Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site.

Abstract

The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.Posttranscriptional regulatory mechanisms play a key role in cell fate decisions in many developmental pathways. In the somatic sexual-development pathway of the fruit fly, Drosophila melanogaster, both the maintenance and elaboration of pathway choice are controlled at the level of alternative pre-mRNA splicing (2, 21). The binary switch gene Sex-lethal (Sxl) sits at the top of the somatic sexualdevelopment pathway and functions in both determination and differentiation. The activity state of the Sxl gene is chosen during early embryogenesis in response to the primary sex determination signal, the ratio of X chromosomes to autosomes (9,14,27,47). Sxl is turned on in females, while it remains off in males. Activation in females triggers an autoregulatory feedback loop in which female Sxl proteins promote their own synthesis by directing the femalespecific splicing of Sxl primary transcripts (3,4 regulates the splicing of transformer (tra) RNA (5). When Sxl is on, it directs the female-specific splicing of tra primary transcripts, producing mRNAs which have an intact open reading frame. The resulting tra protein, together with the constitutively expressed tra-2 protein, then activates the female-specific splice site of doublesex (dsx) to generate female dsx RNA (7,18,24,43). When Sxl is off, the default splicing of tra gives mRNAs with a truncated open reading frame, and in the absence of functional tra protein, dsx is spliced in the male mode.The mechanism for Sxl regulation of tra splicing is now reasonably well understood. As diagrammed in Fig. 1 MATERIALS AND METHODSPlasmid construction and Drosophila transformation. TheSxl miniconstruct diagrammed in Fig. 2 was made by first isolating a 310-bp PstI-HpaII fragment of genomic Sxl which contains exon 4 and upstrea...

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

“…The sequence motif involved in nonsense-mediated mRNA decay is another example of a short RNA repeat involved in posttranscriptional regulation. Short RNA sequences that modulate both mRNA turnover and splicing have been identified (10,27,33,34,36,37,63,68,73,74,77). As with the results presented here, sequences flanking these motifs can influence their activity.…”

Combinatorial control of a neuron-specific exon

“…The differing effects of these sequences are presumably due to variable concentrations of the regulatory proteins that bind to them+ Each cell line has a distinct combination of regulatory proteins that defines its response to a set of elements+ Proteins that act through the exonic element and some that act through the intronic enhancer may be broadly distributed, as these elements function in all the cells+ Other enhancer proteins, as well as proteins that govern repression by the 39 splice site, are restricted in their expression to certain cells+ In this model, the level of exon inclusion will be determined by the particular combination of regulatory elements present in the exon and by the relative concentrations of the cognate regulatory proteins present in a particular cell+ Most systems of regulated splicing employ multiple regulatory elements distributed along the RNA transcript+ Repression of an exon in the Drosophila sxl transcript by the sex lethal protein requires multiple Sxl protein binding sites present on both sides of the regulated exon (Horabin & Schedl, 1993;Samuels et al+, 1994;Deshpande et al+, 1996)+ Activation of a femalespecific exon in the Drosophila doublesex (dsx) transcript requires multiple positive-acting elements that make up a complex exonic splicing enhancer (Hertel & Maniatis, 1998, and references therein)+ In mammalian cells, systems of tissue-specific splicing appear even more complex, employing diverse sequence elements and a combination of positive and negative control+ The neuronally regulated exon in the g2 subunit of the GABA A receptor contains regulatory elements in the upstream 39 splice site and within the exon itself (Zhang et al+, 1996)+ Like the src N1 exon, these elements act both positively and negatively; some sequences are thought to allow splicing in neural cells and others to prevent splicing elsewhere+ Similarly, the neural specific Y exon of the Agrin transcript contains both positive and negative regulatory elements, but in this case they are present in the downstream intron (Wei et al+, 1997)+ A combination of positive and negative control is also seen for exons regulated in muscle and other cell types (Helfman, 1994;Y+ Wang et al+, 1997)+ One protein implicated in the repression of several different tissue-specific splicing patterns is the polypyrimidine tract binding protein (Patton et al+, 1991;Mulligan et al+, 1992;Gooding et al+, 1994;Singh et al+, 1995)+ It appears that repression of the neuron-specific exons src N1 and GABA A g2 is mediated by PTB (Ashiya & Grabowski, 1997;Chan & Black, 1997)+ PTB is ubiquitously expressed and appears to mediate a general repression of splicing that is released in specific cell types+ The protein factors determining the tissue-specific release from inhibition have not been identified+ However, in both the src N1 and g 2 exons, a neural-specific protein related to PTB is seen binding to the repressor elements in neural extracts …”

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

“…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.…”

“…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.…”

“…In the presence of Sxl protein, the late transcripts skip exon 3 and splice in the female pattern. Studies by Horabin and Schedl (19,20) and Sakamoto et al (41) have demonstrated that the late 2-3-4 splicing pattern can be reconstructed by using a minigene limited to those three exons, both in transformant flies and in Drosophila tissue culture cells. In this way, pre-mRNA regions in the introns flanking exon 3 were defined as important for sex-specific splicing.…”