C9orf 72 interacts with coilin and influences Cajal body dynamics and splicing
published: July 21, 2017, recorded: May 2017, views: 8
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Rationale & Hypothesis: The GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the leading cause of amyotrophic lateral sclerosis (ALS). Although the precise disease mechanisms are unclear, haploinsufficiency of C9orf72 has been proposed. Little is currently known about the function of the C9orf72 protein, but a yeast two- hybrid assay found an interaction between C9orf72 and coilin. Coilin is the major protein component of Cajal bodies (CBs), nuclear suborganelles which act as sites of splicing machinery maturation and assembly. C9orf72 ALS patients have splicing defects that correlate with disease severity. We hypothesise that haploinsufficiency of the C9orf72 protein in ALS leads to dysregulation of Cajal bodies and downstream splicing.
Objectives: Confirm the interaction between C9orf72 and coilin and investigate the effect of C9orf72 on Cajal bodies and splicing.
Methodology: An in vitro GST-binding assay was used to establish if there is a direct interaction between GST-C9orf72 and 35S-labelled coilin. Co-immunoprecipitation experiments were performed on Hek293 cell lysates to confirm the cellular interaction between myc-C9orf72 and endogenous coilin. The localisation of the interaction was investigated using an in situ Proximity Ligation Assay (PLA). Immunostaining for coilin and SMN was performed on Hek293 cells following C9orf72 knockdown to investigate the effect of C9orf72 on CBs. Finally, an artificial reporter splicing assay was used to study the effect of C9orf72 knockdown on splicing efficiency.
Findings: The GST-binding assay suggests there is a direct interaction between C9orf72 and coilin in vitro. In cell lysates, myc-C9orf72 was successfully co- immunoprecipitated with endogenous coilin, suggesting the interaction is physiologically relevant. PLA confirmed the interaction and shows it occurs in both the cytoplasm and nucleus. Interestingly, C9orf72 knockdown in Hek293 cells led to an increase in the number of CBs but a decrease in the splicing of both major and minor introns in the artificial reporter splicing assay. The results confirm C9orf72 interacts with coilin and may regulate CBs and splicing.
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