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). Signal transduction through activated Notch involves the CSL group of transcriptional regulators. Notch signals need to be tightly regulated, and in Drosophila they are antagonized by the Hairless (H) protein. H silences the activity of Notch target genes by transforming the Drosophila CSL protein, Suppressor of Hairless [Su(H)], from a transcriptional activator into a repressor while recruiting one of the corepressors dCtBP or Groucho. The H protein has a calculated molecular mass of approximately 110 kDa and contains several functional domains apart from the two small corepressor-binding domains. However, although there is no indication for alternative splicing, two Hairless protein isoforms, H(p120) and H(p150), are observed throughout development. Here, we 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.

Fig 1.

The shorter Hairless H^p120 isoform is translated from the third start codon. (A) Schematic drawing of the H protein with potential start sites M1, M2, and M3. FL represents the full-length construct; the translated region is shown as the thicker bar, and the Su(H)-binding domain is crosshatched. Anti-A and anti-NTH show fragments used for production of respective antisera. C1 is an N-terminal truncation deleting the interval between M1 and M3; C4 is a 50-codon internal deletion in the C-terminal domain and was used as control. The blow-up of the N-terminal region shows the site of mutations that were introduced in respective constructs. The three potential start codons M1, M2 (at position 19), and M3 (at position 148) were altered individually or in combination into isoleucine (GTG). Mutant constructs (Δ) were cloned under heat-shock promoter control and transformed into flies. The same was done for Cfs, which contains a frame shift at position 113 (AflII), resulting in termination of translation 45 codons downstream of M3 (arrow). (B) Two H isoforms, H^p120 and H^p150, are detected in Drosophila proteins of different sources. WT, protein extracts from WT embryos; nuc, nuclear; cyt, cytoplasmic extracts from embryos; iv, in vitro-translated protein derived from an FL-H cDNA clone. Mutation of potential start sites eliminates formation of specific H isoforms. Protein extracts from embryos bearing the respective transgene and induced by a heat pulse were detected in Western blots with anti-A Abs. Mutation of M1 plus M2 (Δ1/2) interferes with H^p150 isoform formation just like the N-terminal truncation construct C1, whereas mutation of M3 (Δ3) eliminates formation of the short H^p120 isoform. Mutation of M2 (Δ2) still gives rise to the long isoform but at a reduced ratio compared to H^p120, suggesting that M1 is used less efficiently as start site. FL-H (full-length construct) and C4 serve as controls. Please note that the WT protein, denoted with a circle, is also detected at low levels. (C) A frame shift at codon 113 in construct Cfs results in a translation stop 45-aa downstream of M3. However, the H^p120 isoform is detected with anti-A antiserum, demonstrating that the short isoform is generated by internal translation initiation at M3 and not by site-specific cleavage or leaky scanning. The full-length construct (FL) serves as control. Anti-NTH Abs detect only the long H isoform in FL and, in addition, the N-terminal peptide derived from Cfs. Two peptides are seen because of translation start at M1 (fsM1) and M2 (fsM2), respectively. S, protein standard; *, unspecific crossreactivity of anti-NTH.

Fig 2.

Hairless contains a bona fide IRES. (A) The interval between M2 and M3 was cloned between lacZ and luciferase (luc) genes under the control of a ubiquitous promoter. In construct IS, the two cistrons were split by converting M2 into a stop codon; this should result in β-galactosidase (arrow) and, only in the case of internal translation initiation at M3, luciferase would be expected (dashed arrow). (B) Enzymatic activity of luciferase (luc) was measured in S2 cells transfected with dicistronic and control constructs. Transfection was controlled by β-galactosidase activity. Experiments were repeated sevenfold; a representative example is shown. IS gives activity similar to construct CI, which contains the Antp-IRES (16) and several times above pA LL lacking the IRES (16) and BTI lacking a promoter. Luciferase also can be detected in Western blots from IS and CI (dotted arrow). IS also produces low amounts of a lac/luc fusion product, most likely resulting from repression of the stop codon (open arrow). However, the levels are rather low; probing the same extracts with anti-β-galactosidase Abs shows that the in-frame construct I produces similar levels of the lac/luc fusion protein (open arrow), as construct IS produces the lac-product (arrow) but no fusion protein. (C) Comparison among defined IRES sequences from polio virus 2 (PV-2), human ornithine decarboxylase (ODC H.s.), and Drosophila Hairless (D.m). Both defined boxes A and B, as well as the spacing, are roughly conserved. Two different mutations were introduced into box A of the H-IRES, here shown in italics (boxA1 and boxA2). In vitro translation products of full-length Hairless (WT) and the mutated constructs boxA1 and boxA2, detected with anti-A antiserum, are shown. Please note that the short H^p120 isoform is only produced from the WT construct and is essentially absent from the mutants, indicating the uridines in box A are essential for the activity of the H-IRES.

Fig 3.

Both H isoforms are required in vivo. Transformant flies carrying the respective mutant heat-shock constructs (Fig. 1) were crossed to Hairless loss of function allele H^P8. Heterozygous H offspring were allowed to develop at ambient temperature and analyzed for dominant loss of large bristles. The H^P8 mutant has ≈10 fewer macrochaetae than WT. An average of at least three independent strains of each construct is shown. Any one is able to rescue this heterozygous phenotype, however, with significantly lower efficiency and greater variability when compared to the FL-H and ΔM1 constructs, which allow the translation of both protein isoforms.

Fig 4.

H^p120 but not H^p150 accumulates in mitotic cells. Transformant larvae carrying the respective constructs, FL-H (A), ΔM3 (B), and ΔM1/2 (C), were subjected to a brief heat shock, and imaginal discs were dissected ≈4 h later. Staining was performed with anti-A H Abs (green). This Ab specifically detects WT H protein in the cytoplasm; only ectopic H protein is detected in the nucleus (4). Thus, ectopically induced H protein can be specifically visualized. Mitotic cells were labeled with anti-phospho-Histone H3 Abs (pink). The blade region of wing imaginal discs is shown in an overview (Left, six sections crossing ≈10 μm; bar = 2 μm) and as close up (A′ and A" traverse ≈16 μm; B′ and B" and C′ and C" each ≈30 μm; bar = 1 μm). Double stainings are shown; the right column shows the respective single stainings of the close-ups. Note that ΔM3 gives rise only to H^p150 and ΔM1/2 gives rise only to H^p120 isoforms (compare to Fig. 2A). H^p150 is largely absent from mitotic cells (see B′ and B”); instead, these cells accumulate H^p120 (see C′ and C”).