Prepro-VIP level was increased significantly in the brain of Eif4ebp1 KO mice (normalized band intensities: KO versus WT, 3.88 ± 0.36 versus 1 ± 0.18, p < 0.05, Student’s t test;

Figure 5C). In contrast, expression of VPAC2 (the VIP receptor expressed in the SCN), and of the precursor proteins of other neuropeptides implicated in the SCN synchrony ( Piggins et al., 1995 and Maywood et al., 2011), including prepro-GRP and prepro-AVP, was not changed ( Figure 5C). In addition to the neuropeptides, we examined other proteins involved in SCN synchrony, including GABAa receptor ( Liu and Reppert, 2000, see more Colwell et al., 2003 and Albus et al., 2005) and gap junction protein Connexin 36 ( Long et al., 2005). The levels of Connexin 36 and the GABAa receptor α subunit were not altered in the Eif4ebp1 KO brain ( Figure 5C). Furthermore, the expression of the 4E-BP1 binding partner, eIF4E, was not changed ( Figure 5C). These results demonstrate specific regulation of prepro-VIP by 4E-BP1. To complement the in vivo data, we studied prepro-VIP expression in mouse Neuro2A and human SHEP neuroblastoma cells (Waschek et al., 1988). Treatment of Neuro2A cells with the specific mTOR active-site inhibitor, PP242, resulted in reduced prepro-VIP levels and in dephosphorylation of 4E-BP1 Ion Channel Ligand Library after 3 hr (Figure S4B). To determine whether the effect of mTOR inhibition on prepro-VIP expression is dependent on 4E-BP1, we knocked down

4E-BP1 in SHEP cells using lentivirus (Figure S4C). Prepro-VIP was increased by ∼1-fold in 4E-BP1 knockdown cells. Rapamycin decreased 4E-BP1 phosphorylation and inhibited prepro-VIP

expression in control cells (scrambled), but not in 4E-BP1 knockdown cells (sh4e-bp1) (Figure S4C). Serum stimulation induced strong prepro-VIP expression in control cells, but to a lesser extent in 4E-BP1 knockdown cells (Figure S4D), indicating that inducible prepro-VIP expression is at least partially dependent on 4E-BP1. Consistent with these data, overexpression of 4E-BP1 led to a reduction in prepro-VIP (Figure S4E). Overexpression of WT eIF4E, but not the W56A mutant, which cannot bind to the mRNA cap (Gingras et al., 1999), increased prepro-VIP (Figure S4F), demonstrating that prepro-VIP synthesis is dependent on eIF4E and cap-dependent translation in neuroblastoma cells. Expression of Vip 5′ UTR-RLuc mRNA, but not RLuc or Grp 5′ Phosphoprotein phosphatase UTR-RLuc mRNA, was enhanced in Eif4ebp1 KO (∼2-fold) as compared to WT mouse embryonic fibroblasts ( Figure S4G; p < 0.05, ANOVA). Grp mRNA 5′ UTR has a similar length but lesser secondary structure than Vip mRNA 5′ UTR. Thus, these results demonstrate that Vip mRNA translation is preferentially enhanced in 4E-BP1 KO cells. Because 4E-BP1 inhibits translation initiation, it was anticipated that prepro-VIP upregulation in the Eif4ebp1 KO brain is at the mRNA translation initiation step. To demonstrate this, we studied Vip mRNA translation by polysome profiling.