Heterochronic genelin-14 has a complex genomic DNA structure with 13 exons spanning over 20kb, and produces at least three different transcripts by alternative splicing of 5' exons. LIN-14 is a novel protein whose biochemical activity remains unknown. Based on a fully functional LIN-14::GFP fusion protein construct we made, a series of deletions of the LIN-14::GFP were then constructed and expressed under the control of the hypodermal-specific
col-10 promoter for the in-vivo assay. Judged by its rescuing activity in hypodermal cell lineages, an exon9-13 construct (
aa292-535) appears to be the minimum rescuing contstruct that we have tested so far. An exon10-13 construct does not rescue, arguing that exon9 is indispensable for executing LIN-14 function. In fact, the exon10-13 construct shows dominant negative activity, suggesting that this truncated LIN-14 polypeptide can interfere with endogenous
lin-14 activity. The data suggests that LIN-14 exons 1-8 are not strictly required for
lin-14 function and at least in the hypodermis, the alternative LIN-14 products predicted are also not essential. The results also shown that LIN-14 has an unconventional, extended NLS domain, rather than a more typical discrete NLS, as the whole region from
aa284-466 is required for efficient nuclear localization. In addition, we observed that deletion constructs that display
lin-14 activity like exon9-13 etc. show strong association with chromosomes during mitosis. Chromosomes show strong LIN-14::GFP fluorescence from metaphase to anaphase. In contrast, non-rescuing constructs like exon10-13 and exon11-13 shows no chromosomal association, suggesting the ability to associating with chromosomes is closely related to LIN-14 function. One model consistent with our data so far is that
lin-14 encodes a transcription factor with DNA binding activity in its more carboxy-terminal exons.