, 2010). Moreover, the Hiw-Wnd pathway does not regulate Dscam promoter activity, because the expression of a Dscam[TM2]::GFP transgene, under the control of the Dscam promoter, was not significantly different between wild-type and hiw mutant brains ( Figure S3C).

These results suggest that Hiw-Wnd pathway regulates Dscam expression possibly at the level of protein translation. The UTRs of mRNAs are key components of protein translational control (Wilkie et al., 2003). In order to determine the requirement of the UTRs in Dscam expressional control, we generated Dscam transgenes fused to GFP with or without Dscam 5′ and/or 3′ UTRs ( Figure 4). The expression of a Dscam transgene lacking both UTRs (Dscam::GFP) was not affected by hiw mutations ( Figure 4A). Similarly, expression GDC-0199 ic50 Fulvestrant purchase of a transgene with only the 5′ UTR (5′-Dscam::GFP) was also unaffected by hiw function

( Figure 4B). In contrast, the expression levels of a transgene with both the 5′ and 3′ UTRs (5′-Dscam::GFP-3′) and those of the transgene with only the 3′ UTR (Dscam::GFP-3′) were significantly elevated in hiw mutant neurons ( Figures 4C and 4D). Consistently, overexpressing Wnd enhanced the expression of the Dscam transgene with only 3′ UTR in C4 da neurons ( Figure 4E) as well as Drosophila Schneider 2 (S2) cells in culture ( Figure 4F). These results denote that Hiw-Wnd pathway controls Dscam expression through the 3′ UTR of Dscam mRNA. Next, we

tested whether the Dscam 3′ UTR is sufficient for translational control by the Hiw-Wnd pathway. We generated reporter transgenes by fusing EGFP cDNA with either the 3′ UTR of Dscam mRNA or that of SV40 as a control ( Figures 5A and 5B). Hiw mutations specifically enhanced the expression of the Dscam 3′ UTR reporter in C4 da neurons ( Figures 5A and 5B). Consistently, expression of Wnd in cultured S2 cells markedly increased expression of the Dscam 3′ UTR reporter ( Figure 5C). We further found that the first 202 nucleotides of Dscam 3′ UTR are sufficient for the Wnd regulation ( Figure 5D). Taken together, these results suggest that the Dscam 3′ UTR is necessary and sufficient for translational regulation by the Drosophila DLK pathway. The RNA-binding protein fragile X mental retardation Meloxicam protein (FMRP) is involved in the posttranscriptional regulation of a number of target mRNAs ( Santoro et al., 2012). FMRP has been reported to bind to Dscam mRNA in mammalian neurons ( Brown et al., 2001; Darnell et al., 2011), but the functional relevance of this binding is unknown. We wondered whether FMRP might also regulate Dscam protein translation. We tested the association between Drosophila FMRP (dFMRP) and Dscam mRNA in larval brain lysates by RNA immunoprecipitation. Compared to a control antibody, anti-dFMRP antibody pulled down more Dscam mRNA as assessed by real-time PCR ( Figure 6A).