Mutation of the DEAD-box helicase belle downregulates the cyclin-dependent kinase inhibitor Dacapo

Abstract

The retinoblastoma protein (pRB) negatively regulates cell proliferation by limiting the activity of the family of E2F transcription factors. In Drosophila, mutation of the DEAD-box helicase belle (bel) relieves an E2F/pRB induced G(1) cell cycle arrest; however, the mechanism of this rescue is unknown. Here, we show that the level of the cyclin-dependent kinase inhibitor Dacapo (Dap), homolog of mammalian p21/p27, is strongly reduced both in bel mutant cells in vivo and in tissue culture cells depleted of Bel by RNA interference. Interestingly, the loss of bel also partially alleviates an ectopically induced G(1) cell cycle arrest. Additionally, we show that Bel undergoes nucleocytoplasmic shuttling. Thus, inactivation of bel renders cells less sensitive to several anti-proliferative signals inducing G(1) arrest.

Figure 1. The nucleocytoplasmic localization of Bel in vivo and in cell culture

(A-B) Eye imaginal discs were dissected from third-instar larvae and stained with anti-Bel (red) and DAPI (blue). Bel is predominantly localized to the cytoplasm in cells of control discs (A), but shows an enrichment in the nucleus in cells of discs treated with LMB (B). (A′) and (B′) are the same corresponding images as (A) and (B) showing only Bel. (A″) and (B″) are the same corresponding images as (A′) and (B′) at a higher magnification. (C-D) S2R+ cells were stained with anti-Bel (red), the nucleolar marker anti-fibrillarin (green), and DAPI (blue). Bel is predominantly localized to the cytoplasm in control cells (C), but shows an enrichment in the nucleus and nucleolus in cells treated with LMB (D). (C′) and (D′) are the same corresponding images as (C) and (D) showing only Bel. (C″) and (D″) are the same corresponding images as (C) and (D) showing only Fibrillarin (Fib) and DAPI.

Figure 2. Dap is down-regulated and the the second mitotic wave (SMW) is partially restored in the absence of Bel

(A) The level of Dap was lower in S2 cells depleted of Bel by RNAi than in control cells. Protein levels were determined by Western blot analysis. Luciferase (luc) and dE2F1 dsRNA were used as nonspecific RNAi controls. Tubulin (Tub) was used as a loading control. (B-G) Eye imaginal discs were dissected from third-instar larvae. Genotypes are shown, the morphogenetic furrow (MF) is marked by an arrowhead and posterior is to the right. (B) Larvae containing the Dap-HB-lacZ reporter were stained with anti-b-galactosidase (red). Dap-HB enhancer activity appears to be ubiquitous throughout the eye disc. (C) In clones of bel mutant cells marked by the absence of GFP (green) the expression of Dap-HB-lacZ (red) is severely reduced. For (C) and (C′) clones of wild-type cells, identified by the presence of GFP (green), and homozygous bel^L4740 mutant cells, identified by the absence of GFP (green) were induced by ey-FLP. (C′) is the same corresponding image as (C) showing only Dap-HB-lacZ. (D) In wild-type eye discs cells are arrested in G₁ and sycnchronously reenter the cell cycle in the SMW as evidenced by the stripe of BrdU (red), a marker of S-phase, immediately posterior to the MF. (E) When expression of p21 is ectopically driven by the GMR promoter the number of cells which enter the SMW is severly reduced as evidenced by the lack of cells incorporating BrdU (red) immediately posterior to the MF. (F) In clones of bel mutant cells marked by the absence of GFP (green) the SMW is patially restored as evidenced by the presence of cells incorporating BrdU (red) immediately posterior to the MF. (G) Cells of bel mutant clones marked by the absence of GFP (green) complete the SMW as evidenced by the presence of phosphorylated histone H3 (pH3, magenenta), a marker of mitosis.