Figure 5. Overexpression of SUGP1 Partially Rescues the Mutant Spliceosome.

(A and B) The indicated plasmids were cotransfected to HEK293T cells in 10-cm plates, followed by affinity purification using the small-scale protocol. The SF3B1-associated proteins were resolved by SDS-PAGE, followed by silver staining (A) or western blotting (B). Marker, Precision Plus Protein marker (Bio-Rad); Vec, empty vector plasmid; SUGP1, expression plasmid for HA-tagged SUGP1; WT, expression plasmid for His6-FLAG-tagged WT SF3B1; K700E, expression plasmid for His6-FLAG-tagged K700E SF3B1.

(C) The indicated plasmids were cotransfected to HEK293T cells in six-well plates. Total RNA was extracted from the cells, followed by RT-PCR of the cryptic 3′ss (open arrowheads) and canonical 3′ss (solid arrowheads) of two select genes (GCC2 and KANSL3). Vec, empty vector plasmid; SUGP1, expression plasmid for HA-tagged SUGP1; WT, expression plasmid for HA-tagged WT SF3B1; K700E, expression plasmid for HA-tagged K700E SF3B1. Note that the PCR products of the cryptic 3′ss of GCC2 and KANSL3 (open arrowheads) in the sixth lane appeared to migrate slightly more slowly than the PCR products in the fifth lane. However, DNA sequencing results indicated that the cryptic 3′ss and canonical 3′ss of GCC2 and KANSL3 in the last two lanes were exactly the same.

(D) Quantification of RT-PCR products in (C). The bar graph was made using Prism (GraphPad). Error bars represent SDs of the means (n = 3, three independent experiments). **p < 0.01 (unpaired, two-tailed, and unequal variance t test, calculated using Microsoft Excel).

(E) Protein extracts from HEK293T cells as in (C) were resolved by SDS-PAGE, followed by western blotting.

See also Figures S5 and S6.