Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism.

Horabin, Jamila I.; Schedl, Paul

doi:10.1128/mcb.13.3.1408

Cited by 58 publications

(45 citation statements)

References 33 publications

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“…The Sxl gene also provides the mechanism for remembering the female determined state (15). This is accomplished through an autoregulatory feedback loop in which the Sxl protein directs its own expression by promoting the female-specific splicing of primary transcripts produced from the Sxl late or maintenance promoter, Sxl-P m (3,4,36,37). Similarly, the male determined state is maintained by the default splicing machinery, which splices primary transcripts from Sxl-P m in the nonproductive, male mode.…”

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confidence: 99%

scute (sis-b) Function in Drosophila Sex Determination

Deshpande

Stukey

Schedl

1995

Molecular and Cellular Biology

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The primary sex determination signal, the X chromosome-to-autosome (X/A) ratio, controls the choice of sexual identity in the Drosophila melanogaster embryo by regulating the activity of the early promoter of the Sex-lethal gene, Sxl-P e . This promoter is activated in females (2X/2A), while it remains off in males (1X/2A). Promoter activation in females is dependent upon X-linked numerator genes. One of these genes, sisterless-b (sis-b), corresponds to the scute (sc) locus of the achaete-scute complex, and it encodes a helix-loop-helix transcription factor. In the studies reported here we have used monoclonal antibodies to study the expression and functioning of the sc(sis-b) protein. Sc is first detected at nuclear cycle 6 to 7, well before Sxl-P e is first active. At this stage, the protein is in the cytoplasm, not the nucleus. Only after the formation of the syncytial blastoderm, at nuclear cycle 10 to 11, does a substantial fraction of the protein enter the nucleus, and this nuclear import closely coincides with the initial activation of Sxl-P e . Consistent with the idea that the dose of sc(sis-b) is critical for its function as an X-chromosome counting element, wild-type syncytial blastoderm embryos could be grouped into two classes based on the level of protein. Western blot (immunoblot) analysis demonstrates that this difference in protein level correlates directly with the activity state of the Sxl gene. Finally, we provide the first direct evidence that Sc forms heteromeric complexes in vivo in early embryos with the maternally derived helix-loop-helix protein Daughterless. This in vivo complex is likely to be critical for Sc function in Sxl-P e activation.The choice of sexual identity in the fruit fly Drosophila melanogaster is determined by the X chromosome-to-autosome (X/A) ratio (1,16,20,48,49). A ratio of 1 (2X/2A) signals the initiation of the female developmental pathway, while a ratio of 1/2 (1X/2A) signals male development. The system that measures the X/A ratio operates only transiently during early embryonic development, and it functions to set the activity state of the binary-switch gene Sex-lethal (Sxl) (17). In 2X/2A animals, the X/A signaling system turns on the Sxl gene, while the gene is not activated in 1X/2A animals. In the on state, the Sxl gene directs female development by controlling several regulatory cascades that function in different aspects of somatic sexual development (14,32,41,44,50). These regulatory cascades include the transformer-doublesex somatic sexual differentiation pathway and the dosage compensation pathway (2,8,44). The Sxl gene also provides the mechanism for remembering the female determined state (15). This is accomplished through an autoregulatory feedback loop in which the Sxl protein directs its own expression by promoting the female-specific splicing of primary transcripts produced from the Sxl late or maintenance promoter, Sxl-P m (3,4,36,37). Similarly, the male determined state is maintained by the default splicing machinery, which splices primary tr...

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scute (sis-b) Function in Drosophila Sex Determination

Deshpande

Stukey

Schedl

1995

Molecular and Cellular Biology

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“…Experimental data from Horabin and Schedl (19) indicate that exons 2 and 3 in the late splice show a preferential relationship. When exon 3 and its flanking sequences were inserted into the intron of the white or ftz gene, this exon was predominantly skipped in male flies, suggesting that appropriate flanking intron and exon sequences are required for default male splicing and that exon 3 is relatively weak when placed in a foreign context.…”

Section: Discussionmentioning

confidence: 99%

“…pBS2N is a derivative of pBluescript KS(ϩ) in which the KpnI site has been replaced with a NotI linker. The BamHI-NotI Sxl fragment was transferred to the same sites of pCaSpeR-HS83 (19,20). The SmaI (from the vector polylinker)-to-BspEI Sxl fragment was then ligated into pT7.BS digested with ScaI and XmaI.…”

Section: Methodsmentioning

confidence: 99%

“…The BamHI-EcoRI fragments from the constructs in pBluescript were then cloned into the BamHI and EcoRI sites of a P-element transformation vector modified from pCaSpeR-HS83 (19,20). The modification involved replacement of the polylinker sequences between the two PstI sites with a NotI linker and replacement of the polylinker XhoI site with a BamHI linker.…”

Section: Methodsmentioning

confidence: 99%

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The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5′ Splice Sites

Zhu

Urano

Bell

1997

Molecular and Cellular Biology

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The Sex-lethal (Sxl) early transcripts have a unique 5 exon and a splicing pattern that differs from that of the late transcripts. While the late transcripts are regulated sex specifically by control of exon 3 inclusion, the early transcripts are not. While the late transcripts include exon 3 by default, the early transcripts skip exon 3. Splicing patterns of a reporter gene that mimics the early transcript, and its variants, were analyzed in Drosophila transformants and tissue culture cells. The results demonstrate that the early, in contrast to the late, splicing pattern is not regulated by stage-specific or sex-specific trans-acting factors, and so the pattern appears to arise from some type of intrinsic splice site preference or compatibility. Inclusion or exclusion of exon 3 is determined by the identity of the upstream 5 splice site region as late or early. The important region of the early exon lies within 233 nucleotides of the immediately adjacent intron.Splicing reactions remove intron sequences from premRNA and join exons together with exquisite precision. These reactions are carried out within the spliceosome, using several snRNA molecules and many associated proteins. Accuracy in splicing requires that the appropriate pre-mRNA splice sites, which may be separated by vast distances, be recognized and distinguished from among many closely related sequences. Three minimum sequence elements have been identified (33)(34)(35). At the 5Ј splice site, there is a short consensus sequence that is complementary to sequences of U1 snRNA; the branch point sequence is recognized by U2 snRNA; at the 3Ј splice site, the last two nucleotides of the intron are invariantly AG (31). In addition, in higher eukaryotic cells, a polypyrimidine tract is usually located between the branch point and 3Ј splice site; it is bound by the protein U2AF, which enhances the binding of U2 snRNA to the branch point (56, 57). To some degree, the likelihood of choosing a particular splice site correlates with how closely it matches the consensus sequence. Nevertheless, these limited sequence requirements are not sufficient to explain the accuracy observed in splice site selection, as sequences matching the consensus are often found unused within introns. Additional features have also been identified as important for selection of the correct pair of 5Ј and 3Ј splice sites (5).Sequence context beyond the splice sites and branch point can influence splice site selection and splicing efficiency. For example, exon sequences have been known for some time to influence splice site selection, and several exonic purine-rich splicing enhancers have recently been characterized (12,24,28,47,53,54). Some of these enhancers have been shown to be bound by members of the SR family of proteins, which as a class appear to be involved in joining 5Ј and 3Ј splice sites across exons as well as introns (14). While SR proteins are essential for splicing in general, they can also differentially influence the choice of splice sites in a concentration-dependent ma...

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“…Intronic inhibitory elements located centrally in an alternatively spliced intron have also been described (e.g. alternative splicing of the sex-lethal exon 3 in Drosophila [22,23]). …”

Section: Figure 1 Schematic Representation Of the β-Globin And Serca2mentioning

confidence: 99%

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

Bosch

Mertens

Gijsbers

et al. 1997

Biochemical Journal

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Expression of the muscle-specific 2a isoform of the sarco\ endoplasmic reticulum Ca# + -ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3h end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3h end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscletype splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand,

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Regulated splicing of the Drosophila sex-lethal male exon involves a blockage mechanism.

Cited by 58 publications

References 33 publications

scute (sis-b) Function in Drosophila Sex Determination

scute (sis-b) Function in Drosophila Sex Determination

The Sex-lethal Early Splicing Pattern Uses a Default Mechanism Dependent on the Alternative 5′ Splice Sites

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript

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