Nonsense-Mediated Decay: Assaying for Effects on Selenoprotein mRNAs

Abstract

If the abundance of a particular selenoprotein mRNA is reduced during selenium deprivation, then the mRNA is likely to be a natural substrate for NMD. One assay for NMD involves changing the TGA Sec codon(s) to either a TGC cysteine codon or a TAA nonsense codon. If selenium deprivation elicits NMD and has no other effect on selenoprotein gene expression, then, regardless of selenium concentration, the level of UGC-containing mRNA should be most abundant, the level of UGA-containing mRNA should be intermediate in abundance, and the level of UAA-containing mRNA should be least abundant. Furthermore, the level of UGA-containing mRNA should be decreased by a decrease in selenium concentration, while the levels of UGC- and UAA-containing mRNAs should be unaffected by selenium concentration. A different assay for NMD involves coexpression of the particular selenoprotein gene and a vector expressing a dominant-negative version of hUpf1p. This assay is simpler and more versatile than the first assay because it can be used to assay any cellular gene in situ provided the cells can be stably transfected with the hUpf1p expression vector.