Abstract
Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.
Highlights
Understanding tissue- and stage-specific gene regulation remains one of the central issues in developmental biology
We identify PPS as a novel component of the machinery required for Sxl splicing autoregulation by showing that the lack of pps function interferes with Sxl expression and that the PPS protein is physically linked to the Sxl pre–mRNA, the SXL protein and components of the general splicing machinery
Together with the observation that the PPS protein contains four signature motifs typically found in proteins that function in transcriptional regulation, our data suggest that linking transcription to splicing regulation is important for controlling Sxl expression
Summary
Understanding tissue- and stage-specific gene regulation remains one of the central issues in developmental biology. The Drosophila sex-determination gene Sex-lethal (Sxl) is a prime example of a developmental switch gene regulated by alternative splicing. Throughout most of development and in adult tissues, Sxl is controlled by sex-specific alternative splicing to produce mRNAs with different coding potentials [1]. The third exon is always skipped to generate protein encoding mRNAs. The mechanism leading to exon skipping is autoregulatory and depends on the SXL protein binding to multiple intronic sites located both upstream and downstream of the regulated exon. Current models, based on both biochemical and genetic studies, suggest that SXL forces the male exon to be skipped by interacting with and antagonizing a set of general splicing factors, including the U1 snRNP, the U2AF heterodimer, FL(2)d and SPF45 [2,3,4]
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