Nucleolin binds to a subset of selenoprotein mRNAs and regulates their expression.
Selenium, an essential trace element, is incorporated into selenoproteins as selenocysteine (Sec), the 21st amino acid. In order to synthesize selenoproteins, a translational reprogramming event must occur since Sec is encoded by the UGA stop codon. In mammals, the recoding of UGA as Sec depends on the selenocysteine insertion sequence (SECIS) element, a stem-loop structure in the 3' untranslated region of the transcript. The SECIS acts as a platform for RNA-binding proteins, which mediate or regulate the recoding mechanism. Using UV crosslinking, we identified a 110 kDa protein, which binds with high affinity to SECIS elements from a subset of selenoprotein mRNAs. The crosslinking activity was purified by RNA affinity chromatography and identified as nucleolin by mass spectrometry analysis. In vitro binding assays showed that purified nucleolin discriminates among SECIS elements in the absence of other factors. Based on siRNA experiments, nucleolin is required for the optimal expression of certain selenoproteins. There was a good correlation between the affinity of nucleolin for a SECIS and its effect on selenoprotein expression. As selenoprotein transcript levels and localization did not change in siRNA-treated cells, our results suggest that nucleolin selectively enhances the expression of a subset of selenoproteins at the translational level.
Miniard
Angela C
AC
Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA.
Middleton
Lisa M
LM
Budiman
Michael E
ME
Gerber
Carri A
CA
Driscoll
Donna M
DM
D020413:3' Untranslated Regions; D000818:Animals; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D005979:Glutathione Peroxidase / Q000235:genetics / Q000378:metabolism; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D051140:Selenoproteins / Q000235:genetics* / Q000378:metabolism; D000097796:Nucleolin
D020413:3' Untranslated Regions; D005979:Glutathione Peroxidase; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D051140:Selenoproteins; D000097796:Nucleolin
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