Worm Breeder's Gazette 14(5): 55 (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 Dartmouth College, Hanover, NH 03755
lin-14 has a complex genomic DNA structure with 13 exons
spanning over 20kb, and produces at least three different transcripts.
The predicted proteins encoded by the three transcripts are all
approximately 540aa but differ in their N-terminal regions. LIN-14 is a
novel protein whose biochemical activity remains unknown.
To investigate the structure and function of LIN-14, we tagged
the LIN-14 carboxy terminus with GFP to make a LIN-14::GFP fusion
protein, and the fusion protein was expressed under the control of
lin-14 native upstream genomic sequences (approximately 12kb). This
LIN-14::GFP construct was found to fully rescue a lin-14(null) mutant,
indicating that the LIN-14::GFP fusion protein is fully functional. A
series of deletions of the LIN-14::GFP were then constructed and
expressed under the control of the hypodermal-specific col-10 promoter.
(The smaller and better-characterized col-10 promoter was used instead
of the lin-14 promoter sequences for the convenience of making the
constructs).
We first asked what is the minimum segment of LIN-14 that is
capable of rescuing lin-14(n179ts) at nonpermissive temperature. Judged
by its rescuing activity in hypodermal cell lineages, an exon9-13
construct (containing aa292-535 of LIN-14) appears to be the minimum
rescuing contstruct that we have tested so far. An aa284-466 construct,
which contains an amino acid sequence well conserved between C. elegans,
C. briggsae and C. vulgarus, can also rescue n179 , though not as
strongly as the exon9-13 construct. 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, perhaps by interacting with
targets, or with endogenous LIN-14 protein.
The exon9-13 construct, expressed by the col-10 promoter, can
rescue a lin-14(null) mutation in the hypodermis, suggesting that LIN-14
exons 1-8 are not strictly required for lin-14 function. This result
suggests that, at least in the hypodermis, the alternative LIN-14
products predicted to be generated by alternative utilization of 5'
exons are also not essential.
We also measured the efficiency of nuclear localization of the
various truncated LIN-14::GFP proteins in transgenic worms. The results
show that LIN-14 has an unconventional, extended NLS domain, rather than
a more typical discrete NLS. The whole region from aa284-466 is
required for efficient nuclear localization and deletions from either
end of this domain significantly reduce the efficiency of nuclear
localization. Basic Arg/Lys clusters similar to a typical NLS consensus
sequence can be found at both ends of this domain, but non of them is
sufficient to bring LIN-14 to the nucleus. LIN-14 activity is not
required for its nuclear localization since a mutant exon9-13 construct
(containing the n179 point mutation 303R->G*) is localized in nucleus
equally well as the corresponding wild type exon9-13 construct, even
though it fails to show any rescue activity.
In the course of these experiments, we observed that deletion
constructs that display lin-14 activity (exon8-13, exon9-13, aa284-466)
show strong association with chromosomes during mitosis. Chromosomes
show strong LIN-14::GFP fluorescence from metaphase to anaphase. As the
chromosomes decondense after mitosis, LIN-14::GFP becomes strongly
re-localized back to the reformed nucleus. In contrast, the mutant
exon9-13 construct containing the n179 mutation does not associate with
metaphase or anaphase chromosomes (although it is nuclear localized).
Other non-rescuing constructs (exon10-13 and exon11-13) also show 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.
*Thanks to Brenda Reinhart and Gary Ruvkun for sharing n179 sequence
data.