Sxl promotes female-specific alternative splicing - Ssx stabilizes the male- male-specific splicing pattern

In contrast to other systems, flies do not encode a Y-specific protein like SRY in humans (Sinclair et al. 1990) or Xol-1 in C. elegans. These male-specific factors promote development of male individuals by initiating the production of male hormones by establishing male sexual characteristics (reviewed in Lucchesi et al. 2005; Ercan and Lieb 2009). In Drosophila, however, male hormones do not govern sexual development and the Y chromosome does not participate in sex determination. The sex decision in the early embryo is made by a syncytium, which is guided exclusively by the ratio of X-chromosomes to autosomes (X:A) (Gonzalez et al. 2008). This elementary decision is implemented by the

60 3. DISCUSSION

auto-regulatory positive feedback loop of the female-specific protein Sxl. This positive feedback loop converts a transient and weak signal into an all-or-nothing response that is continuously maintained in female flies, committing to female development. In contrast, Sxl protein production remains shut-off in males (reviewed in Salz and Erickson 2010). The role of Sxl in alternative splicing has long been appreciated, although numerous molecular details are still unclear. However, the question whether the paralog Ssx participates in this pathway remained unanswered. The analysis of the role of Ssx in alternative splicing was initiated due to the detection of binding events between Ssx and the Sxl pre-mRNA by iCLIP experiments in Drosophila male cells (Fig. 2.14). The expression of the Ssx protein was analysed in both sexes (Fig. 2.7) and our data excluded that Ssx promotes exon skipping similarly to Sxl in males, since this would cause deleterious consequences in sex determination and dosage compensation. The erroneously exon skipping event would enable the expression of functional Sxl protein in male animals. Functional Sxl protein in males would lead 1) to alternative splicing of sex-specific mRNAs triggering the female development cascade and 2) an inactivation of the dosage compensation complex by translational downregulation of msl-2, which would in turn trigger the repression of the hypertranscription of the single X chromosome and would result in a reduced gene dose in males.

Intriguingly, both proteins bind similar sequences and exhibit comparable affinities to the investigated binding sites (Fig. 2.11; 2.16; 2.25). Moreover, both proteins compete with each other for binding to a shared binding motif. In every competition set-up, the highest concentrated protein was binding the RNA the most efficient, displacing the lower concentrated protein from the RNA (Fig. 2.16). Irrespective of similar binding motifs (Fig.

2.14) and affinities, overexpression of FlagHA-Ssx in flies showed no impact on the splicing patterns of several well-characterized and sex-dependent Sxl targets (Fig. 2.18).

Since the overexpression of Ssx was rather mild (Fig. 2.7 B and C), we hypothesized that the amount of FlagHA-Ssx protein was not sufficient to interfere with sex-dependent splicing.

Moreover, the increased lethality which we observed in heterozygous flies of the UASt::ssx;da::GAL4 strain, might be due to translational miss-regulation of several mRNA targets by the overexpressed protein. In addition, flies homozygous for the constitutive FlagHA-Ssx expression were not viable at all (Fig. 2.1).

Surprisingly, splicing analysis of the fly strain ssx indicated amounts of the female-specific Sxl splice variant in male flies (Fig. 2.20). Unlike its closely related paralog, Ssx does not promote the skipping of the poison exon within Sxl mRNA in females. Instead, Ssx rather promotes functional exon 3 splicing in males by antagonizing the regulatory function of Sxl in this context (Fig. 2.21 and 2.22). For the establishment and the maintenance of Sxl female-specific splicing, numerous factors have been identified which act together with the Sxl protein to ensure correct pattern formation and female development. However, the question

3. DISCUSSION 61

of how male flies can protect themselves against low-level expression of Sxl protein which could inadvertently initiate the feedback loop, remains enigmatic. Presumably, Sxl expression in male flies occurs at a non-zero rate which could accidentally trigger the Sxl expression cascade resulting in erroneous activation of Sxl protein expression (Figure 3.1 A).

We have identified the protein Ssx as an antagonist of the Sxl auto-regulatory feedback loop.

It competes with Sxl for binding to the regulatory elements in the Sxl primary transcript and thus prevents it from exerting its auto-regulatory function in splicing. By this, Ssx establishes a threshold that prevents small amounts of Sxl protein from initiating the splicing cascade, protecting male flies from a runaway protein production (Fig. 3.1 A blue dashed line).

Conversely, loss of Ssx sensitizes male flies to the auto-regulatory activity of Sxl resulting in production of detectable amounts of female-specific Sxl transcripts (Fig 3.1 A magenta dashed line).

In sum, Ssx acts as a safeguard in male flies by preventing small amounts of aberrantly produced Sxl protein from initiating the auto-regulatory, positive feedback loop. It therefore contributes to the development of male individuals by stabilization of a male-specific gene expression pattern. Robustness in development and in cell fate decisions was initially described by Waddington in 1957 who established the term channelling. He described the robustness of biological systems which are buffered against external influences and fluctuations to propagate and ensure stable pathways throughout the development (Waddington 1957).

Figure 3.1: Ssx ensures the male-specific splice pattern of Sxl transcripts in male flies. A) Model for the protection in male flies from the accidental activation of the developmental cascade regulated by Sxl.

Fluctuations of the non-zero background expression of Sxl are shown as a black curve. The threshold for the activation of the Sxl splicing cascade depicted as blue dashed line. Loss of Ssx is associated with a lowered threshold and an elevated risk of productive Sxl splicing (shown as dashed line in magenta). B) In females, a burst of early Sxl expression activates female-specific Sxl splicing and a continuous productive Sxl protein expression (depicted as a curve in magenta).

62 3. DISCUSSION