The down regulation of
lin-14 by
lin-4 in the late L1 is critical for the expression of successive larval developmental events. Both
lin-4 and
lin-14 3'UTR sequences are required in this down-regulation. However, the reporter genes gfp or luciferase fused to the
lin-14 3'UTR are not down-regulated efficiently by
lin-4, suggesting that features of the
lin-14 coding sequence or protein product are also important for sensitivity to
lin-4 action. We recently identified a 5.7kb
lin-14 genomic DNA sequence,
lin-14R, that (under the transcriptional control of the
col-10 promoter) rescues
lin-14(
n179ts) precocious phenotypes without causing a retarded phenotype, indicating that
lin-14R, which contains only exons 4-13 of
lin-14 sequence, produces functional LIN-14 that is properly down regulated. To visualize down-regulation of a transgene, a
lin-14R::gfp fusion was made by inserting GFP into the carboxy end of LIN-14. The
col-10::
lin-14R::gfp also rescues
lin-14(
n179ts) and the GFP expression appears to be down-regulated in hypodermal cells after the L1. We are going to use this
lin-14::gfp fusion to precisely define the cis-acting elements of the
lin-14 3'UTR required for
lin-14 down-regulation by
lin-4. To identify those
lin-14 coding sequences that influence the developmental regulation of
lin-14 protein level and those
lin-14 coding sequences that mediate the regulation of target gene expression, various N-terminal deletions of LIN-14 were fused to the
col-10 promoter and are being tested for rescue of
lin-14(
n179ts) and for proper down-regulation. So far, we have observed that a deletion construct (pVT322) containing only the last six exons (exon 8 to exon 13) is still capable of rescuing
lin-14(
n179ts) . pVT322 causes a partial retarded phenotype, even though the construct includes the wild type
lin-14 3'UTR and therefore was expected to be regulated by
lin-4. It is possible that the N-terminal exons of
lin-14 affect LIN-14 stability and therefore contribute to the down-regulation of LIN-14, while the last six closely clustered exons contain activity for regulation of target genes. We are investigating this model by converting all the
lin-14R deletion constructs to GFP fusions so that their expression and regulation can be compared and quantified by measuring the strength of GFP expression throughout development.