Biotinylated RNA Pull-Down Assay for SYNGAP1 mRNA

To assess the interaction of SYNGAP1 mRNA 3′UTR with endogenous RNA-binding proteins, a biotinylated RNA pull-down assay was performed as described in previous studies (Udagawa et al., 2015 (link); Yokoi et al., 2017 (link)). The SYNGAP1 3′UTR sequence was amplified from the plasmids produced in the 3′ rapid amplification of cDNA ends (RACE) of wild-type (WT) iPSC-derived motor neurons. The mutation was introduced using the PrimeSTAR Mutagenesis Basal kit (Takara), according to the manufacturer's instructions. DNA templates were amplified using RT-PCR and the primers 5′-TAATACGACTCACTATAGGGCCCACCCAGCATCAGAGACC-3′ and 5′-GTCCCTGGGGGTCAAAGAGA-3′, and were purified using a PCR purification kit (QIAGEN) as a template for in vitro transcription. T7 RNA polymerase (Takara) with biotin RNA labeling mix (Roche) was used for in vitro transcription according to the manufacturer's instructions. The lysate containing 300 μg of the proteins from the motor neuron cultures was incubated with 3 μg biotinylated RNA for 1 h at room temperature; then, streptavidin Dynabeads (Invitrogen) were added. After 1 h of incubation at 4°C, the beads were washed with Brain IP buffer three times, boiled with 4× NuPAGE LDS-PAGE sample buffer (Novex) containing β-mercaptoethanol for 5 min at 95°C, and analyzed with Western blotting. The band intensities of the input and pull-down were quantified.