The selenocysteine incorporation machinery: interactions between the SECIS RNA and the SECIS-binding protein SBP2.
The decoding of UGA as a selenocysteine (Sec) codon in mammalian selenoprotein mRNAs requires a selenocysteine insertion sequence (SECIS) element in the 3' untranslated region. The SECIS is a hairpin structure that contains a non-Watson-Crick base-pair quartet with a conserved G.A/A.G tandem in the core of the upper helix. Another essential component of the Sec insertion machinery is SECIS-binding protein 2 (SBP2). In this study, we define the binding site of SBP2 on six different SECIS RNAs using enzymatic and hydroxyl radical footprinting, gel mobility shift analysis, and phosphate-ethylation binding interference. We show that SBP2 binds to a variety of mammalian SECIS elements with similar affinity and that the SBP2 binding site is conserved across species. Based on footprinting studies, SBP2 protects the proximal part of the hairpin and both strands of the lower half of the upper helix that contains the non-Watson-Crick base pair quartet. Gel mobility shift assays showed that the G.A/A.G tandem and internal loop are critical for the binding of SBP2. Modification of phosphates by ethylnitrosourea along both strands of the non-Watson-Crick base pair quartet, on the 5' strand of the lower helix and part of the 5' strand of the internal loop, prevented binding of SBP2. We propose a model in which SBP2 covers the central part of the SECIS RNA, binding to the non-Watson-Crick base pair quartet and to the 5' strands of the lower helix and internal loop. Our results suggest that the affinity of SBP2 for different SECIS elements is not responsible for the hierarchy of selenoprotein expression that is observed in vivo.
Fletcher
J E
JE
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Copeland
P R
PR
Driscoll
D M
DM
Krol
A
A
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C121530:SECISBP2 protein, human; C498488:Secisbp2 protein, rat; D017279:Selenocysteine; D012313:RNA