The Exosome Complex Is Reduced in Skeletal Muscle and Fibroblasts
Immunoblotting for components of the exosome complex was performed on cultured fibroblasts of individual 1:II-1 and skeletal muscle of individual 2:II-1. EXOSC9 was less abundant in both affected fibroblasts and skeletal muscle than in controls. Additionally, in fibroblasts from individuals harboring mutations in EXOSC3, EXOSC8, and RBM7, EXOSC9 was reduced; the reduction was most pronounced in cells from the EXOSC3 and EXOSC8 cell lines (Figure 3A). These data suggest that a primary reduction in one component of the exosome complex, or in a related protein such as RBM7, leads to destabilization of the whole complex. BN-PAGE with protein lysates from affected individual’s fibroblasts probed with an antibody for EXOSC3 supported this hypothesis. Variants in EXOSC3, EXOSC8, EXOSC9, and RBM7 resulted in reduction of the exosome complex irrespective of which subunit carried the primary variant (Figure 3B).
Figure 3 The Exosome Complex Is Reduced in Affected Individuals’ Fibroblasts
(A) Immunoblotting of fibroblasts from affected individuals with variants in different components of the exosome complex (the homozygous c.92G>C in EXOSC3, the homozygous c.5C>T in EXOSC8, the homozygous c.41T>C in EXOSC9 [individual 1:II-1], and the homozygous c.236C>G in RBM7) shows reduced EXOSC9, but other components of the exosome complex were also reduced. Actin was used as a loading control.
(B) Blue native polyacrylamide gel electrophoresis (BN-PAGE) shows that there is a reduction of the assembly of the whole exosome complex in affected individuals’ fibroblasts. GAPDH was used as a loading control.
(C) Immunoblot on muscle extracts from affected individual 2:II-1 and four controls confirms that EXOSC9 was severely reduced in affected individual 2:II-1.