lin-4 encodes a small RNA that acts as an antisense supressor of
lin-14 and
lin-28 translation. Based on Genefinder analysis of the genomic sequence,
lin-4 was predicted to be located in an 1.29kb intron between exon 1 and exon 2 of a host gene, which we have tentatively named
lho-1(
lin-4 host gene). As yet the function of the
lho-1 is not known, but
lho-1 was found to contain weak similarity to a small region of precursors of vasotocin and mammalian vasopressin. To clarify the structural and regulatory relationship between
lin-4 and
lho-1, we performed RT-PCR to determine the actual intron/exon structure of
lho-1 in the
lin-4 region, and to identify its 5' terminal sequence. We also used
lho-1::GFP fusions to identify
lho-1 promoter sequences. cDNA complementary to the 5' sequences of
lho-1 mRNA was synthesized from total C.elegans RNA using Tth DNA polymerase at 67 C (high temperature was required owing to apparent secondary structure in
lho-1 5' sequences). cDNA sequences were amplified by PCR using a SL1 primer and another primer specific to
lho-1 exon 3. Sequence analysis of the resulting PCR product showed that, contrary to Genefinder predictions, exon 1 of
lho-1 is 4.11 kb upstream of exon 2, located between bp 2229 to 2403 of cosmid F59G1.
lin-4 is located between bp 6168-6189 of F59G1, between exons 1 and 2. Exon 2 and 3 are as expected from Genefinder predictions. There is no in-frame ATG in exons 1 or 2, while in exon 3, there are three in-frame ATG codons, suggesting that the translation start site of
lho-1 is in exon 3. To identify the location of
lho-1promoter sequences with respect to the
lin-4 promoter, three different GFP fusion protein constructs were made with various lengths of
lho-1 5' sequences upstream of exon 2. (GFP was inserted inframe into
lho-1 exon3) These constructs were injected into
dpy-20 worms along with
dpy-20 cosmid and GFP expression was assayed in transformed lines. The shortest construct, containing only 750 bp upstream of exon 2 (essentially just the
lin-4 promoter sequence), showed no GFP expression. A longer construct, which includes 3.5kb upstream of exon 2, had weak GFP expression in HSN neurons, intestine, and a few neurons in the head and tail of adults. Animals transformed with the longest construct, which includes 4.48kb upstream of exon 2 including exon 1 and 370bp upstream of exon 1, displayed strong GFP expression in HSNs, vulva, intestine, neurons in the tail, and numerous neurons in the head along the pharynx. Expression was also seen in adults in a cell tentatively identified as ALA. From these results, we concluded that
lin-4 is located within a 4.11kb intron of
lho-1. The expression of
lho-1::GFP requires greater than 3.5kb upstream of
lho-1 exon 2, while
lin-4 expression requires only about 700bp of sequences upstream of exon 2, indicating that the expression of
lho-1 and
lin-4 are regulated seperately. The expression pattern of
lho-1::GFP fusions suggests that it may encode a protein that functions in the nervous system. To identify sequences responsible for the developmental regulation of
lin-4, we made a series of constructs with deletions in the
lin-4 upstream sequences contained in the 693bp
lin-4 rescuing fragment. The constructs were tested for
lin-4 function by transformation into
lin-4(0) worms along with
col-19::GFP plasmid. Deletions from bp 7-89 or from bp 96-117 of the rescuing fragment were found to eliminate
lin-4(0) rescuring activity, suggesting that positive cis-elements exist in the 5' terminal region of the 693bp fragment. Deletions from bp 294-470 or from bp 466-498 don't affect
lin-4 rescuring activity. In fact, these two deletions result in a precocious phenotype consistent with precocious and/or elevated expression of
lin-4. These results suggest that negative regulatory elements are contained in the regions from bp 294-470 and from bp 466-498. Future analysis of the precocious transgenic lines is required to confirm whether
lin-4 RNA is expressed precociously. Additional deletion analysis is being used to further localize the negative regulatory elements, and a yeast one-hybrid screen will be used to search for trans-acting regulators of
lin-4 transcription.