Genetics, Vol 139, 631-648, Copyright © 1995

INVESTIGATIONS

Transposon Insertions Causing Constitutive Sex-Lethal Activity in Drosophila melanogaster Affect Sxl Sex-Specific Transcript Splicing

M. Bernstein, R. A. Lersch, L. Subrahmanyan and T. W. Cline
Department of Biology, Princeton University, Princeton, New Jersey 08544, Department of Biology, Yale University, New Haven, Connecticut 06520-8103 Present address: Yale University, New Haven, CT 06520-8103.

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle ``on'' vs. ``off'' reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. ``Male-lethal'' (Sxl(M)) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, Sxl(M) haplo-X animals (chromosomal males) die because of improper dosage compensation, and Sxl(M) chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous Sxl(M) alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: Sxl(M1), Sxl(M3), and Sxl(M4). Sxl(M1) is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. Sxl(M3) is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and Sxl(M4) is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with Sxl(M4) being the strongest and Sxl(M1) the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable Sxl(M) derivatives in which the Sxl(M) insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these Sxl(M) alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of Sxl(M) alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

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