Functioning of the Drosophila Wilms'-tumor-1-associated protein homolog, Fl(2)d, in Sex-lethal-dependent alternative splicing

Abstract

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. Here we attempt to identify factors that interact genetically and physically with fl(2)d. We begin by analyzing the Sxl-Fl(2)d protein-protein interaction in detail and present evidence suggesting that the female-specific fl(2)d(1) allele is antimorphic with respect to the process of sex determination. Next we show that fl(2)d interacts genetically with early acting general splicing regulators and that Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, we could not detect Fl(2)d complexes containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, our findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly.

Figure 1.—

Monoclonal antibody recognizes Fl(2)d protein on Western blots and whole-mount embryos. (A) Monoclonal anti-Fl(2)d 9G2 recognizes His-tagged purified Fl(2)d protein and 80-kDa protein in adult and embryonic extracts on Western blot. (B and C) Whole-mount embryos: Blue, DNA; Green, Fl(2)d. Wild-type embryos have Fl(2)d protein while the protein is absent from fl(2)d²/fl(2)d² embryos. The higher magnification in C shows that Fl(2)d is not distributed evenly on the DNA in the nucleus.

Figure 2.—

Interactions between Sxl and Fl(2)d proteins in vivo and in vitro. (A) Fl(2)d-probed immunoprecipitations from wild-type nuclear embryonic extract. Lane 1, extract; lane 2, Sxl immunoprecipitation; lane 3, RNase-A-treated Sxl immunoprecipitation; lane 4, β-gal immunoprecipitation. (B) Fl(2)d-probed in vitro immunoprecipitations. Lane 1, Fl(2)d protein; lane 2, Sxl immunoprecipitation with Fl(2)d protein alone; lane 3, Sxl immunoprecipitation with Fl(2)d and Sxl proteins; lane 4, β-gal immunoprecipitation with Fl(2)d and Sxl proteins. (C) GST-probed Fl(2)d in vitro immunoprecipitations. Lane 1, R1R1-GST protein; lane 2, Fl(2)d immunoprecipitation with R1R1-GST and Fl(2)d proteins; lane 3, Fl(2)d immunoprecipitation with R1R1-GST protein alone. (D) GST-probed Fl(2)d in vitro immunoprecipitations. Lane 1, NSxl-GST protein; lane 2, Fl(2)d immunoprecipitation with NSxl-GST and Fl(2)d proteins; lane 3, Fl(2)d immunoprecipitation with NSxl-GST protein alone.

Figure 3.—

Fl(2)d and Sxl form protein–protein complexes in wild-type and fl(2)d¹ ovaries. An asterisk indicates an additional band picked up by Fl(2)d antibody in ovarian extract. (A) Fl(2)d-probed Western blot. Lane 1, embryonic extract; lane 2, ovarian extract. (B) Fl(2)d-probed immunoprecipitations from wild-type ovarian extract. Lane 1, ovarian extract; lane 2, Sxl immunoprecipitation; lane 3, Scute immunoprecipitation. (C) Fl(2)d-probed immunoprecipitations from fl(2)d¹ ovarian extract. Lane 1, ovarian extract; lane 2, Sxl immunoprecipitation; lane 3, Scute immunoprecipitation.

Figure 4.—

Fl(2)d and Snf form a protein–protein complex in embryos. (A) Fl(2)d-probed immunoprecipitations from wild-type nuclear embryonic extract. Lane 1, extract; lane 2, Snf immunoprecipitation; lane 3, RNase-A-treated Snf immunoprecipitation; lane 4, β-gal immunoprecipitation. (B) Fl(2)d-probed immunoprecipitations from wild-type (top), snf¹⁴⁸ (middle), and snf^5mer (bottom) nuclear embryonic extracts. Lane 1, extract; lane 2, Snf immunoprecipitation; lane 3, β-gal immunoprecipitation.

Figure 5.—

Fl(2)d forms protein–protein complexes with the early acting general splicing regulators U1-70K, U2AF50, and U2AF38. Immunoprecipitations of wild-type nuclear embryonic extract probed for U1-70K (top), U2AF50 (middle), and U2AF38 (bottom). Lane 1, extract; lane 2, Fl(2)d immunoprecipitation; lane 3, Scute immunoprecipitation.

Figure 6.—

Fl(2)d and Sxl do not interact with the mid- and late-acting splicing regulators SKIP and U5-40k. (A) Immunoprecipitations from wild-type nuclear embryonic extract. (Left) Probed for SKIP (top), U5-40k (top middle), U2AF50 (bottom middle), and Fl(2)d (bottom). Lane 1, extract (lane 2, blank); lane 3, Scute immunoprecipitation; lane 4, Sxl immunoprecipitation; lane 5, Fl(2)d immunoprecipitation. (Right) Probed for Fl(2)d (top), U2AF50 (middle), and Sxl (bottom). Lane 1, extract (lane 2, blank); lane 3, Scute immunoprecipitation; lane 4, U5-40k immunoprecipitation. (B) GST pull downs from embryonic extract probed for SKIP (top) and Snf (bottom). (Left) Lane 1, embryonic extract input; lane 2, GST pull down with GST-Sxl; lane 3, GST pull down with GST protein alone. (Right) Lane 1, embryonic extract input; lane 2, GST pull down with GST-U2A′.