Worm Breeder's Gazette 13(5): 57 (February 1, 1995)
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, Dartmouth College, Hanover NH 03755. lin-14 activity is temporally down-regulated during wild type larval development by the action of a small antisense RNA product of lin-4. This down-regulation appears to be post-transcriptional. As a first step to investigate the mechanism by which lin-4 may be regulating Lin-14 protein levels, we have begun an analysis of the distribution of lin-14 mRNA on size-fractionated polysomes. If lin-4 inhibits lin-14 at the level of translational initiation, we would expect to find a developmental decrease in the fraction of large lin-14 RNA-containing polyribosomes. However, our principal finding is that there is no change in the size distribution of lin-14 polysomes between the L1, when lin-4 RNA is absent, and the L2, when lin-4 RNA is abundant. This is remarkable since the steady-state level of Lin-14 protein drops dramatically, and becomes essentially undetectable, by the L2 stage. Two possible explanations emerge from this data: First, Lin-14 protein may be made in the L2 but is tagged for rapid degradation, a process signaled by lin-4 RNA. Alternatively, the lin-14 polysomes may no longer be translationally competent in the presence of lin-4 RNA. (We have not yet eliminated the formal, yet seemingly unlikely possibility that lin-14 mRNA in L2 animals shifts from polyribosomes to some other macromolecular complex with an identical sedimentation profile.) A second (and expected) finding from our polysome studies is that the 22 nucleotide lin-4 RNA is detected in polysome fractions in approximately the expected molar ratio to lin-14 mRNA. Since lin-4 RNA is not translated, its cosedimentation with polysomes would seem to result from its association with lin-14. A test of whether this lin-4 polysome association is via the lin-14 3' UTR led to a third finding (and now nothing is surprising!): lin-4 cosediments with polysomes made from C. elegans extracts containing a lin-14 (n355n679) allele that reportedly lacks putative lin-4 binding sites. If the interaction of lin-4 RNA with polysomes is specific in these n355n679 extracts, the implication is that lin-4 is associating with a) other sequences in the lin-14 mRNA, b) with mRNA's besides lin-14 , or c) directly with ribosomes. This putative ribosome binding by lin-4 RNA, together with an antisense lin-4/lin-14 association, suggests an appealing model in which translation by ribosomes containing the lin-4/lin-14 complex is halted by a direct inhibition of the machinery of translational elongation or termination. So far, direct tests of the various models mentioned here have proved to be elusive. We are continuing cell fractionation studies to further characterize the translationally incompetent lin-14/lin-4 polysomes. We have also begun a thorough mutagenesis of the lin-4 transcribed region to identify structural features of lin-4 RNA involved in interactions with lin-14 RNA and with translational inhibition.