We are interested in the molecular basis by which heterochronic genes control the temporal progression of C. elegans development.
let-7 mutations result in a retarded heterochronic phenotype in the hypodermis, partially suppress the precocious hypodermal heterochronic phenotype of
lin-14,
lin-28, and
lin-42 mutations (see Reinhart et al. this meeting), and do not permit proper activation of LIN-29 expression (Bettinger and Rougvie, pers comm).
let-7 hermaphrodites also die at the L4 to adult molt by bursting at the vulva, and do not lay a brood. Precocious mutations in
lin-14,
lin-28, and
lin-42 partially suppress this lethality by an unknown mechanism. We reasoned that additional precocious heterochronic mutations could be identified as suppressors of the lethality of
let-7(
n2853ts). In an F2 screen, we isolated 40 suppressor mutants from approximately 105 mutagenized genomes. One class of suppressors, consisting of 7 alleles, semi-dominantly suppress the lethality and the retarded heterochronic phenotype of
let-7 null mutants, and display a recessive precocious heterochronic phenotype. In addition we studied one mutation causing a similar phenotype,
n2914 that came from a screen for dominant
let-7 suppressors conducted by Basson and Horvitz (WBG 14(4)
p62). Two of the 8 alleles (
n2914 and
mg187) additionally displayed a sterile phenotype, potentially the null phenotype.
n2914, was mapped near
mec-8 on LGI (Basson and Horvitz, WBG 14(4)
p62). We confirmed and refined this map position using deficiency mapping with deletions that extend into this region. We showed that
n2914 failed to complement a heterochronic mutation
lin-41(
ma104), previously identified and broadly mapped to this region in the Ambros lab (Liu, PhD thesis), and that the
lin-41(
ma104) allele semi-dominantly suppressed
let-7. Animals homozygous for the canonical
lin-41(
ma104) allele are fertile, while
lin-41(
ma104)/nDf24 animals are sterile, indicating that
ma104 is probably not a null allele. In contrast,
lin-41(
n2914) acts genetically as a null allele. Cosmid C12C8 rescues the heterochronic phenotype of
lin-41(
n2914), and partially rescues the sterility. In addition, approximately one half of the
lin-41(
n2914) animals carrying the C12C8 array display a retarded phenotype, resembling that of
let-7(lf) mutants and opposite to the precocious phenotype of
lin-41(lf) alleles. This suggests that
lin-41 may function as a genetic switch to regulate developmental timing. We identified polymorphisms associated with
lin-41 alleles in the gene corresponding to C12C8.3. The
lin-41(
n2914) null allele is a small deletion that results in a frame shift predicted to truncate the C12C8.3 protein near the N-terminus, while the remaining identified mutations fall in the C-terminus.
lin-41 is predicted to code for 2 similar proteins, LIN-41A and LIN-41B, of 1143 and 1146 amino acids respectively, the result of an alternate splice. Both LIN-41 products contain an N-terminal RING finger (a zinc chelating domain thought to be involved in protein protein and/or protein nucleic acid interactions), a pair of B boxes (additional zinc chelating domains), a coiled coil domain and a C-terminal domain that contains six copies of a 26 amino acid repeat. Thus LIN-41 is a member of a growing family of RBCC (Ring finger-B box- Coiled coil) proteins, many of which have been implicated in disease. For example PML, TIF1, and Rfp were all identified as oncogenic translocations, SS-A/Ro is an auto-antigen in lupus, XPRF is required for proper midline development, and HT2A binds to the HIV Tat transactivator. LIN-41 is also similar to a number of C. elegans proteins including NCL-1, a cytoplasmic protein that regulates ribosomal RNA and rRNA synthesis.
let-7 encodes a small RNA (Reinhart et al, this meeting). We do not yet comprehend how the LIN-41 RING finger protein may interact with
let-7 RNA most directly, and other heterochronic genes in temporal patterning. The genetic data constrain possible models. Since haploinsufficient
lin-41 bypasses the need for
let-7,
lin-41 probably acts dowstream or in parallel to
let-7. Perhaps high dose of
lin-41 down regulates the target of
let-7, ie
lin-41 may negatively regulate a target that is positively regulated by
let-7 (or vice versa). We are currently using epistasis analysis to order
lin-41 with respect to other heterochronic genes in the pathway and are analyzing a LIN-41/GFP fusion construct to determine the expression pattern.