Figure 6. RNPS1 and CASC3 Differ in Their Requirement for Efficient NMD.

(A) Fold change in levels of endogenous NMD-targeted transcripts (bottom) in HEK293 Flp-In TRex cells depleted of core or alternate EJC factors. (EIF4A3 knockdown: 48 hr, alternate EJC factor knockdown: 96 hr). Shown are the average values normalized to TBP levels from three biological replicates ± standard error of means (SEM). *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Percent knockdown of each protein in a representative experiment is in the legend.

(B) An EJC core crystal structure (PDB: 2J0S) showing the EIF4A3 residues that interact with CASC3 (enlarged below).

(C) Real-time PCR analysis as in (A) from HEK293 cells depleted of EIF4A3. The EIF4A3 knockdown data are the same as in (A) and is plotted on the left y axis. Either wild-type EIF4A3 or a mutant with reduced CASC3 interaction were exogenously expressed in EIF4A3 knockdown cells as indicated in the legend on the top right, and these data are plotted on the right y axis.

(D) Northern blots showing levels of wild-type (bwt, lane 1) or PTC-containing (codon 39; β39; lanes 2-5) β-globin mRNA and a longer internal control β-globin (βUAC-GAP) mRNA from HeLa Tet-off cells treated with siRNAs indicated on the top. Tables below indicate percentage of normalized β39 mRNA as compared to normalized βwt mRNA (top) or fold-increase in β39 mRNA upon knockdown as compared to control.

See also Figure S6.