Two isoforms of the Notch antagonist Hairless are produced by differential translation initiation

Abstract

The Notch-signaling pathway controls cellular differentiation, including proliferation and cell death in all higher metazoans (including flies and men show that the smaller isoform derives from an internal ribosome entry site (IRES) within the ORF. The IRES is active in a heterologous assay and contains an essential, conserved structural element. The two Hairless isoforms have residual activity in vivo which is, however, reduced compared to a combination of both, which implies that both protein isoforms are necessary for WT function. In larval tissues, translation of the two isoforms is cell-cycle regulated: whereas the H p150 isoform is translated during interphase, H p120 is enriched during mitosis. Thus, the presence of either H isoform throughout the cell cycle allows efficient inhibition of Notch-regulated cell proliferation.

Hairless-Mediated Repression of Notch Target Genes Requires the Combined Activity of Groucho and CtBP Corepressors

“…For immunoprecipitation, we used rabbit anti-Hairless A antibodies at 1:250 dilution; for detection we used rat anti-Hairless A at 1:500 (28,29), mouse anti-Gro antibodies at 1:50 (14), and rat anti-CtBP antibodies at 1:100. In vitro transcription-translation was performed with a TNT T7/T3-coupled reticulocyte lysate system (Promega) using Hairless, Gro, and CtBP cDNAs cloned in BT vector (Stratagene).…”

“…H⌬G deletes amino acids 490 to 712. Counts begin with the first methionine (29). In the H*C mutation, the CtBP binding site was altered by in vitro mutagenesis from PLNLSKH to VIQITKR; in H*G, the wild-type GBD was replaced by GBD-2.…”

“…Staining of imaginal disks and Western blot analysis were performed as described before (34) using the following antisera: anti-Gro and anti-beta-galactosidase (developed by C. Delidakis and J. Sanes, respectively; obtained from Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242); anti-H-A and anti-NTH (28,29); and anti-CtBP and anti-Su(H) (18). Secondary antibodies coupled to alkaline phosphatase, fluorescein, Cy3, or Cy5 were purchased from Jackson Laboratory.…”

A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

“…A putative translation start is found at Met-113, which would produce an FNR S with the right size (34 kDa) and an N-terminal sequence matching the one found by Matsuo et al (18) for an FNR that copurifies with the NDH-1 complex in Synechocystis. Although this may sound curious, many examples of internal ribosome entry sites (IRESs) within coding regions have been described in various organisms, although rarely in prokaryotes (26)(27)(28). To test this hypothesis, we introduced missense and frame-shift mutations in petH by a procedure similar to that described in ref.…”

A molecular link A molecular link between Hairless and Pros26.4, a member of the AAA-ATPase subunits of the proteasome 19S regulatory particle inDrosophila

“…Mutations in Hairless are haplo-insufficient and cause a dominant loss of bristles and wing venation defects. This phenotype was rescued to some extent by simultaneously removing one copy of Pros26.4 by using deficiency Df(3R)mbc-R1: flies heterozygous for H P8 in a wild-type background lacked 12.2 macrochaetae on average (see Maier et al, 2002), whereas in the background of Df(3R)mbc-R1, they lacked only 9.7 bristles on average. This slight amelioration of the Hairless lossof-function phenotype by concurrent loss of Pros26.4 argues for a negative regulation of Hairless by Pros26.4.…”