The relationship between SMN protein levels and KIF5A mRNA stability was investigated. (A) Experimental schematic. At 12 days after induction, SMN knockdown (SMN-KD) and scrambled control (SCR) i³Ns were collected for the assays shown in panels B and E–H. (B) SMN-KD and SCR i³Ns were treated with actinomycin D (Act-D, 10 μg/mL) to inhibit de novo RNA synthesis, and cells were harvested at the indicated time points for qPCR analysis. KIF5A mRNA declined more rapidly in SMN-KD neurons than in SCR controls, with differences at 3 hours and 6 hours. Statistical significance was determined by 2-way ANOVA followed by Šídák’s multiple comparisons test. n = 3 independent experiments. (C) Schematic of the dual-luciferase reporter assay used to assess UTR-dependent regulation of KIF5A expression. Luciferase reporters containing no UTR, the KIF5A 5′-UTR, or the KIF5A 3′-UTR were transfected into stable SCR or SMN-KD HEK293T cells (Supplemental Figure 6, A–C). (D) Quantification of luciferase activity. Reporter activity from control and 5′-UTR constructs was unchanged between SCR and SMN-KD cells, whereas the KIF5A 3′-UTR reduced reporter activity in SMN-KD cells. (E) Immunoprecipitation (IP) using an anti-SMN antibody confirming efficient pulldown of SMN protein from i³N lysates. (F) RNA immunoprecipitation (RIP) followed by RT-PCR analysis demonstrating preferential enrichment of KIF5A mRNA over KIF5B mRNA in SMN immunoprecipitants. (G) Schematic of the RNA pulldown assay using biotinylated 5′ and 3′-UTRs of KIF5A, KIF5B, and KIF5C incubated with i³N lysates. (H) Immunoblot analysis of RNA pulldown fractions showed recovery of SMN with KIF5A 3′-UTRs and, to a lesser extent, with the KIF5B 3′-UTR, but not with any 5′-UTRs or KIF5C UTRs. FUS was not detected. Beads-only samples without RNA (No RNA) were used as a negative control, and blank lanes contained no sample.