Regulation of the sex determination gene
tra-1 is necessary for proper sexual development in C. elegans .
tra-1 is the terminal regulator in the somatic sex determination pathway, and is also necessary for germline sex determination.
tra-1 produces two proteins, TRA-1A and TRA-1B. TRA-1A is a member of the GLI family of transcription factors, and contains five zinc-fingers. TRA-1B is co-linear with the amino terminus of TRA-1A, but only contains the first two zinc fingers. (Unless otherwise stated, both proteins will be referred to as TRA-1). We find that the nuclear levels of TRA-1 correlate with sexual cell identity. Immunofluorescence analysis indicates that the levels of nuclear TRA-1, in both adult intestines and germlines, is higher in hermaphrodites than in males. By western analysis, the amounts of TRA-1 are similar in both males and hermaphrodites. Thus, sub-cellular localization, not protein levels, is responsible for the differences in nuclear levels of TRA-1 between the sexes. The importance of TRA-1 localization patterns in sexual development is further supported by the finding that XX
tra-2(lf) mutants, which are masculinized, show less nuclear TRA-1 than XX wild-type animals. In addition, XX
tra-2(lf);
fem-1(lf) double mutants, which are feminized, show similar nuclear levels of TRA-1 as wild-type XX animals. The sex-specific sub-cellular localization of TRA-1 is also dependent upon the interaction of TRA-1 with the
tra-2 mRNA. Previously, we found that nuclear export of the
tra-2 mRNA is regulated by TRA-1 binding to the
tra-2 3' untranslated region (3'UTR). TRA-1 binds the
tra-2 3'UTR, and promotes
tra-2 mRNA export to the cytoplasm (Graves et. al., 1999). Here we show that the nuclear levels of TRA-1 and sexual cell identity are regulated by nuclear export, and that this is mediated by TRA-1 binding the
tra-2 3'UTR. Treatment of XO animals with Leptomycin B, (an inhibitor of CRM1 mediated nuclear export), increases nuclear TRA-1. Leptomycin B treatment also results in aberrant activation of the vitellogenin-2 promoter in wild-type XO animals but not in
tra-1(null) animals. Therefore, inappropriate female development occurs when nuclear export is inhibited. This indicates that nuclear export of TRA-1 inhibits its activity, and consequently, female development. Export of TRA-1 requires binding to the
tra-2 3'UTR: a genetic mutation in the
tra-2 3'UTR that deletes the TRA-1 binding site results in an overabundance of nuclear TRA-1 and inappropriate female development in both XX and XO animals. Both increased nuclear TRA-1 and female development are suppressed by over-expression of RNA containing the TRA-1 binding site, emphasizing the importance of RNA binding for TRA-1 nuclear export and sexual development. The mutual dependence of TRA-1 and
tra-2 mRNA on one another for nuclear export suggests they export the nucleus as a TRA-1/
tra-2 mRNA complex. This work identifies a novel RNA based mechanism for controlling PolII transcriptional regulatory activity and cell fate determination. Graves. et al. (1999) Nature . (399), 802-805.