The Sex-lethal amino terminus mediates cooperative interactions in RNA binding and is essential for splicing regulation.

Abstract

Sex-lethal (Sxl~ acts as a binary switch that regulates Drosophila sexual differentiation and dosage compensation and also maintains a stable female state through autoregulation. As part of a cascade of genes that are regulated by sex-specific splicing, Sx/controls the sex-specific splicing of transformer (tra) RNA and also its own RNA. Sxl contains two RNP-CS (RNA-binding) domains and is known to bind tra pre-mRNA near the alternative 3' splice site, thus blocking use of that site to give the female-specific splicing pattern. Here, we test how Sxl protein interacts with Sx/RNA during autoregulation. We show that Sxl not only binds Sx/pre-mRNA near the alternative 3' splice site but also at distant, multiple sites surrounding the Sx/ alternative exon. Moreover, Sxl binds cooperatively at these multiple sites. The Sxl amino terminus is essential for the cooperative interaction and is also required for regulatory activity in vivo. It appears that this region of Sxl protein, which resembles regions in some other RNA-binding proteins, is a domain that mediates protein-protein interactions during RNA binding and plays an important role in splicing regulation.

Sex-lethal Interacts with Splicing Factors In Vitro and In Vivo

“…The results presented here suggest a model (Fig. 11) to explain how Sxl proteins bound to intron sequences far from the male exon are able to block the utilization of the male exon splice sites (29,45,61). Sxl proteins would bind to the poly(U) tracts in the introns upstream and downstream of the Sxl male exon (exon 3), possibly utilizing cooperative interactions among adjacent Sxl molecules to stabilize the complex (49,61).…”

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB)

“…PTB2 . PTB1 repressive hierarchy upon TM exon 3 was maintained in all experiments, there were some apparent discrepancies that are noteworthy+ Principal among these is the observation that PTB1 overexpression in SM cells led to decreased skipping of exon 3+ Although it is formally possible that overexpressed PTB1 acts as an activator of exon 3, the simplest interpretation is that it is less repressive than PTB4 (or PTB4-containing heterodimers), and that by displacing PTB4 from regulatory elements, it leads to lower levels of exon skipping+ Although many alternative splicing events are influenced equally by PTB1 and PTB4 (Fig+ 5;Wagner et al+, 1999;Jin et al+, 2000), our results suggest that a specific subset may be influenced by this ratio+ Ironically, although we have demonstrated this potential using TM minigene constructs, we do not believe that the PTB1/4 ratio is a major deciding factor between inclusion or skipping of TM exon 3+ First, the ratio of PTB1 and PTB4 shows no correlation with alterations in TM splicing between fully differentiated and dedifferentiated rat aorta cells (C+ Gooding & C+W+J+ Smith, unpubl+ observations)+ Secondly, overexpression of PTB4 was not able to cause complete switching to the differentiated SM-specific splicing pattern (Fig+ 4)+ Moreover, when added back to PTB-depleted extracts, the PTB isoforms were equally effective at restoring the original splicing pattern at physiological concentrations (Fig+ 7)+ It was only at higher concentrations (5-10-fold excess) that PTB4 was more effective at inducing TM exon 3 skipping+ In addition to PTB binding sites, clusters of CUG motifs on either side of TM exon 3 are required for regulation+ The putative corepressor factors that interact at these sites have yet to be identified+ It is possible that in the presence of an appropriate corepressor, PTB4 would prove to be a more potent repressor of TM exon 3 at lower concentrations+ Despite our incomplete understanding of the mechanism of TM splicing regulation, the clear implications of our results are that the alternative splicing activity of PTB1 and PTB4 are distinct in a subset of PTB-regulated splicing events and that changes in this ratio could play a role in regulating these events+ Early models for repression by PTB suggested that it may compete with U2AF 65 for binding at certain polypyrimidine tracts (Mulligan et al+, 1992;Lin & Patton, 1995;Singh et al+, 1995), in an fashion analogous to the competition between U2AF 65 and sex-lethal protein at the transformer-regulated 39 splice site (Valcár-cel et al+, 1993)+ However, most exons that are regulated by PTB have multiple binding sites for PTB within and flanking the exons (Ashiya & Grabowski, 1997;Chan & Black, 1997;Perez et al+, 1997a;Gooding et al+, 1998;Zhang et al+, 1999;Carstens et al+, 2000)+ In the case of the n-src exon, cooperative binding of PTB to sites on both sides of the exon is required for repression + This is reminiscent of the cooperative binding of SXL protein to its own pre-mRNA (Wang &am...…”

Modulation of msl-2 5′ splice site recognition by Sex-lethal

“…sion+ Indeed, cooperative interactions between SXL molecules bound to different sites within the same RNA molecule-albeit at closer distances than in msl-2-have been documented and shown to play a role in Sex-lethal autoregulation (Wang & Bell, 1994)+ A second possibility is that SXL binding at the 59 end of the intron prevents 59 splice site recognition by splicing factors, thereby directly contributing to splicing inhibition+ The results shown below indicate that SXL binding to the 59 end of the intron does indeed antagonize factors involved in early 59 splice site recognition that strongly influence spliceosome assembly and that are critical for msl-2 splicing+ SXL binding to the (U)11 sequence inhibits U1 snRNP recruitment to the 59 splice site…”