Figure 5.

RIOK3 X2 variant mRNA encodes a truncated protein product and is targeted by nonsense-mediated decay (NMD). (A) Schematic of the normal and X2 variant alternative splicing on the precursor RIOK3 RNA transcript together with a representation of the full-length and X2 variant RIOK3 protein isoforms. (B) Alternatively spliced RIOK3 mRNA is efficiently degraded by nonsense mediated decay. HEK293 cells were mock treated or treated with cycloheximide (CHX) for the times indicated. Total RNA was harvested and subjected to RT-PCR using primers flanking the spliced exons. PCR products were run on agarose and stained using ethidium bromide. As CHX inhibits NMD, X2 and X1/X2 products are preserved with treatment. Actin B was used as a control. (C) Expression of RIOK3 full-length and X2 variant proteins from transfected expression plasmids. RIOK3 full-length and X2 constructs were transfected into HEK293 cells and expression was analyzed by Western blot. The presence of the same FLAG tag at the N-terminus of both proteins allowed us to compare their expression efficiencies.