(A) Immunoblotting showed that the PTBP1 knockdown reduced the translation of co-expressed human FGF13 isoform 2 in HEK293 cells (n = 4, p=0.0077 for FGF13, p=0.0032 for PTBP1). The mRNA level of FGF13 remained unchanged when PTBP1 was silenced (n = 3, p=0.8100 for FGF13, p=0.0066 for PTBP1). (B) Immunoblotting showed that the PTBP2 knockdown reduced the FGF13 (isoform 4) expression in cultured mouse neurons (n = 4, p=0.0425 for FGF13, p=0.0018 for PTBP2). Three different small interference RNAs (siRNAs) targeting PTBP2 were mixed to enhance the knockdown efficiency in neurons. (C) Schematic graph shows that MCP-fused PTBP2 is recruited to the MS2-inserted FGF13 5′-UTR. (D) Quantitative real-time PCR showed that exogenous MS2-FGF13 mRNA exhibited an increased binding to Flag-MCP-PTBP2, after RNA immunoprecipitation (RIP) by Flag beads in HEK293 cells (n = 3, p=0.0262), while GAPDH mRNA was not enriched (n = 3, p=0.3807). (E) Immunoblotting showed that MS2-PTBP2 overexpression increased the protein level of FGF13 in HEK293 cells compared to PTBP2 (n = 4, p=0.0054). (F) Schematic graph shows that engineered PTBP2 protein with PUF scaffold recognizes 8 nt RNA sequence and is recruited to the Fgf13 5′-UTR. Due to the difference in designed recognition RNA sequence (the mutation site of Fgf13 is highlighted in red color), wPUF recognizes and binds to wildtype (WT) 5′-UTR, while mPUF binds to mutant (Mut) 5′-UTR. However, the PTBP2 within fused proteins normally binds to WT Fgf13 5′-UTR but has a lower binding affinity with Mut Fgf13 5′-UTR. (G) Immunoblotting showed that wPUF-PTBP2 with Flag tag at the N-terminus had lower binding level with synthesized Mut RNA compared to synthesized WT RNA (n = 3, p=0.0099), but mPUF-PTBP2 reversed the binding difference between synthesized WT and Mut RNAs (n = 3, p=0.2386). Proteins of input were simultaneously immunoblotted. ##p<0.01 and not significant (n.s.) versus indicated group by Student’s t-test. (H) The engineered PTBP2 with PUF scaffold could increase the FGF13 expression in cultured neurons of Mut mice. Lentivirus expressing PUF-Vector, wPUF-PTBP2, and mPUF-PTBP2 were used to transfect cortical neurons cultured from Mut mice. Immunoblotting showed that the FGF13 expression was increased by mPUF-PTBP2, but not by wPUF-PTBP2 (n = 8). p=0.0357 (*) versus Flag-PUF-Vector and p=0.0008 (###) versus indicated group by Student’s t-test. (I) Neurons from the Mut mice exhibited a lower ratio of polarized neurons with single axon and an increase in axon branching compared to that of WT mice. Introducing wPUF-PTBP2 did not alter the aberrant neuronal morphogenesis, but mPUF-PTBP2 could increase the ratio of polarization and decrease the axon branching. Neurons were immunostained with axon marker SMI-312. n = 100, 92, 78, and 95 neurons for four groups, respectively, from three experiments. For polarization ratio: p=0.0500 (*) and p=0.0500 (*) versus Flag-wPUF-PTBP2, while p=0.0500 (#) versus indicated group by Mann–Whitney U test. For axon branching: p=1.67E-10 (***) and p=1.88E-08 (***) versus Flag-wPUF-PTBP2, while p=3.65E-09 (###) versus indicated group by Mann–Whitney U test. Scale bar, 50 μm. Data are represented as mean ± SEM (A, B, D, E, G–I). *p<0.05 and **p<0.01 by Student’s t-test (A, B, D and E).