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First published online 31 March 2004
doi: 10.1242/dev.01078

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Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis

Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA

View larger version (62K): [in a new window]   Fig. 1. Sqd and Hrb27C interact. (A) A schematic of the SqdA protein [321 amino acids (aa)] containing two RNA-binding domains (RRMs) (striped boxes), an M9-like nuclear transport motif (black box) and a glycine rich C-terminal region (hatched box). The underlined region represents the region used as bait in the yeast two-hybrid screen. The Hrb27C protein (422 aa) also contains two RNA-binding motifs (striped boxes). We isolated four different clones of Hrb27C: two contain all but the first 11 amino acids, one contains all but the first 28 amino acids and one is truncated to exclude the first 109 amino acids (underlined). (B) The Hrb27C-activation domain (AD) fusion interacts with the LexA-SqdA fusion but not with LexA alone or a LexA-p53 fusion. The AD alone does not interact with any LexA fusion proteins. (C) Western blot probed for Hrb27C and Sqd after immunoprecipitation (IP) from ovarian lysate with either Sqd or SpnF antibody in the absence (–) or presence (+) of RNAse A. Hrb27C co-purifies in an RNA-dependent manner with Sqd but not SpnF. WCL, whole cell lysate (one-tenth volume).

View larger version (60K): [in a new window]   Fig. 2. hrb27C mutant germline clones display a dorsalized phenotype because of defects in Grk expression. (A) Wild-type egg with two dorsal appendages that mark the dorsal anterior surface. (B-D) hrb27C mutants lay a range of dorsalized eggs. For the two strongest alleles hrb27Ck02814 and hrb27CrF680, the appendage defects observed range from being (B) widely spaced, with an expansion of the operculum in 28-62% of the eggs, to (D) a crown of appendage material that surrounds the anterior circumference of the egg in 5-6% of the eggs. More intermediate phenotypes were also observed (C) with a broad appendage that spans at least the width of the normal appendages in 31-60% of the eggs (n=86 for hrb27Ck02814 and n=262 for hrb27CrF680). (E,F) grk in situ hybridizations of stage 9 egg chambers. In wild-type (E) grk is tightly localized to the dorsal anterior region of the oocyte, but is detected in a ring around the anterior circumference of a fraction of the egg chambers in hrb27C mutants (F). (G-I) Grk antibody staining of stage 9 egg chambers from hrb27C mutants reveals that Grk protein has a variable distribution that includes (G) tight localization to the dorsal anterior region as in wild-type, (H) diffuse localization throughout the oocyte with an enrichment in the dorsal anterior region, and (I) an anterior ring around the oocyte.

View larger version (33K): [in a new window]   Fig. 3. Hrb27C can bind to the 3'UTR of grk. (A) Schematics of the entire 3'UTR of grk, osk and nos with the regions used as probes or competitors underlined. (B) UV crosslinking analysis of ovarian proteins that can interact with grk, osk or nos. The banding profiles reveal that the same proteins bind to grk and osk RNAs, but these proteins do not bind to nos RNA. The identity of the protein that binds to grk and osk is Hrb27C because it can be immunoprecipitated with Hrb27C antibodies, but not with CycE antibodies. (C) UV crosslinking analysis of osk (labeled probe) in the presence of grk, osk (positive control) or nos (negative control) as unlabelled competitor. A 200-fold excess grk or osk is able to compete binding of Hrb27C (arrow) from osk, while excess nos is not.

View larger version (52K): [in a new window]   Fig. 4. sqd and hrb27C mutants have nurse cell chromosomes that fail to disperse and this phenotype is rescued by isoform-specific transgenes of Sqd and Otu. (A) Wild-type ovariole stained with Hoechst to show the polytene (arrowhead) nurse cell chromosomes that have a blob-like appearance (double arrowhead) at stage 5 and are fully dispersed (arrow) by stage 6. (B) Wild-type stage 8 egg chamber stained with Hoechst with properly dispersed nurse cell chromosomes. (C,D) sqd (C) and hrb27C (D) mutant egg chambers at stage 8 stained with Hoechst reveal that the nurse cell chromosomes fail to disperse throughout oogenesis. (E) The percentage of polytene nurse cell nuclei in stage 6 and later egg chambers from sqd1 mutants and from sqd1 mutants carrying transgenes expressing the specified Sqd isoforms (A, B or S) (n>400 for each genotype). (F) The percentage of polytene nurse cell nuclei in stage 6 and later egg chambers from sqd1 mutants and from sqd1 mutants carrying one (n=339) or two copies (n=818) of the Otu104 transgene. (G) RT-PCR analysis of otu transcripts in previtellarial egg chambers. Although the larger otu transcript is absent from hfp mutants, it is present in sqd mutants. (H) Western analysis using an Otu antibody of previtellarial egg chambers from wild type (WT), sqd1 and hfp mutants. sqd mutants show a reduction in the amount of the 104 kDa Otu isoform. Western analysis of the samples using a Tubulin antibody reveals equal loading of the sqd and wild-type samples.

View larger version (65K): [in a new window]   Fig. 5. Otu interacts with Hrb27C and plays a role in regulating grk localization. (A) Western blot probed for Hrb27C after immunoprecipitation (IP) of ovarian lysate using either Otu or Odd skipped (Odd) antibody. Only the Otu-IP shows a specific interaction. WCL, whole cell lysate (one-tenth volume). (B) A representative dorsalized egg laid by an otu7 mutant. (C) grk in situ hybridization of a late stage 9 egg chamber from an otu7 mutant. In wild type, grk is tightly localized to the dorsal anterior region of the oocyte (see Fig. 2E), but is mislocalized along the anterior circumference of the egg chamber in otu7 and otu11 mutants.

View larger version (19K): [in a new window]   Fig. 6. Model for the dual roles of a RNP complex containing Hrb27C, Sqd and Otu in oogenesis. In the oocyte nucleus, Sqd, Hrb27C and additional factors (rectangle and oval) form a complex that binds to grk mRNA prior to export. Once in the cytoplasm, Sqd and Hrb27C remain part of the complex, some factors dissociate and Otu, along with different accessory factors (rounded rectangle), associate to form a distinct complex to facilitate grk RNA localization, anchoring and translational regulation in the dorsal anterior region of the oocyte. In the nurse cells, a distinct complex also containing Hrb27C, Sqd and Otu in addition to other accessory factors (trapezoid) function in the cytoplasm to mediate the processing, localization, translational regulation or stabilization of an unidentified RNA target (X), which can then regulate nurse cell chromosome dispersal at the appropriate time in oogenesis.