Figure 3.

Functional analysis of DCPS variants. (A) DCPS in vitro decapping assay. mRNA decapping was assayed using recombinant full-length DCPS wild-type (DCPS^WT) or altered [splice mutation/45 bp insertion (DCPS^Ins15) and p.Thr316Met(DCPS^T316M)] DCPS protein in pET28a (Novagen, EMD Biosciences) vector expressed and purified from Escherichia coli BL21(DE3) cells. Migration of the input cap structure (m⁷GpppG) or m⁷GMP product (m⁷Gp) is designated, with the red ‘p’ denoting the location of the ³²P. The percent decapping is indicated at the bottom of the panel. (B) DCPS activity in lymphoblastoid cell lines. Lymphoblastoid cell lines from homozygous affected (III-2 and IV-3) and heterozygous unaffected (III-3 and IV-2) family members were used for decapping assays. Decapping products and labeling are as in (A). (C) RT–PCR analysis for DCPS. RT–PCR across the DCPS splice mutation (from exons 3 to 6) was performed using mRNA from patients (III-2 and IV-3) and unaffected family member (III-3), plus cartoon indicating exon usage. (D) Western Blot of DCPS. Wild-type and variant DCPS protein from the indicated lymphoblast cell lines were detected by western blotting to demonstrate migration of the variant DCPS protein in homozygous and heterozygous individuals. A wild-type lymphoblastoid cell line is shown to indicate the position of wild-type DCPS.