The heterochronic gene
lin-4 encodes a 22-nt RNA that represses the expression of
lin-14 and
lin-28 via their 3' untranslated regions after the L1. This activity of
lin-4 is necessary for the succession of diverse somatic developmental events through the first three larval stages. However,
lin-14 and
lin-28 can be repressed at the normal time even in the absence of
lin-4 activity if either one of the two genes is inactivated by mutation. Thus there is a
lin-4 -independent repression of
lin-14 and
lin-28 that mediates a positive regulatory relationship between them. Interestingly, this 'feedback loop' is also stage-specific. Like the
lin-4 repression, it is not observed in the early L1 but is evident by the L2. We have found that the
lin-4 -independent repression of
lin-28 shows the same unusual phenomenon seen for wildtype regulation of
lin-14 and
lin-28 . We have observed that despite a decrease in LIN-28 protein level of about 10-20 fold, no change in the mRNA level nor its association with polyribosomes occurs from early to late stages. This is true both in wildtype and in
lin-4 (0);
lin-14(ts) . We and others have interpreted the unusual nature of this regulatory phenomenon in the heterochronic pathway to mean that the repressive event occurs after translation initiation, during peptide synthesis or shortly after (1). The fact that this phenomenon occurs in the absence of
lin-4 indicates that the 22-nt
lin-4 RNA is not a core component of the mechanism. We believe that
lin-4 rather initiates or potentiates the post-initiation repression. The
lin-4 -independent repression of
lin-28 apparently involves sequences in the
lin-28 3' untranslated region that are distinct from the
lin-4 site. We also sought to determine whether
lin-4 activity is sufficient to repress
lin-28 when the
lin-4 -independent mechanism is not active. We have found by immnuoblot of synchronized late-stage populations that LIN-28 protein is as high in
lin-14(
n355sd) and
daf-12(
rh61) as it is in
lin-4(
e912) . We have determined for the
lin-14(
n355sd) strain that the
lin-4 RNA is indeed expressed. Thus, in strains in which
lin-4 is present,
lin-28 is not repressed if
lin-14 levels are high. We have attempted to establish an in vitro translation system for C. elegans in which to investigate the mechanism of this regulation. We have been successful in creating a mixed system in which C. elegans polyribosomes translate in a reticulocyte extract. With this system we have obtained evidence that the heterochronic gene mRNAs are actively translated at late stages. 1 Olsen and Ambros, 1999, and K. Seggerson, L. Tang, and E.G. Moss, submitted.