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MacNicol AM
,
Hardy LL
,
Spencer HJ
,
MacNicol MC
.
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BACKGROUND: There is increasing evidence of a pivotal role for regulated mRNA translation in control of developmental cell fate transitions. Physiological and pathological stem and progenitor cell self-renewal is maintained by the mRNA-binding protein, Musashi1 through repression of translation of key mRNAs encoding cell cycle inhibitory proteins. The mechanism by which Musashi1 function is modified to allow translation of these target mRNAs under conditions that require inhibition of cell cycle progression, is unknown.
RESULTS: In this study, we demonstrate that differentiation of primary embryonic rat neural stem/progenitor cells (NSPCs) or human neuroblastoma SH-SY5Y cells results in the rapid phosphorylation of Musashi1 on the evolutionarily conserved site serine 337 (S337). Phosphorylation of this site has been shown to be required for cell cycle control during the maturation of Xenopus oocytes. S337 phosphorylation in mammalian NSPCs and human SH-SY5Y cells correlates with the de-repression and translation of a Musashi reporter mRNA and with accumulation of protein from the endogenous Musashi target mRNA, p21(WAF1/CIP1). Inhibition of Musashi regulatory phosphorylation, through expression of a phospho-inhibitory mutant Musashi1 S337A or over-expression of the wild-type Musashi, blocked differentiation of both NSPCs and SH-SY5Y cells. Musashi1 was similarly phosphorylated in NSPCs and SH-SY5Y cells under conditions of nutrient deprivation-induced cell cycle arrest. Expression of the Musashi1 S337A mutant protein attenuated nutrient deprivation-induced NSPC and SH-SY5Y cell death.
CONCLUSIONS: Our data suggest that in response to environmental cues that oppose cell cycle progression, regulation of Musashi function is required to promote target mRNA translation and cell fate transition. Forced modulation of Musashi1 function may present a novel therapeutic strategy to oppose pathological stem cell self-renewal.
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25888190
???displayArticle.pmcLink???PMC4369890 ???displayArticle.link???BMC Dev Biol ???displayArticle.grants???[+]
Figure 1.
Musashi undergoes regulatory phosphorylation in response to neural differentiation. (A) Primary embryonic rat neural stem/progenitor cells (NSPCs) were maintained as neurospheres in proliferation media (prolif) or plated on adherent substrate in differentiation media for 2Â hrs (diff). Protein lysates were processed for western blotting with the indicated antibodies. (B) Human neuroblastoma SH-SY5Y cells were maintained as neurospheres in proliferation media (prolif), or under differentiation conditions (diff) for the indicated times and analyzed by western blotting with the indicated antibodies.
Figure 2.
De-repression of a Musashi-dependent reporter mRNA during differentiation of SH-SY5Y cells. (A) SH-SY5Y cells were transfected with plasmids encoding luciferase reporter mRNAs under control of a Musashi binding element (MBE) or a control 3â² UTR (Control) and luciferase activity was assessed, relative to a co-transfected Renilla luciferase standard, following culture under proliferation or differentiation conditions for 24 hours. The dot plot shows the relative luciferase activity of six independent experiments as a percent of the 3â² UTR control (100%) for both proliferation and differentiation, as indicated. Luciferase expression was repressed in proliferating cells (mean of 77% with a 95% confidence interval: 73 to 81%; pâ<â0.0001, one sample t-test) but not in differentiated cells (mean of 97.7% with a 85% confidence interval 90.6 to 105.4%, pâ>â0.47, one sample t-test). (B) Western blot demonstrating persistence of Musashi1 in SH-SY5Y cells 24 hours after induction of differentiation. In this experiment, 10, 20 and 30 μg total protein (as indicted) were analyzed for Musashi1 and GAPDH protein levels from proliferating neurosphere culture (Prolif) or cells cultured in neuronal differentiation media for 24 hours (Diff). A representative experiment is shown.
Figure 3.
Overexpression of Musashi1 blocks neural differentiation. NSPCs (panels A-D) and SH-SY5Y cells (panels E-H) were transfected with a plasmid encoding the eGFP moity alone (A and E), wild-type Musashi1 (B and F) or Musashi1 encoding a non-phosphorylatable S337A mutation (C and F) fused to a C-terminal eGFP epitope tag and incubated at low cell density under differentiation conditions for 4Â days (NSPCs) or 7Â days (SH-SY5Y) and scored for initiation of differentiation phenotype (hypertrophic cells with neuritesâ>â2 soma lengths). Representative photos shown. Some variations in the intensity and subcellular distribution of Musashi1 or Musashi1 S337A were noted in transfected cells within the same experiment. Over the course of three experiments, a total of 70 NSPC cells (A) or 136 SHSY5Y cells (E) transfected with eGFP alone were counted. (D and H) represent histograms showing quantitation of the differentiation data (mean of three experiments with SEM) at 14Â days (D) or 7Â days (H). ****,pâ<â0.0001 for (D); **,pâ<â0.01 for (H)) as assessed by one-way ANOVA.
Figure 4.
Inhibition of Musashi phosphorylation promotes cell survival. (A) NSPCs and (B) SH-SY5Y cells were transfected with the eGFP tagged Musashi proteins described in Figure 3 and fluorescent cells were photographed and counted 1 day after transfection then maintained under conditions of serum deprivation for 14 days (NSPC) or 4 days (SH-SY5Y) and counted relative to cell expressing the control, eGFP tag alone. Histogram represents the mean of the three experiments with SEM. The difference in surviving cell numbers were significant (**,pâ<â0.01 for NSPCs; ****,pâ<â0.0001 for SH-SY5Y cells). (C) SH-SY5Y cells were maintained as neurospheres in control proliferation media (Con) or plated on adherent substrate under conditions of serum starvation for 3 days (SS). Protein lysates were processed for western blotting with the indicated antibodies.
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