Worm Breeder's Gazette 13(3): 37 (June 1, 1994)

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.

The laf-1 Gene Functions in the 3'UTR-mediated Translational Repression of the Sex Determination Gene, tra-2 .

Elizabeth Goodwin[1], Judith Kimble[2]

[1]Dept. of CMS Biology, Northwestern Univ. Medical School, Chicago Ill.
[2]University of Wisconsin, Madison, WI

The sex determination gene tra-2 specifies female cell fates in C. elegans. Loss of tra-2 activity transforms XX hermaphrodites into males. Six dominant gain-of-function (gf) alleles of tra-2 have been identified [Doniach, (1986) Genetics 114;53-76; Schedl and Kimble, (1988) Genetics 119;43-61] which cause enhanced tra-2 activity. All six tra-2 (gf)alleles feminize the XX germline; the germline produces only oocytes. The strongest gf allele also feminizes males: the intestine produces yolk and the germline produces a few oocytes. Therefore, the gf alleles identify a negative regulation of tra-2 that is necessary for hermaphrodite development and continued specification of proper sexual cell fates in the male. The tra-2 (gf)alleles disrupt a 60nt direct repeat located in the tra-2 3' untranslated region (3'UTR). We have shown that this direct repeat is necessary for inhibiting the translation of tra-2 mRNA, and that a binding factor specifically associates with the direct repeat (Goodwin et al., ( 1993) Cell 75,329-339). Our working model is that the binding of factor to the direct repeat inhibits translation of the tra-2 mRNA.

In order to identify trans-acting factors and further delineate cis-acting elements that are involved in translational control of tra-2 ,we have developed a transgenic assay in which expression of lac-Z is controlled by the tra-2 3'UTR. We have integrated into the wild-type genome transgenes that contain the HSP16 .41promoter, the lac-Z coding region, either the wild-type tra-2 3'UTR or a tra-2 (gf)3'UTR, and 1kb of tra-2 3' flanking region. After heat-shock, transgenes with the wild-type 3'UTR show little ß-gal activity. However, transgenes with a tra-2 (gf)3'UTR show strong ß-gal activity. By RNAse protection analysis, the wild-type and mutant transgenes produce approximately equivalent amounts of RNA, indicating that the differences in ß-gal activity are due to translational regulation.

The laf-1 gene (for lethal and fog) may control tra-2 translation. laf-1 maps about 1.5 m.u. to the left of daf-2 on III. All five laf-1 alleles cause a semi-dominant feminization of the germ line (the Fog phenotype); in addition, all five are homozygous larval lethal. Finally, at 25°C, two laf-1 alleles ( q21 7 and q80 )show defects in germline meiosis. The larval lethality and meiotic defects suggest an involvement of laf-1 in controlling other processes other than sex determination.

Four lines of evidence suggest that laf-1 activity influences tra-2 translational regulation. First, in a laf-1 /+mutant background, translation of the transgene carrying the tra-2 wild-type 3'UTR is increased. However, in fem-3 , fog-1 ,or fog-2 mutant backgrounds, translation of the transgene is not affected, indicating that the enhanced ß-gal activity is specific to laf-1 rather than feminization per se. Second, at 20°C, about 25% of the laf-1 /+animals produce only oocytes. Germline feminization was predicted for a defective translational regulator of tra-2 .However, the semi-dominance was not predicted, but might be explained by complete translational inhibition requiring that each element of the direct repeat be bound by a binding factor. Third, double mutant analysis places laf-1 upstream of tra-2 in a genetic hierarchy, as expected for a repressor of tra-2 .Fourth, a laf-1 mutant alters the activity of the binding factor in RNA gel retardation analysis.

We suggest that laf-1 is centrally involved in the translational repression of tra-2 and might itself encode the repressor. We are currently investigating the nature of laf-1 mutations (loss-of-function or gain-of-function?) and attempting to clone the laf-1 gene.