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. 2021 Sep 20;13(11):e14163. doi: 10.15252/emmm.202114163

Figure 5. SRPK1 inhibitors selectively suppress RAN translation.

Figure 5

  • A
    Schematic of SRPK1 signaling pathway that regulates subcellular SRSF1 localization, which in turns can impact export and translation of repeat RNAs in the cytoplasm. SRPK1 may also act directly on protein translation pathways (dotted arrow). Known pharmacological compounds that inhibit SRPK1 can disrupt this pathway.
  • B
    Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters. β‐Actin is used as a loading control. To prevent signal saturation, AUG‐nLuc lysate was diluted 1:3 in sample buffer prior to loading (n = 3 biological replicates). Schematics of the AUG‐nLUC‐3xFLAG and +1CGG(100)‐nLuc‐3xFLAG reporters presented on top.
  • C
    Relative expression of AUG‐nLuc and CGG‐nLuc reporters in HEK293T cells (n = 8–9 biological replicates) following treatment with DMSO and SRPIN340.
  • D
    Anti‐FLAG immunoblot of DMSO and SRPIN340 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters (n = 3 biological replicates). Schematics of the GA70 (GGGGCCx70) and +2CGG reporters presented on top.
  • E
    Immunoblot of DMSO and SPHINX31 pre‐treated HEK293T cells expressing AUG‐nLuc‐3xFLAG control or CGG‐nLuc‐3xFLAG RAN translation reporters (n = 3 biological replicates).
  • F
    Anti‐FLAG of DMSO and SPHINX31 pre‐treated HEK293T cells expressing GGGGCC‐nLuc‐3xFLAG (GA70) and +2CGG‐nLuc‐3xFLAG (FMRpolyA) RAN translation reporters (n = 3 biological replicates).

Data information: Error bars represent mean ± SD. Statistical analysis was performed using two‐tailed Student’s t‐test with Welch’s correction. *P < 0.05; **P < 0.01, ***P < 0.001, and ****P < 0.0001. To prevent over‐exposure, the AUG‐nLuc lysate was diluted 1:3 in sample buffer.

Source data are available online for this figure.