Protein Interaction Mapping of Translational Regulators Affecting Expression of the Critical Stem Cell Factor Nos

Abstract

The germline stem cells of the Drosophila ovary continuously produce eggs throughout the life- span. Intricate regulation of stemness and differentiation is critical to this continuous production. The translational regulator Nos is an intrinsic factor that is required for maintenance of stemness in germline stem cells. Nos expression is reduced in differentiating cells at the post-transcriptional level by diverse translational regulators. However, molecular mechanisms underlying Nos repression are not completely understood. Through three distinct protein-protein interaction experiments, we identified specific molecular interactions between translational regulators involved in Nos repression. Our findings suggest a model in which protein complexes assemble on the 3' untranslated region of Nos mRNA in order to regulate Nos expression at the post-transcriptional level.

Keywords: Drosophila; Nos; Oogenesis; Ovary; Stem cells.

Fig. 1. Bam physically interacts with Mei-P26 and Brat.

(A) Yeast two-hybrid assay, with protein-protein interactions monitored using β-galactosidase assays. Bam full- length sequence was fused with the LexA DNA-binding domain (denoted as LexA-Bam FL). Other constructs were fused to the Gal4 transcriptional activation domain (TAD). FL, N, NHL indicates full-length, the N-terminus, and the C-terminal NHL domain of Mei-P26 or Brat. Mean±SD values were obtained from three independent experiments. P values were determined by Student’s t-test in Sigma Plot. ^*** P<0.001. (B) Pull-down assay. S2 cell lysates co-expressing Flag-Bam with Myc-Mei-P26 FL, Myc-Mei-P26 N, and Myc-Mei-P26 NHL were subjected to immunoprecipitation with Flag antibody (α-Flag). The extract (1/10 of the lysate) and the resulting immunoprecipitates (IPs) were subjected to immunoblot analysis with Flag and Myc an- tibodies. Three independent experiments were carried out with similar results. (C) Fragment complementation assay. Confocal images of HEK293 cells expressing proteins fused to either N-terminal (GFP-N) or C-terminal (GFP-C) fragments of GFP. P50/p65 was used as a positive control, with p50 fused to GFP-N and p65 fused to GFP-C. For assaying Bam/Mei-P26 FL, Bam/Mei-P26 N, and Bam/Mei-P26 NHL, Bam was fused to GFP-N and others fused to GFP-C. Three independent experiments were carried out with similar results. The scale bar indicates 10 μm.

Fig. 2. Sxl physically interacts with Mei-P26, Brat, Pum, and Bgcn.

(A, B) Yeast two-hybrid assay, with protein-protein interactions monitored by β-galactosidase assays. (A) Sxl full- length sequence was fused with the transcriptional activation domain (TAD) of Gal4 (TAD-Sxl). The other constructs were fused to the LexA DNA-binding domain (LexA). (B) Sxl full-length sequence was fused with the LexA DNA-binding domain (LexA-Sxl). The other con- structs were fused to the TAD of Gal4. FL, N, NHL respectively indicate full-length, N-terminus, and the C-terminal NHL domain of Mei-P26 or Brat. Mean±SD values were obtained from three independent experiments. P values were obtained by Student’s t-test in Sigma Plot. ^*** P<0.001. (C, D) Pull-down assay. (C) S2 cell lysates coexpressing Flag-Sxl with Myc-Brat were subjected to immunoprecipitation with Flag antibody (α-Flag). The extract (1/10) and the resulting immunoprecipitates (IPs) were subjected to immunoblot analysis with Flag and Myc anti- bodies. (D) S2 cell lysates co-expressing Flag-Sxl with Myc-Bgcn FL (upper) and Myc- Mei-P26 FL (lower) were subjected to immunoprecipitation with Flag antibody. FL denotes full-length. The extract (1/10) and the resulting IPs were subjected to immunoblot analysis with Flag and Myc antibodies.

Fig. 3. Model illustrating protein assembly on nos 3’UTR.

(A) Schematic drawing of a map of interactions revealed by our protein-protein interaction assays. (B) A model illustrating how these proteins are assembled on the nos 3’UTR.