Worm Breeder's Gazette 14(5): 54 (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.
Department of Biological sciences, Gilman Hall, Dartmouth College, Hanover, NH 03755
The steady state level of LIN-14 protein decreases at least 10-fold from
the early L1 to the L2 stage as the result of a post-transcriptional
mechanism that somehow involves the 22 nucleotide regulatory RNA encoded
by lin-4. We are doing experiments designed to determine the mechanism
of lin-4 action. Surprisingly, lin-14 mRNA and lin-4 RNA are both found
associated with polysomes in the L2 and later stages though the level of
LIN-14 protein is reduced at least 10 fold compared to the L1 (WBG v13,
#5, p57). In the simplest interpretation, this suggests that the
mechanism of LIN-14 protein down regulation must be after the step of
translation initiation. However, as may often be the case when examining
an apparently novel regulatory process, so far we have been better able
to rule out several possible regulatory mechanisms rather than to prove
a specific mechanism.
Recent experiments demonstrate that:
1) there is no detectable change in the sedimentation profile of lin-14
mRNA from the L1 to the L2 in higher resolution experiments,
corroborating earlier results;
2) there is no evidence for sequestration of lin-14 mRNA in the nucleus;
3) there is no evidence for lin-14 mRNA accumulation in mRNPs
(classified as particles of size on the order of 20-40S), suggesting
that the site of lin-4 RNA action is at the polysomes;
4) the amounts of lin-14 mRNA on polysomes in the L1 vs. the L2 stages
differ by no more than a factor of two, suggesting that lin-4 RNA does
not have a significant effect on the level of lin-14 RNA;
5) the rate of lin-14 transcription in the L2 is similar to that in the
L1 stage as shown by nuclear run-ons, showing that lin-14 is regulated
entirely post-transcriptionally;
6) the lin-14 mRNA polyA tail length by RT-PCR is 12-30 nucleotides in
the L1 and remains unchanged in the L2, suggesting that regulatory
models correlating mRNA translation efficiency with polyA tail length
are not applicable in this case.
Provided there is no decrease in the half-life of lin-14 mRNA in
the L2, these results are consistent with the hypothesis that LIN-14
protein down regulation is a result of a lin-4 RNA mediated inhibition
of lin-14 mRNA translation at or after elongation. However, these data
are also consistent with a dramatic decrease in LIN-14 protein stability
- an intriguing possibilty since this would suggest that lin-4 RNA is
targeting a component of a protein degradation pathway to lin-14 mRNA
polysomes.