Worm Breeder's Gazette 14(5): 56 (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.
Biological Sciences, Dartmouth College, Hanover, NH 03755
Mutations in the heterochronic gene lin-28 cause precocious development affecting a variety of lineages where diverse events specific to the second larval stage are skipped and later stage-specific events occur precociously. lin-28 encodes a protein with two potential RNA-binding domains: a cold-shock domain, which contains RNP1 and RNP2 motifs, and a pair of retroviral-type (CCHC) zinc finger motifs. We have constructed a lin-28:GFP fusion and have found that it is expressed in the cytoplasm of cells in diverse tissues, including hypodermis, muscle, and neurons, cell types affected in lin-28 mutants. Cold-shock domain proteins from Xenopus are known to be cytoplasmic RNA-binding proteins that mask mRNAs from translation. LIN-28 may act to negatively regulate its downstream targets by a similar mechanism. lin-28:GFP expression decreases from early to late postembryonic development. It is most intense in late embryos and early L1 larvae, dim in L2 larvae, and dim in some cells or undetectable in the L3 and L4 stages. When the fusion is crossed into a lin-4 mutant background, fluorescence is continuous throughout larval development. lin-4 encodes a small regulatory RNA that has been shown to down-regulate the expression of the heterochronic gene lin-14 after the L1 stage. LIN-4 is believed to bind seven complementary elements in the 3!UTR of lin-14. We used a double mutant allele of lin-14 to determine whether lin-4 has other targets in developmental timing regulation. The allele lin-14(n355n679ts) lacks the seven LIN-4 complementary elements (LCEs), so it is therefore insensitive to LIN-4 negative regulation, but it also has a ts mutation in the coding region that reduces its activity. An animal carrying this mutation grown at 20!C, produces adult alae at the appropriate time at the end of larval development. However, if a double mutant is made, lin-4; lin-14(n355n679ts), adult alae development is retarded, indicating that LIN-4!must have a target or targets other than lin-14. We compared the sequences of 3!UTRs of lin-28 from C. elegans, C. remanei, and C. vulgaris, and found that among the contiguous conserved sequences, which are less that 20% of the UTR, is a 15nt element that is complementary to the LIN-4 RNA. Computer folding predicts that LIN-4 and the complementary element in lin-28 would form a structure like those predicted to form between LIN-4 and the seven complementary elements in the lin-14 3!UTR: two stretches of contiguous base-pairing flanking a 6nt looped-out sequence of LIN-4. Deleting the LIN-4 complementary element (LCE) causes deregulated lin-28:GFP expression in late larval stages, and produces a dominant gain-of-function allele that causes a retarded phenotype. This retarded phenotype resembles those of lin-4 and lin-14(gf) mutants in the retarded development of the vulva and lateral hypodermis. However, unlike lin-4 or lin-14(gf), lin-28(gf) causes a proliferation of the lateral hypodermal seam cells, where as many as sixty seam cells exist on one side of the animal, which we interpret as reiteration of the double-cell division pattern specific to the L2 that lin-28(lf) mutants lack. This lin-28(gf) retarded phenotype demonstrates that the regulation of lin-28 activity is critical to normal developmental timing and indicates that lin-28 activity is part of a switch that controls choices of stage-specific fates. We have also found that lin-14 activity is required for the maintenance of lin-28:GFP expression in a lin-4 mutant. lin-4; lin-14(n179ts); lin-28:GFP animals at 25!C develop mostly normally and show diminished fluoresce in late stages. This finding indicates that lin-14 controls the timing of developmental events at least in part by controlling the level of lin-28 activity. It remains to be demonstrated whether or not lin-14 acts exclusively through lin-28 to control the succession of L2- to L3-specific fates. When we replaced the lin-28 3!UTR with the unc-54 3!UTR, then the expression of lin-28:GFP in lin-14(n179ts) continued to be expressed at late stages and caused retarded development. This raises the interesting possibility that lin-14 acts directly or indirectly through the lin-28 3!UTR to control developmental timing after the L1.