrate.  The 
modified oligos have been successful in inhibiting gene expression in 
Xenopus embryos and trypanosames.  The numerous modifications 
available are designed to either increase the intracellular half life 
of the oligo or cause functionally irreversible duplexes to form.  
Modified oligos appear to be the best available method for transient 
anti-sense mediated inhibition.