dE2F2-Independent Rescue of Proliferation in Cells Lacking an Activator dE2F1

Abstract

In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G 1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor.Cell proliferation and differentiation are precisely coordinated during the development of a multicellular organism. The loss of such control may ultimately lead to defects in development and cancer. E2F transcription factors are important regulators of the cell cycle and critical downstream targets of the retinoblastoma (pRB) tumor suppressor protein (12). In spite of remarkable progress, dissecting the pRB pathway in mammalian cells remains a challenging task. One issue arises from functional redundancy and compensation among E2F and pRB family members. In the past years, Drosophila melanogaster has been recognized as a valuable tool for understanding various aspects of E2F biology. This is mainly due to the high conservation in cell cycle regulation between flies and mammals and the relative simplicity of the E2F/pRB module in Drosophila. In Drosophila, there are two E2F genes. As mammalian counterparts, their products can be classified as repressors (dE2F2) and activators (dE2F1).Analysis of dE2F single-and double-mutant animals has provided insights into the normal function of dE2Fs during development. de2f1 mutant larva are severely retarded in larval growth and show a strong reduction in S phases and the expression of E2F targets. Unexpectedly, these defects are largely suppressed in de2f1 de2f2 double mutants. This suggests that the de2f1 mutant phenotype is not entirely due to the absence of activator dE2F1 but rather, to some extent, is due to the presence of the "unchecked" repressor dE2F2 (18). This result is particularly striking since de2f2 mutants are viable and develop normally (5, 18). Why "unchecked" dE2F2 has such a strong effect on cell proliferation in the absence of dE2F1 is not clear. A possible explanation, supported by gene expression profiling (11), is that, in de2f1 mutant cells, dE2F2 inappropriately represses dE2F1-specific genes that are critical for cell proliferation. ...

DEAD-box RNA helicase Belle/DDX3 and the RNA interference pathway promote mitotic chromosome segregation

“…To address this possibility, we analyzed Belle, a DEAD-box RNA helicase, which is the closest somatic paralog of Vasa (32,33). Belle is expressed in both germline and somatic cells and has been indicated to regulate cell cycle progression, mRNA splicing, and RNAi (33)(34)(35)(36)(37). Consistent with the findings of previous studies, we detected high levels of Belle, but not Vasa, in Drosophila somatic S2 cells, although both proteins are expressed in Drosophila ovaries (Fig.…”

“…In Drosophila, Belle, a DEAD-box RNA helicase, is the closest somatic paralog of Vasa (32,33). Belle is expressed in both germline and somatic cells and has been indicated to regulate cell cycle progression, mRNA splicing, and RNA interference (RNAi) (33)(34)(35)(36)(37). Furthermore, the endo-siRNA pathway functions similarly to the piRNA pathway in somatic cells (24)(25)(26)(27).…”

Pumilio facilitates miRNA regulation of the E2F3 oncogene

“…S2B). Depletion of Cullin 4 , a known regulator of dE2F1 ubiquitination and degradation (Shibutani et al 2008), similarly increased dE2F1 levels; in contrast, RNAi of belle (bel), an RNA-binding protein that has previously been reported not to regulate dE2F1 (Ambrus et al 2007), had no effect (Fig. 1D).…”

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells

“…Although Bel appears to function in the siRNA pathway (Zhou et al, 2008), we found that the siRNA pathway is not involved in regulating Notch in follicle cells. A few reports have also identified several phenotypes associated with disruption of bel that indicate that Bel functions in the 1743 RESEARCH ARTICLE microRNAs regulate Notch in follicle cells G1/S transition in the eye disc by affecting Hedgehog signaling and Dacapo expression (Ambrus and Frolov, 2010;Ambrus et al, 2007), as well as identifying a role for Bel with the zinc-finger protein Zn72D in regulating the splicing and translation of maleless transcripts (Worringer et al, 2009). It will be interesting to determine whether the function of Bel in these other important cellular processes is also related to a role in RNAi pathways.…”

“…Bel was recently found to be necessary for small interfering RNA (siRNA) silencing in Drosophila cell culture and to be in a complex with several components of both the siRNA and miRNA pathways, including the small non-coding RNAs that mediate both pathways (Zhou et al, 2008). Bel has also been linked to other cellular processes and this might be related to its role in RNAi (Ambrus and Frolov, 2010;Ambrus et al, 2007;Worringer et al, 2009 (Ambrus et al, 2007;Bender et al, 1987;Johnstone et al, 2005;Jones and Rawls, 1988). Both bel 74407 and bel 47110 failed to complement any of the other bel alleles, indicating that these two lines represent strong mutations in bel.…”