Functioning of the Drosophila integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf(+) gene dose effects

Abstract

Snf, encoded by sans fille, is the Drosophila homolog of mammalian U1A and U2B" and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf's functioning within snRNPs. Sxl is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, we show that Snf is rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic snf(+) dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a(60), has no effect. Increased snf(+) enhances Sxl autoregulation even when U1-70k and SF3a(60) are reduced by mutation to levels that, in the case of SF3a(60), demonstrably interfere with Sxl autoregulation. The observation that increased snf(+) does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a(60) is additional evidence that snf(+) dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.

Figure 1

Molecular lesions associated with four gain-of-function (M-type, male-lethal) and one loss-of-function (f-type, female-lethal) Sxl alleles used in this study. Inverted triangles represent insertions of DNA drawn to scale. The part of Sxl shown includes the region of sex-specific alternative splicing. Sxl protein imposes the exon 2–4 (female) splicing mode, which generates more full-length Sxl protein. Without Sxl, the exon 2–3-4 (male, “nonproductive”) splicing mode ensues, with exon 3 aborting translation. Because these four incompletely constitutive Sxl^M alleles relax but do not eliminate this autoregulatory requirement for Sxl, the level of female splicing reached in males and thus the degree of developmental disruption caused by the mutants is influenced by factors like Snf that affect positive autoregulation.

Figure 2

Males showing the reciprocal, tissue-specific feminizing effects of Sxl^M12 and Sxl^Mf1. For each pair, the male on the right carries a msl-2^cnstv transgene to suppress dosage compensation upsets and hence more fully reveal the extent of feminization (see text). Sxl^M12 predominately affects abdominal histoblast derivatives (tergites and sternites) whereas Sxl^Mf1 predominately affects imaginal disc derivatives (e.g., forelegs and genitalia). Sexual phenotype was quantified by using the scale shown in C for the foreleg sexcomb region. The sexcomb is a row of distinctive male-specific bristles, with each comb-tooth bristle being the product of a single differentiating cell. In these cases, intersexuality was of the mosaic type (see text). Forelegs of Sxl^Mf1 males from the various crosses in Fig. 3 illustrate the full range of sexual transformation observed: (1) none, fully male with at least eight teeth and no breaks; (2) slight, mostly male but with one break and no fewer than seven comb teeth; (3) intermediate, more than three comb teeth and either multiple breaks or fewer than seven teeth; (4) severe, mostly female but with one to three comb teeth; (5) complete, entirely female.

Figure 3

Effects of changes in the dose of genes encoding the U1 snRNP protein U1-70k or the U2 snRNP protein SF3a⁶⁰ (noi) on the sexual differentiation of Sxl^Mf1/Y male forelegs. Phenotype classes are defined in Fig. 2. Crosses: (A) w Sxl^Mf1ct⁶/FM7c, B ☿☿ × ♂♂ w/Y; P{snf⁺w^+mC}108/+. (B) w Sxl^Mf1 ct⁶/w; P{U1-70K⁺w^+mC}Hcter/+ ☿☿ × ♂♂ w/Y; P{U1-70K⁺w^+mC}Hcter/+. (C) w Sxl^Mf1ct⁶/w; P{noi⁺w^+mC}A71/+ ☿☿ × ♂♂ w/Y; P{snf⁺w^+mC}108. (D) w Sxl^Mf1ct⁶/w; U1-70K⁶²/SM6β Cy, Roi ☿☿ × ♂♂ w/Y; U1-70K⁶² P{snf⁺w^+mC}108/+. (E) w Sxl^Mf1 ct⁶/w; noi^2-P{w+mC}/TM2,Ubx ☿☿ × ♂♂ w/Y; P{snf⁺w^+mC}108/+; Ki/Df(3R)noi-D. (F) y w/w Sxl^Mf1ct⁶; TM3, Sb/noi^2-P{w+mC} ☿☿ × ♂♂ w/Y; P{snf⁺w^+mC}108 and P{snf⁺w^+mC}19/+; Df(3R)noi-D./TM3, Sb.