A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in targeting the 3'-UTR.
Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3'-untranslated regions (3'-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3'-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown 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, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone.
Yoo
Min-Hyuk
MH
Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland 20892, USA.
Xu
Xue-Ming
XM
Turanov
Anton A
AA
Carlson
Bradley A
BA
Gladyshev
Vadim N
VN
Hatfield
Dolph L
DL
D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D010641:Phenotype; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011836:Radiation Tolerance / Q000235:genetics; D051140:Selenoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014466:Ultraviolet Rays; D000094765:Glutathione Peroxidase GPX1
D020413:3' Untranslated Regions; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051140:Selenoproteins; D005979:Glutathione Peroxidase; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase; C516125:Txnrd1 protein, mouse; D010770:Phosphotransferases; C499551:Sephs2 protein, mouse; D000094765:Glutathione Peroxidase GPX1; C000722068:Gpx1 protein, mouse