Specific isoforms of squid, a Drosophila hnRNP, perform distinct roles in Gurken localization during oogenesis

Abstract

Heterogeneous nuclear RNA-binding proteins, hnRNPs, have been implicated in nuclear export of mRNAs in organisms from yeast to humans. A germ-line mutation in a Drosophila hnRNP, Squid (Sqd)/hrp40, causes female sterility as a result of mislocalization of gurken (grk) mRNA during oogenesis. Alternative splicing produces three isoforms, SqdA, SqdB, and SqdS. Here we show that these isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in grk mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. We show that this differential nuclear accumulation is correlated with a differential association with the nuclear import protein Transportin. Finally, we provide evidence that grk mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. These results demonstrate the isoform-specific contributions of individual hnRNP proteins in the regulation of a specific mRNA. Moreover, these data suggest a novel role for hnRNPs in localization and translational regulation of mRNAs.

Figure 1

Schematic diagram of Sqd protein isoforms. The three Sqd isoforms, SqdA, SqdB, and SqdS, are identical to amino acid position 285 (A). Within this conserved region are the two RNA-binding domains RBD-1 and RBD-2 (depicted by the hatched boxes) and an M9-like nuclear-import domain (represented by the black box). At the carboxyl termini of the proteins, all three contain a glycine-rich domain, which in the SqdS and SqdB isoforms continues to the end of the protein. The SqdB and SqdS isoforms use the same 3′ exon; however, the splice site in the SqdB isoform results in a protein with 27 fewer amino acids than SqdS. In addition, SqdS contains another M9 nuclear import motif (amino acids 300–338) that is not present in either SqdA or SqdB. The SqdA isoform uses an alternative 3′ exon and encodes a protein with a unique carboxyl terminus. A comparison of the human hnRNPA1 M9 domain with the M9 domains found in Sqd is shown in B. The M9 motif found only in SqdS shares 16 of 38 amino acid identity with human hnRNPA1 M9 (top), whereas the M9-like motif found in all three Sqd isoforms shares 13 of 38 amino acid identity with hnRNPA1 (bottom). Alignment of the M9 motifs was accomplished using the ClustalW Multiple Sequence Alignment Program.

Figure 2

Rescue of sqd¹ D-V patterning defects by individual Sqd isoforms. Females of the indicated genotypes were placed in laying blocks and eggs were collected on days 2 and 3 after blocking. (Top) The series of dorsalized eggshell phenotypes: wild-type egg with two dorsal appendages (A), slightly dorsalized egg with a broad fused dorsal appendage (B), a more dorsalized egg with widely spaced dorsal appendages attributable to the expansion of the dorsal midline fates (C), and a severely dorsalized egg with expanded dorsal appendage material around the anterior circumference of the eggshell (D). For each maternal genotype the percentage of eggs with the indicated phenotypes were calculated (n = total number of eggs examined). In the case of Sqd transgene expressing females, a single female laid the range of eggshell phenotypes shown.

Figure 3

grk mRNA localization and protein distribution within Sqd transgenic egg chambers. In wild-type egg chambers, grk mRNA is tightly localized to the dorso-anterior corner of the oocyte in close proximity to the oocyte nucleus (A). Within egg chambers from SqdA females, grk mRNA is distributed around the anterior circumference of the oocyte, although it is more concentrated on the dorsal side (B). Egg chambers from SqdS females show a grk mRNA localization pattern indistinguishable from wild-type (C), and in contrast, in egg chambers from SqdB females, grk mRNA is mislocalized in an anterior ring in a pattern that is indistinguishable from sqd¹ egg chambers (D). Grk protein localization within egg chambers from Sqd transgenic females (E–H). In stage 10 wild-type egg chambers, Grk protein accumulates in the dorso-anterior corner of the oocyte in a location coincident with the grk mRNA (E). Within SqdA and SqdS egg chambers, Grk protein is localized to the dorsoanterior corner of the oocyte (F,G), much like that in wild type, and in SqdB egg chambers Grk protein is mislocalized along the anterior circumference of the oocyte (H). The granular staining observed in the nurse cells in F and H is nonspecific background staining that is seen in grk mutant ovaries as well.

Figure 4

Expression of Sqd isoforms within the ovary. Stage 10 egg chambers from sqd¹ females carrying the individual Sqd transgenes were examined for Sqd protein expression. Sqd protein is in red, and cortical actin detected with phalloidin is in green. Within egg chambers from Sqd transgenic females Sqd protein is undetectable in the germ line of SqdA females (A), but is detected within the nurse cell nuclei of both SqdS and SqdB females (B,C). Furthermore, in egg chambers from SqdS females, Sqd protein is detected within the oocyte nucleus (B). Higher magnification view of the somatic follicle cells is shown, in which endogenous protein and the transgene-derived protein, is detected. In SqdS egg chambers the follicle cell Sqd protein is predominantly nuclear (E). However, in SqdA egg chambers diffuse, cytoplasmic Sqd protein is detected (D). The differential nuclear accumulation of SqdB, SqdS, and SqdA is associated with a differential interaction with the nuclear import protein Transportin (F). [³⁵S]methionine-labeled Drosophila Transportin protein was incubated with a series of GST fusion proteins (GST only, GST–SqdA, GST–SqdS, GST–SqdB, and GST–Encore) and precipitated with glutathione-coated-agarose beads. Transportin binding to the GST-fusions was assessed with SDS-PAGE and autoradiography. Transportin protein is brought down by both SqdS and SqdB, but not with GST only, SqdA, or Encore protein.

Figure 5

Sqd protein distribution is altered in fs(1)K10 egg chambers and physically interacts with K10 protein. Expression of Sqd protein in wild type, sqd¹ and fs(1)K10 stage 10 egg chambers, with Sqd in red and cortical actin detected with phalloidin in green. In wild-type egg chambers, Sqd protein is detected in the follicle cell nuclei, the nurse cell nuclei, and the oocyte nucleus (A). In contrast, in sqd¹ egg chambers, Sqd protein is undetectable in the germ line (B). Within fs(1)K10 egg chambers, Sqd protein is detected in the nurse cell nuclei, but is undetectable within the oocyte nucleus (C). To determine whether Sqd protein and K10 protein can physically associate with one another, in vitro binding studies were conducted. ³⁵S-labeled K10 protein was incubated with a series of GST fusion proteins, and after incubation with glutathione-coated–agarose beads, K10 binding was assessed by SDS-PAGE and autoradiography (D). The input ³⁵S-K10 protein is shown in lane 1. Association with the following GST-fusion proteins was tested: (lane 2) GST only; (lane 3) GST–SqdA; (lane 4) GST–SqdS; (lane 5) GST–SqdB; (lane 6) GST–Grk.

Figure 6

Sqd protein binds grk mRNA directly. (A) The regions of the grk 3′ UTR and the nos 3′ UTR that were used as probes are depicted. (B) Wild-type ovarian lysates were subjected to UV cross-linking to the indicated RNA probes, after cross-linking the RNA–protein complexes were precipitated with anti-Sqd and resolved by SDS-PAGE. Sqd protein associates with the grk 3′ UTR (lane 1), whereas it does not interact with the +3 region of the nos 3′ UTR (lane 2). (C,D) To test the specificity of the Sqd–grk interactions, UV cross-linking analyses were repeated in the presence of excess, cold competitor RNAs. The interaction between whole ovarian protein extracts and grk 3′ UTR fragments 2 and 3 were tested. Binding in the presence of no competitor (lane 1), or with increasing amounts (∼20×, 100×, or 200× excess) of specific competitor (lanes 2–4), or with increasing amounts of nonspecific competitor (lanes 5–7) are shown.

Figure 7

Sqd protein associates with the translational repressor protein Bruno. (A) ³⁵S-labeled Bruno protein (lane 1) was incubated with a series of GST fusion proteins, and after incubation with glutathione-coated–agarose beads, Bruno’s association was assessed by SDS-PAGE and autoradiography. The following GST fusions were tested for their ability to bind Bruno: (lane 2) GST only; (lane 3) GST–SqdA; (lane 4) GST–SqdS; (lane 5) GST–SqdB; and (lane 6) GST–Encore. (B) To further investigate the interaction between Sqd protein and Bruno protein, coimmunoprecipitation experiments were done. Whole cell lysates from wild-type ovaries were subjected to immunoprecipitation with a mouse anti-Sqd monoclonal or with a mouse anti-Wind antisera. The blots were probed with a rabbit anti-Bruno antisera: (lane 1) whole cell ovarian extract; (lane 2) anti-Wind immunoprecipitate; (lane 3) anti-Sqd immunoprecipitate.

Figure 8

Model for the role of Sqd in grk mRNA localization and translation. In the oocyte nucleus SqdS protein binds to grk mRNA and to K10 protein, possibly as a complex. Then SqdS protein in a complex with grk mRNA and potentially other factors, exits the nucleus. Once in the cytoplasm, SqdS delivers grk mRNA to the SqdA isoform and the translational regulatory protein Bruno. The RNA–protein complex is localized and anchored, whereupon positive factors, such as Encore, Vasa, (Orb)oo18 RNA-binding, or (SpnE), Spindle E, relieve the translational repression by Bruno (and possibly interact with Sqd), leading to efficient translation of grk mRNA.