SiRNA knockdown‐mRNA knock‐in as a means of assessing elenoprotein function

Abstract

A knockdown/knock-in strategy was designed for studying selenoprotein (SP) function. The technique entails the initial knockdown of gene expression using RNAi technology wherein the siRNA target is directed to the SP mRNA 3′-untranslated region (3′-UTR). Gene expression is then knocked-in circumventing the siRNA by mutations within the target region of the knock-in construct. We tested this strategy by targeting thioredoxin reductase 1 (TR1), which is an essential Sec-containing oxidoreductase that controls intracellular redox state in mammals. TR1 knockdown cells grew more slowly, were more sensitive to UV irradiation and had increased apoptosis in response to UV than control cells. Cells transfected with a construct encoding the wild type TR1 gene and having mutations in the sequences targeted by siRNA, restored TR1 expression and catalytic activity. Replacement of TR1 reversed the sensitivity of TR1 knockdown cells by UV irradiation. The data demonstrate that the RNAi-based knock-in technology provides a means of studying SP function and this approach provides an alternative means for examining many of the subtleties of protein function not available using RNAi technology alone. In addition, by targeting the 3′-UTR, the coding sequence is not altered and can be changed in subsequent knock-in experiments. This research was supported by the Intramural Research Program of the NIH, NCI, CCR, and by NIH GM065204.