Down-Regulation of RpS21, a Putative Translation Initiation Factor Interacting with P40, Produces Viable Minute Imagos and Larval Lethality with Overgrown Hematopoietic Organs and Imaginal Discs

Abstract

Down-regulation of the Drosophila ribosomal protein S21 gene (rpS21) causes a dominant weak Minute phenotype and recessively produces massive hyperplasia of the hematopoietic organs and moderate overgrowth of the imaginal discs during larval development. Here, we show that the S21 protein (RpS21) is bound to native 40S ribosomal subunits in a salt-labile association and is absent from polysomes, indicating that it acts as a translation initiation factor rather than as a core ribosomal protein. RpS21 can interact strongly with P40, a ribosomal peripheral protein encoded by the stubarista (sta) gene. Genetic studies reveal that P40 underexpression drastically enhances imaginal disc overgrowth in rpS21-deficient larvae, whereas viable combinations between rpS21 and sta affect the morphology of bristles, antennae, and aristae. These data demonstrate a strong interaction between components of the translation machinery and showed that their underexpression impairs the control of cell proliferation in both hematopoietic organs and imaginal discs.

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

“…In contrast to aub, the phosphorylation of short Osk is not affected in vls. The same blot was successively probed for ribosomal P40, as loading control (Török et al, 1999). (H,I) Vas is localized at the posterior pole of the oocyte in wild type (H) and vls 3 (I) stage 10 egg chambers.…”

Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

“…Some co-chaperones such as CHIP, display a single TPR domain, which binds alternatively Hsp70 or Hsp90 (15,17). In contrast, HOP harbors three TPR domains, which enable it to transfer clients from Hsp70 to Hsp90 (23).…”

“…Polyclonal anti-dHsp90 antibody was kindly given by Renato Paro. Proteins were detected as follows: Rpb1 detected with mouse monoclonal PB7-C2 antibody; Rpb2 with goat S20 from Santa Cruz Biotechnology; Nop58 with polyclonal antibodies generated from rabbits immunized with an KKLQEVD-SLWKEFETPEK peptide (14) IA); polyclonal anti-15.5K antibodies from Santa Cruz Biotechnology (SC-86760); polyclonal anti-P40 antibodies have been described previously (15), and anti-FLAG M2 monoclonal antibody was from Sigma. Tissue Analysis-Larvae were dissected in PBS, and tissues were fixed with 4% paraformaldehyde and mounted in Vectashield mounting medium (Vector Laboratories).…”

“…The pBD-(GAL4)-spag transformed yeast cells were retransformed with embryonic (0 -5 h) or ovarian cDNA libraries, in Hibry-Zap II phagemid AD(GAL4) vector (15). Clones were selected for growth on ϪLeu, ϪTrp, and ϪHis SD plates and ␤-galactosidase filter assay.…”

“…The induction was performed with 400 M isopropyl 1-thio-␤-D-galactopyranoside for 5 h, and GST fusion proteins were purified as described earlier (15). GST fusion proteins (ϳ1-2 g) bound to ϳ20 l of glutathione-Sepharose 4B (GE Healthcare) were incubated with 35 Slabeled Spag, Spag⌬TPR, or Hsc70-3 previously synthesized by TNT coupled reticulocyte lysate in vitro translation system (Promega) or with 2 g of unlabeled recombinant bovine Hsc70-4 protein (StressGen) in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40, and protease inhibitor mixture (complete, Roche Applied Science) buffer for 3 h at 4°C by rotation of the tubes.…”