DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA

a Cap-independent translational regulation contributes to DSCR1.4 protein expression. SHSY5Y cells were treated with DMSO or 200？μM rapamycin or 50？mg/ml cycloheximide for the indicated times. The levels of endogenous proteins were measured by Western blotting (WB) using anti-DSCR1.4, anti-phosphoS6RP, anti-GAPDH antibodies. GAPDH was used as a loading control. The activity of rapamycin was analyzed by the phosphorylation status of S6RP. The numbers at the bottom indicate the fold increases relative to control. The amount of DSCR1.4 was normalized to GAPDH. b Schematic representation of pRF bicistronic luciferase plasmids used for observing cap-independent translation activity of human and mouse DSCR1.4 5′UTR. c, d hDSCR1.4 5′UTR and mDSCR1.4 5′UTR induce cap-independent translation initiation. c SHSY5Y and d N2A cells were transfected with the bicistronic reporter plasmids and were incubated for 24？h. pRF β-globin was used as a negative control. pRF EMCV and pRF p53 were used as the positive control. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean？±？SEM (n？=？3, n？=？4). e, f hDSCR1.4 5′UTR and mDSCR1.4 5′UTR prefer cap-independent translation to cap-dependent translation. e SHSY5Y cells were transfected with in vitro transcribed m7G capped or ApppG capped hDSCR1.4 5′UTR-FLUC. f N2A cells were transfected with in vitro transcribed m7G capped or ApppG capped mDSCR1.4 5′UTR-FLUC. Transfected cells were incubated for 6？h and were harvested. Translation activity is shown as the ratio of FLUC to FLUC mRNA. Translation activity of m7G capped transcripts was set as 1. The bars represent the mean？±？SEM (n？=？4, n？=？3). g, i The 5′ proximal 119 nucleotides sequence of hDSCR1.4 5′UTR is important for cap-independent translation activity of hDSCR1.4 5′UTR. pRF plasmids with serial deletion constructs and six nucleotides mutant construct were transfected into g SHSY5Y cells and i mouse primary hippocampal neurons. Luciferase activity is shown as the ratio of FLUC to RLUC. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean？±？SEM (n？=？3). h, j The 5′ proximal 136 nucleotides are essential for cap-independent translation activity of mDSCR1.4 5′UTR. Indicated pRF plasmids were transfected into h N2A cells and j mouse hippocampal neurons. Luciferase activity of pRF Mock plasmid transfected cells was set as 1. The bars represent the mean？±？SEM (n？=？3, n？=？3). Data information: In c–j, *P？<？0.05, **P？<？0.01,