PMID- 17468436
OWN - NLM
STAT- MEDLINE
DCOM- 20070710
LR  - 20221207
IS  - 1355-8382 (Print)
IS  - 1469-9001 (Electronic)
IS  - 1355-8382 (Linking)
VI  - 13
IP  - 6
DP  - 2007 Jun
TI  - A new strategy for assessing selenoprotein function: siRNA knockdown/knock-in 
      targeting the 3'-UTR.
PG  - 921-9
AB  - 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.
FAU - Yoo, Min-Hyuk
AU  - Yoo MH
AD  - Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center 
      for Cancer Research, National Institutes of Health, Bethesda, Maryland 20892, 
      USA.
FAU - Xu, Xue-Ming
AU  - Xu XM
FAU - Turanov, Anton A
AU  - Turanov AA
FAU - Carlson, Bradley A
AU  - Carlson BA
FAU - Gladyshev, Vadim N
AU  - Gladyshev VN
FAU - Hatfield, Dolph L
AU  - Hatfield DL
LA  - eng
GR  - R37 GM065204/GM/NIGMS NIH HHS/United States
GR  - GM065204/GM/NIGMS NIH HHS/United States
GR  - R01 GM065204/GM/NIGMS NIH HHS/United States
GR  - R01 GM061603/GM/NIGMS NIH HHS/United States
GR  - GM061603/GM/NIGMS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20070427
PL  - United States
TA  - RNA
JT  - RNA (New York, N.Y.)
JID - 9509184
RN  - 0 (3' Untranslated Regions)
RN  - 0 (RNA, Messenger)
RN  - 0 (RNA, Small Interfering)
RN  - 0 (Selenoproteins)
RN  - EC 1.11.1.9 (Glutathione Peroxidase)
RN  - EC 1.8.1.9 (Thioredoxin Reductase 1)
RN  - EC 1.8.1.9 (Thioredoxin-Disulfide Reductase)
RN  - EC 1.8.1.9 (Txnrd1 protein, mouse)
RN  - EC 2.7.- (Phosphotransferases)
RN  - EC 2.7.9.3 (Sephs2 protein, mouse)
RN  - EC 1.11.1.9 (Glutathione Peroxidase GPX1)
RN  - EC 1.11.1.9 (Gpx1 protein, mouse)
SB  - IM
MH  - 3' Untranslated Regions
MH  - Animals
MH  - Cell Line
MH  - Glutathione Peroxidase/genetics/metabolism
MH  - In Vitro Techniques
MH  - Mice
MH  - Mutation
MH  - NIH 3T3 Cells
MH  - Phenotype
MH  - Phosphotransferases/genetics/metabolism
MH  - RNA Interference
MH  - RNA, Messenger/genetics/metabolism
MH  - RNA, Small Interfering/*genetics
MH  - Radiation Tolerance/genetics
MH  - Selenoproteins/antagonists & inhibitors/*genetics/*metabolism
MH  - Thioredoxin Reductase 1
MH  - Thioredoxin-Disulfide Reductase/deficiency/genetics/metabolism
MH  - Transfection
MH  - Ultraviolet Rays
MH  - Glutathione Peroxidase GPX1
PMC - PMC1869036
EDAT- 2007/05/01 09:00
MHDA- 2007/07/11 09:00
PMCR- 2007/12/01
CRDT- 2007/05/01 09:00
PHST- 2007/05/01 09:00 [pubmed]
PHST- 2007/07/11 09:00 [medline]
PHST- 2007/05/01 09:00 [entrez]
PHST- 2007/12/01 00:00 [pmc-release]
AID - rna.533007 [pii]
AID - 10.1261/rna.533007 [doi]
PST - ppublish
SO  - RNA. 2007 Jun;13(6):921-9. doi: 10.1261/rna.533007. Epub 2007 Apr 27.